<%BANNER%>

Chemical and Economic Analysis of a Value-Added Product from Muscadine Grape Pomace

Permanent Link: http://ufdc.ufl.edu/UFE0021495/00001

Material Information

Title: Chemical and Economic Analysis of a Value-Added Product from Muscadine Grape Pomace
Physical Description: 1 online resource (106 p.)
Language: english
Creator: Cardona Ponce, Jorge Alfredo
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: analysis, anthocyanins, antioxidant, economic, fermentation, grape, isolation, muscadine, phytochemical, polyphenolics, pomace, stability
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Polyphenolics are recognized for their antioxidant capacity and their contribution to flavor and color of several fruits and vegetables. Health benefits of these compounds are still under investigation and their use is currently based on observed experiments both in vitro and in vivo. The sources of natural antioxidants such as polyphenolics include fruits, vegetables, spices, and herbs. Muscadine grapes (Vitis rotundifolia) are an important fruit crop in the southeastern region of U.S. due to their tolerance to Pierce?s disease. These grapes have a unique polyphenolic profile compared to other Vitis species that has sustained efforts to develop a value-added product from them. By-products (skin and seeds) from muscadine grape processing have presented processors with an inexpensive source of polyphenolics to produce valuable food ingredients. This research study evaluated isolation techniques and subsequent stability of compounds recovered from muscadine grape pomace. Methods to reduce or eliminate sugars were also explored by the use of yeast fermentation and solid-phase isolation. Following fermentation, methods to remove residual water were explored using freeze drying, spray drying and vacuum evaporation. Additionally, processes were assessed to determine the efficiency and profitability for the muscadine grape industry. Target compounds extracted from muscadine grape pomace showed high antioxidant activity (34.3 ? 0.57 micromol Trolox Equivalents/g). Although processing positively influenced polymerization and stability of polyphenolics, antioxidant capacity was significantly decreased. Initially, high temperature, low concentration of polyphenolics, oxygen exposure, and high pH environment were considered major factors that affected polyphenolic content and bioactive characteristics. Latter information indicated that such factors had a significant effect on the polyphenolics antioxidant capacity but only a small change on their concentration. Vacuum drying showed the best results for maintaining polyphenolics concentration and preserving their antioxidant capacity following fermentation. Fermentation proved to be a more practical procedure than solid phase isolation to eliminate or reduce sugars without putting the valuable nutritional characteristics of the final product at risk. Fermentation following a simple concentration step was an economical way to obtain polyphenolics from muscadine grape skins. Results from three isolation techniques suggested that a simple muscadine grape by-product concentration followed by a drying operation can be suitable and profitable for a typical muscadine producer. This process could be adjusted and implemented by any fruit or vegetable processor to estimate the potential increase in profit of their additional by-product operation.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jorge Alfredo Cardona Ponce.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Sims, Charles A.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021495:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021495/00001

Material Information

Title: Chemical and Economic Analysis of a Value-Added Product from Muscadine Grape Pomace
Physical Description: 1 online resource (106 p.)
Language: english
Creator: Cardona Ponce, Jorge Alfredo
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: analysis, anthocyanins, antioxidant, economic, fermentation, grape, isolation, muscadine, phytochemical, polyphenolics, pomace, stability
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Polyphenolics are recognized for their antioxidant capacity and their contribution to flavor and color of several fruits and vegetables. Health benefits of these compounds are still under investigation and their use is currently based on observed experiments both in vitro and in vivo. The sources of natural antioxidants such as polyphenolics include fruits, vegetables, spices, and herbs. Muscadine grapes (Vitis rotundifolia) are an important fruit crop in the southeastern region of U.S. due to their tolerance to Pierce?s disease. These grapes have a unique polyphenolic profile compared to other Vitis species that has sustained efforts to develop a value-added product from them. By-products (skin and seeds) from muscadine grape processing have presented processors with an inexpensive source of polyphenolics to produce valuable food ingredients. This research study evaluated isolation techniques and subsequent stability of compounds recovered from muscadine grape pomace. Methods to reduce or eliminate sugars were also explored by the use of yeast fermentation and solid-phase isolation. Following fermentation, methods to remove residual water were explored using freeze drying, spray drying and vacuum evaporation. Additionally, processes were assessed to determine the efficiency and profitability for the muscadine grape industry. Target compounds extracted from muscadine grape pomace showed high antioxidant activity (34.3 ? 0.57 micromol Trolox Equivalents/g). Although processing positively influenced polymerization and stability of polyphenolics, antioxidant capacity was significantly decreased. Initially, high temperature, low concentration of polyphenolics, oxygen exposure, and high pH environment were considered major factors that affected polyphenolic content and bioactive characteristics. Latter information indicated that such factors had a significant effect on the polyphenolics antioxidant capacity but only a small change on their concentration. Vacuum drying showed the best results for maintaining polyphenolics concentration and preserving their antioxidant capacity following fermentation. Fermentation proved to be a more practical procedure than solid phase isolation to eliminate or reduce sugars without putting the valuable nutritional characteristics of the final product at risk. Fermentation following a simple concentration step was an economical way to obtain polyphenolics from muscadine grape skins. Results from three isolation techniques suggested that a simple muscadine grape by-product concentration followed by a drying operation can be suitable and profitable for a typical muscadine producer. This process could be adjusted and implemented by any fruit or vegetable processor to estimate the potential increase in profit of their additional by-product operation.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jorge Alfredo Cardona Ponce.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Sims, Charles A.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021495:00001


This item has the following downloads:


Full Text





CHEMICAL AND ECONOMIC ANALYSIS OF A VALUE-ADDED PRODUCT FROM
MUSCADINE GRAPE POMACE





















By

JORGE ALFREDO CARDONA PONCE


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2007

































O 2007 Jorge Alfredo Cardona Ponce

































To my family and all the people that have believed in me









ACKNOWLEDGMENTS

I want to thank my first advisor Dr. Stephen Talcott to guide me through this experience,

make this work possible, and for giving me the opportunity to be in graduate school. Also, I

extend special gratitude to Dr. Charles A. Sims who guided me through the second part of this

research and accepted me as his student to finish my graduate student experience at the

University of Florida. I also thank Dr. Murat Balaban and Dr. Allen Wysocki for their countless

advices and time to help in the production of this work. And last but not least, I thank all my

friends and lab mates who have graduated with Dr. Talcott: Flor, Youngmok, Chris, Lisbeth,

Joon, David, Lanier, and Kristine for sharing good times, helping me out in every way possible,

and sharing their knowledge and time.

I also want to thank my family (Fernando, Susana, and Diego) for supporting me in my

decisions that have guided me all the way from Bolivia to Florida. I owe them all the knowledge

and respect I have for others. I truly appreciate their schooling and philosophy of life. Finally,

my most sincere and deepest appreciation to Thelma, for all the patience and support during my

experience in college, graduate school, and for all the happiness we share.












TABLE OF CONTENTS


page

ACKNOWLEDGMENT S .............. ...............4.....


LI ST OF T ABLE S ........._._.. ....._. ...............7.....


LI ST OF FIGURE S .............. ...............8.....


AB S TRAC T ............._. .......... ..............._ 10...


CHAPTER


1 INTRODUCTION ................. ...............12.......... ......


2 LITERATURE REVIEW ................. ...............15................


2. 1 Muscadine Grape ................. ...............15................
2.2 Polyphenols................ ...................1
2.2.1 Structure and Classification............... ............1
2.2.1.1 Phenolic acids and simple phenols............... ...............18
2.2. 1.2 Flavonoids ................. ...............18.......... ....
2.2. 1.3 Tannins ................ ........ ..... ...............20 ...
2.2.2 Polyphenolics as Antioxidants .............. ...............21....
2.2.3 Polyphenolics in Mucscadine Grapes ................. ...............23........... ...
2.3 Anthocyanins ............... ...............24....
2.3.1 Structure and Occurrence .............. ...............24....
2.3.2 Color Stability ............... .... ...............26.
2.3.3 Anthocyanins in Muscadine Grape .............. ...............31....
2.4 Ellagic Acid ................. ...............32...
2.4.1 Structure and Occurrence .............. ...............32....
2.4.2 Ellagic Acid Derivatives............... .. ..................3
2.4.3 Ellagic Acid and Its Derivatives in Muscadine Grape .............. ....................3
2.5 Processing Effects on Polyphenolics ................. ...............35........... ..
2.5.1 Heat Procedures ................. ...............36...............
2.5.2 Drying Procedures .................. ...............37..............
2.5.3 Extraction Procedures and Storage ................. ...............40........... ..
2.5.4 Enzymatic Procedures .............. ...............41....


3 PHYTOCHEMICAL, ANTIOXIDANT AND PIGMENT STABILITY OF
MUSCADINE GRAPE POMACE AS AFFECTED BY CONCENTRATION and
DEHYDRATION ............ ..... .__ ...............42...


3 .1 Introducti on ............ ..... .._ ...............42..
3.2 Materials and Methods .............. ...............43....
3.2. 1 Materials and Processing ............ ......__ ...............43.
3.2.2 Solid Phase Isolation .............. ...............45....












3.2.3 Chemical Analysis................. .... ...... ...................4
3.2.3.1 Spectrophotometric determination of total anthocyanins.............................4
3.2.3.2 Determination of polymeric anthocyanins ................ ....._._ ..............48
3.2.3.3 Determination of total soluble phenolics .............. ...............49....
3.2.3.4 Quantification of antioxidant capacity .............. ...............49....
3.2.3.5 Half life determination .........____........_.__ ....___ ......_... ........50
3.2.3.6 Analysis of polyphenolics by HPLC ....._____ ..... ...___ ...........__....50
3.2.4 Statistical analysis .............. ...............51....
3.3 Results and Discussion ............ _....... .._ ...............51..
3.3.1 Anthocyanin Color Stability ........._...._ ............. ...............52....
3.3.2 Polyphenolic Concentration and Stability ................. ...............57..............
3.3.3 Polyphenolics by HPLC .............. ...............58....
3.3.3.1 Anthocyanins by HPLC .................. ...............58..
3.3.3.2 Ellagic acid and flavonols by HPLC .............. ...............61....
3.3.4 Antioxidant Capacity ........._._._..... ..... ...............63...
3.4 Conclusions............... ..............6


4 ECONOMIC ANALYSIS OF AN ISOLATED PRODUCT OBTAINED FROM
MUSCADINE GRAPE POMACE ................. ...............67........... ....


4.2 M materials and M ethods .............. ...............68....
4.2.1 Data Collection............... ...............6
4.2.2 Economic Analysis............... .. ...............6
4.2.2. 1 Description of the operation ................. ...............69...............
4.2.2.2 Economic assumptions ................. ...............69........... ....
4.3 Results and Discussion .............. ...............72....
4.3.1 Economic Analysis............... ...............72
4.3.1.1 Spray drying operation ................. ...............73........... .
4.3.1.2 Freeze drying operation ................. ...............78........... ..
4.3.1.3 Vacuum drying operation............... ...............8
4.4 Conclusions............... ..............9


5 SUMMARY AND CONCLUSIONS ................ ...............92................


APPENDIX


A PRELIMINARY STUDY I .............. ...............94....


B PRELIMINARY STUDY II ................. ...............95................


C PRELIMINARY STUDY III............... ...............96..


LIST OF REFERENCES ................. ...............97........... ....


BIOGRAPHICAL SKETCH ................. ...............106......... ......










LIST OF TABLES


Table page

2-1 Estimated muscadine grape acreage in the southeastern United States ................... ..........16

2-2 Classes and dietary sources of flavonoids. ............. ...............20.....

3-1 Quality analyses of muscadine pomace extract polyphenolss) as affected by various
processing protocol s............... ...............5

3-2 Anthocyanidin concentrations in the muscadine pomace extract as affected by
various processing protocol s. ................. ...............61......_......

3-3 Ellagic acid and flavonol concentrations in the muscadine pomace extract as affected
by various processing protocols............... ...............6

4-1 Generalities of drying equipment. .............. ...............72....

4-2 Loan payment plan for ten years at a fixed rate of 7.5%. ............. .....................7

4-3 Capital expenditure to initiate a marginal process obtaining extract and dried skins
from muscadine grape skins using spray drying as the isolation technique. .....................74

4-4 Sensitivity analysis for a marginal process obtaining extract and dried skins from
muscadine grape skins using spray drying as the isolation technique ............... ...............77

4-6 Capital expenditure to initiate a marginal process obtaining extract and dried skins
from muscadine grape skins using freeze drying as the isolation technique. ....................78

4-5 Cash flow analysis for a marginal process obtaining extract and dried skins from
muscadine grape skins using spray drying as the isolation technique ............... ...............79

4-7 Sensitivity analysis for a marginal process obtaining extract and dried skins from
muscadine grape skins using freeze drying as the isolation technique. .............................83

4-8 Cash flow analysis for a marginal process obtaining extract and dried skins from
muscadine grape skins using freeze drying as the isolation technique. .............................84

4-9 Capital expenditure to initiate a marginal process obtaining extract and dried skins
from muscadine grape skins using vacuum drying as the isolation technique. .................85

4-10 Sensitivity analysis for a marginal process obtaining extract and dried skins from
muscadine grape skins using vaccum drying as the isolation technique. ..........................88

4-11 Cash flow analysis for a marginal process obtaining extract and dried skins from
muscadine grape skins using vacuum drying as the isolation technique. ..........................90










LIST OF FIGURES


Figure page

2-1 Basic flavonoid structure. ............. ...............19.....

2-2 General structure of hydrolyzable tannins (left) and condensed tannins (right) ................21

2-3 Chemical structures of anthocyanidins .............. ...............25....

2-4 Anthocyanin equilibria: quinonoidal base (A), flavylium cation (B), carbinol base or
pseudobase (C) and chalcone (D). ............. ...............27.....

2-5 Effect of pH value on anthocyanin equilibria ................. ........___ ....................28

2-6 Anthocyanin diglycoside structure (Cyanidin-3,5-diglucoside). ................... ...............31

2-7 Chemical structure of ellagic acid............... ...............32..

2-8 Ellagic acid glycosides A) Ellagic acid-4-arabinoside, B) Ellagic acid-4-
acetylarabinoside, C) Ellagic acid-4-acetylxyloside ................. ............................33

2-9 Ellagitannins: Tellimagrandin II (monomeric ET) (left), Sanguiin H-6 (oligomeric
ET) (right). ............. ...............34.....

2-10 Phase diagram of water ................. ...............39......__. ...

3-1 Total anthocyanin content of muscadine pomace extract as affected by various
processing methods. Error bars represent the standard error of each mean, n=3. ............53

3-2 Polymeric anthocyanins (%) in muscadine pomace extract as affected by various
processing methods. Error bars represent the standard error of each mean, n=3. ....._.....55

3-3 Half life (min) of muscadine pomace extract as affected by various processing
methods. Error bars represent the standard error of each mean, n=3. .........._... ..............56

3-4 Total phenolic content in muscadine pomace extract as affected by various
processing methods. Error bars represent the standard error of each mean, n=3. ............58

3-5 HPLC chromatogram of anthocyanidins present in muscadine pomace: delphinidin
(A), cyanidin (B), petunidin (C), pelargonidin (D), peonidin (E), malvidin (F).
Identification (520 nm) was done based on spectral characteristics and comparison to
cyanidin aglycone. ............. ...............59.....

3-6 HPLC chromatogram of polyphenolics present in muscadine pomace: ellagic acid
(A), myricetin (B), and quercetin (C). Identification (360 nm) was done based on
spectral characteristics and comparison to authentic standards of ellagic acid and
quercetin ................. ...............62.......__.....










3-7 Antioxidant capacity of muscadine pomace extract as affected by various processing
protocols. Error bars represent the standard error of the mean, n=3 ................. ...............65

4-1 Operation flow for a typical grape juice processor planning to process its byproduct......70

4-2 Volume break-even point for a facility using a spray dryer as a final step for product
isolation. Abbreviations: FC Fixed costs, VC variable costs, TC total costs, I
income e. ................. ...............75....... ......

4-3 Break-even point as affected by price for a facility using a spray dryer as a final step
for product isolation. Abbreviations: I income, TC total costs. ................. ......._........77

4-4 Volume break-even point for a facility using a freeze dryer as a final step for product
isolation. Abbreviations: FC Fixed costs, VC variable costs, TC total costs, I
income e. ................. ...............81....... ......

4-5 Break-even point as affected by price for a facility using a freeze dryer as a final step
for product isolation. Abbreviations: I income, TC total costs. ................. ......._........82

4-6 Volume break-even point for a facility using a vacuum dryer as a final step for
product isolation. Abbreviations: FC Fixed costs, VC variable costs, TC total costs, I
income e. ................. ...............86....... ......

4-7 Break-even point as affected by price for a facility using a vacuum dryer as a final
step for product isolation. Abbreviations: I income, TC total costs. ............. .................88

A-1 Total anthocyanins during a 3-day extraction procedure ................. ................ ...._.94

A-2 oBrix values during a 3-day concentration procedure. ........._..._ .......... ...............94

B-1 Total anthocyanin content in a 1-day extraction procedure ................. ......................95

B-2 oBrix values in a 1-day extraction procedure. ....._.__._ .... ... .__. .....__. ........9

C-1 Total anthocyanin content after a 5-hour concentration procedure. ............. ..................96









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Master of Science

CHEMICAL AND ECONOMIC ANALYSIS OF VALUE-ADDED PRODUCT FROM
MUSCADINE GRAPE POMACE

By

Jorge Alfredo Cardona Ponce

December 2007

Chair: Charles A. Sims
Maj or: Food Science & Human Nutrition

Polyphenolics are recognized for their antioxidant capacity and their contribution to flavor

and color of several fruits and vegetables. Health benefits of these compounds are still under

investigation and their use is currently based on observed experiments both in vitro and in vivo.

The sources of natural antioxidants such as polyphenolics include fruits, vegetables, spices, and

herbs. Muscadine grapes (Vitis rotundifolia) are an important fruit crop in the southeastern

region of U.S. due to their tolerance to Pierce's disease. These grapes have a unique

polyphenolic profile compared to other Vitis species that has sustained efforts to develop a value-

added product from them. By-products (skin and seeds) from muscadine grape processing have

presented processors with an inexpensive source of polyphenolics to produce valuable food

ingredients. This research study evaluated isolation techniques and subsequent stability of

compounds recovered from muscadine grape pomace. Methods to reduce or eliminate sugars

were also explored by the use of yeast fermentation and solid-phase isolation. Following

fermentation, methods to remove residual water were explored using freeze drying, spray drying

and vacuum evaporation. Additionally, processes were assessed to determine the efficiency and

profitability for the muscadine grape industry. Target compounds extracted from muscadine

grape pomace showed high antioxidant activity (34.3 + 0.57 Cpmol Trolox Equivalents/g).









Although processing positively influenced polymerization and stability of polyphenolics,

antioxidant capacity was significantly decreased. Initially, high temperature, low concentration

of polyphenolics, oxygen exposure, and high pH environment were considered maj or factors that

affected polyphenolic content and bioactive characteristics. Latter information indicated that

such factors had a significant effect on the polyphenolics antioxidant capacity but only a small

change on their concentration. Vacuum drying showed the best results for maintaining

polyphenolics concentration and preserving their antioxidant capacity following fermentation.

Fermentation proved to be a more practical procedure than solid phase isolation to eliminate or

reduce sugars without putting the valuable nutritional characteristics of the final product at risk.

Fermentation following a simple concentration step was an economical way to obtain

polyphenolics from muscadine grape skins. Results from three isolation techniques suggested

that a simple muscadine grape by-product concentration followed by a drying operation can be

suitable and profitable for a typical muscadine producer. This process could be adjusted and

implemented by any fruit or vegetable processor to estimate the potential increase in profit of

their additional by-product operation.









CHAPTER 1
INTTRODUCTION

Polyphenlics are plant metabolites that are recognized for their antioxidant capacity and

their contribution to flavor and color of several fruits and vegetables (Croft 1999). In recent

years, polyphenolic characteristics such as enzyme inhibition and radical scanvenging have

captured consumer' s attention because of the association of these compounds, and their activity,

with long-term human health (Parr and Bowell 2000).

Since the knowledge of how polyphenolics behave in the body is still limited, the

efficiency of natural antioxidant products is not easy to estimate. Furthermore, phenolic

compounds could act synergistically or antagonistically which complicates the antioxidant

product assessment. Therefore, the use of most natural antioxidant products is currently based

on empirical knowledge from research conducted in model systems and some existing products

(Meyer and others 2002).

The sources of natural antioxidants such as vitamin C, tocopherols, polyphenolics, and

organic acids include fruits, vegetables, spices, and herbs. The share of polyphenolics in the

market of antioxidants has increased as the demand for antioxidants from natural sources grows

steadily. In 1996, 26% of the food antioxidant market was occupied by natural antioxidants with

a yearly growing rate of 6-7% (Meyer and others 2002). In berries and fruits, the most abundant

antioxidants are vitamin C and polyphenolics. Companies such as Optiture (USA), Chr. Hansen

(Denmark), Overseal Natural Ingredients (GB), Quim Dis (France), Inheda (France), and

Folexco (USA) share the market of extracts and concentrates from these sources (Meyer and

others 2002).

Muscadine grapes (Vitis rotundifolia) are unique among grape species due to the presence

of polyphenolics such as anthocyanin diglucosides and ellagic acid and its derivatives (Lee and










Talcott 2004). Moreover, muscadine grapes are an important native fruit crop to the south and

southeastern U.S. due to their remarkable tolerance to Pierce's disease. Pierce's disease is caused

by a bacterium (Xyllela fa;stidiosa)) that invades the vascular system of grape vines causing

decay, and this microorganism is commonly transported by glassy-winged sharpshooters

(Pooling, 1996; Mizell and others 2003). Due to the resistance to Pierce's disease, muscadine

grapes have sustained a commercial industry in the southeast region of the U. S. (Ruel and

Walker 2006).

A growing concern for grape juice and wine producers is the handling of by-products (skin

and seeds) and the production of food-grade products with added value. The importance of

compounds in grape pomace that may have significance to the food industry due to their

association to human health has sustained efforts to produce valuable food ingredients (Sort

2003). In the case of muscadine grapes, almost half of the fresh fruit weight constitutes skin that

is a very rich source of phytochemicals such as resveratrol, ellagic acid and some flavonols

(Pastrana-Bonilla and others 2003; Morris and Brady 2004). However, unique characteristics of

muscadine grapes have presented technological challenges to extract polyphenolics from the

pomace.

This research study assessed isolation techniques and subsequent stability of polyphenolics

recovered from muscadine grape pomace (target compounds from the grape skins only) using

various extraction and processing techniques to obtain a dry powder or a low water concentrate.

Methods to reduce or eliminate sugars were explored by the use of aerobic yeast fermentation

and partitioning from solid-phase supports with specific affinities to the compounds of interest.

Processing methods to reduce or remove residual water were explored using freeze drying, spray

drying and vacuum evaporation techniques. It was hypothesized that extract processing would










affect polyphenolic stability. Processes were sought to optimize the concentration and stability

of target polyphenolics in an effort to determine the most efficient and profitable process for the

industry. The specific objectives of this study were:

* To evaluate the antioxidant capacity, polyphenolic composition and pigment stability of
muscadine grape pomace extract as affected by concentration, sugar elimination, and
dehydration processes.

* To propose protocols suitable for the Muscadine grape industry to develop an extract rich
in polyphenolics.

* To evaluate the profitability of best proposed protocols to manufacture an extract from the
muscadine grape pomace.









CHAPTER 2
LITERATURE REVIEW

2.1 Muscadine Grape

Muscadine grapes (Vitis rotundifolia) are an important native fruit crop to the south and

southeastern U.S and have been cultivated and utilized by people in these regions before

European colonization (Poling 1996). Due to the high pressure of insect vectors and

environmental routes for plant diseases, it is not possible to commercially cultivate most Vitis

species, other than Vitis rotundifolia. Because of their adaptation, many muscadine grape

cultivars have shown remarkable tolerance to pests and diseases (Poling 1996). More

specifically, muscadine grapes are resistant to Pierce's disease caused by Xylella fa;stidiosa,, a

bacteria that is commonly spread by glassy-winged sharpshooters (Homalodisca coagulate) that

invade the vascular system of grape vines and cause a significant vine decline over time (Mizell

and others 2003). Due to the severity of Pierce's disease in the southeastern region of U.S., Vitis

rotundifolia and Vitis arizonica, both native to these regions, have demonstrated resistance to

Pierce's disease sufficient to create a commercial industry (Ruel and Walker 2006).

Muscadine grapes are found in tight small clusters of 3 to 10 berries that may not ripen

uniformly, thus, they are harvested as single berries instead of bunches (Himelrick 2003; Takeda

and others 1983). The fruit possess a much thicker skin than other grape species, has large seeds,

is very turgid, and has musky-flavored pulp. The fruit is found in black to bronze colors (Croker

and Mortensen 2001; Himelrick 2003). The production area of muscadine grapes is around

5,000 acres. Georgia and North Carolina encompass more than half of the total acreage, and,

Arkansas and Florida also hold an important portion of the cultivated area (Table 2-1).

Commercial production of muscadine grapes is divided mainly into fresh fruit and wine.

According to Halbrooks (1998), muscadine juice manufacture has been positively evaluated, but










its production has still not been exploited. Fresh fruit is marketed as pick-your-own and

packaged berries. In 1979, 95% of the grape production of Florida was sold directly to

customers (Degner and others 1981). A recent report divided the muscadine market into juice,

wine, vinegar, sweet spreads, dry products, and by-products and nutraceuticals (Morris and

Brady 2004). The most common product manufactured with muscadine grapes is wine, which is

attractive due to its fruity flavor. The shelf life of these wines may be shorter than other wines

due to changes in their pigments during aging. If there is an oversupply of muscadine wine, high

quality vinegar might be produced to create an extra product of this industry (Sims and Morris

1985; Morris and Brady 2004).

Table 2-1. Estimated muscadine grape acreage in the southeastern United States (Cline and Fisk
2006).
State Acreage
Alabama < 75
Arkansas 400 500
Florida 600 1,000
Georgia 1,400
Lousiana 70
Missisipi 300
North Carolina 1,300
Oklahoma < 50
South Carolina 300
Tennessee 160
Texas < 50
Virginia < 50

Since 40% of the fruit is skin (Pastrana-Bonilla and others 2003), only about half of the

fruit is used in conventional products such as juice and wine. After pressing, processors must

decide the best way to handle their waste and such a substantial volume of residue can only mean

significant costs to the overall procedure. Further processing of pomaces rich in polyphenolics,

such as muscadine grapes, can lead to an increase in economic value per ton of fruit and the

decrease of waste material (Ector 2001). Pigments could be extracted from the skins and be used









as food ingredients; these compounds could then contribute to the overall product color and

increase its nutraceutical content (Morris and Brady 2004).

2.2 Polyphenols

There is substantial interest in polyphenolic compounds in foods due to their effects on

food quality and their association with human health benefits against coronary heart disease and

cancer (Parr and Bowell 2000). Polyphenolic compounds are not only recognized for their

bioactive properties but also for their contribution to flavor and color of several fruits and

vegetables (Croft 1999). Approximately 8000 phenolic compounds have been identified that

possess a common aromaticring structure with at least one hydroxyl group (Robbins 2003).

These phenolic compounds originate as secondary plant metabolites, from phenylalanine and

tyrosine precursors and the phenylpropanoid pathway, and are essential for plant reproduction,

stability, and growth processes in plants (Croft 1999; Shahidi and Naczk 2003).

2.2.1 Structure and Classification

According to the number of phenol subunits, phenolic compounds can be divided into

simple phenols and polyphenols and are further divided into other categories depending on their

structure and activity (Robbins 2003; Shahidi and Naczk 2003). The term "polyphenolics" is

commonly used to describe compounds of this nature. Phenolic compounds are formed by the

release of ammonia from phenylalanine and tyrosine due to the action of phenylalanine ammonia

lyase (PAL) and tyrosine ammonia lyase (TAL) to form trans-cinnamic acid and p-coumaric

acid, respectively. Subsequently, these two compounds serve as precursors in the formation of

several phenolic compounds (Shahidi and Naczk 2003). De Bruyne and others (1999) explained

that phenolic compounds are products of a plant aromatic pathway: the shikimate section that

generates the aromatic amino acids phenylalanine, tyrosine and tryptophan; the phenylpropanoid

pathway that produces the cinnamic acid derivatives; and the flavonoid route that produces a









diversity of flavonoid compounds. Phenolic compounds are divided into the following groups:

hydroxylated derivatives of benzoic or cinnamic acids (phenolic acids); coumarins; flavonoids

and stilbenes; lignans and lignins; suberins and cutins; and tannins (Shahidi and Naczk 2003).

2.2.1.1 Phenolic acids and simple phenols

Phenylpropanoids are typically known as "phenolic acids". These compounds are a group

of aromatic secondary plant metabolites that have one carboxylic acid functional group. While

the basic skeleton in all these compounds is similar, the number and positions of the hydroxyl or

carboxyl groups on the aromatic ring generate a diversity of compounds (Shahidi and Naczk

2003; Robbins, 2003).

Current research relates phenolic acids with various plant functions, including nutrient

uptake, protein synthesis, enzyme activity, photosynthesis, among others (Wu and others 2000).

In foods, phenolic acids are related to sensory characteristics and nutritional properties.

Moreover, these compounds are intimately related with enzymatic browning thus affecting food

quality and shelf life (Robbins 2003).

2.2.1.2 Flavonoids

Flavonoids are widely known for their red, purple, and blue color and their association

with health in diets rich in fruits and vegetables. Currently more than 6,400 flavonoids have

been identified from diverse plant species. Flavonoids include a broad group of compounds that

share a similar diphenylpropane (C6-C3-C6) basic structure (Figure 2-1) and depending on the

position of the association of the aromatic ring and the benzopyrano, flavonoids could be divided

in three main groups: flavonoids, isoflavonoids, and neoflavonoids (Marais and others 2006;

Winkel 2006).

Flavonoids are formed from the condensation of phenylpropane with coenzyme A to form

chalcones that will then form other end products. The differences within flavonoids are dictated









by the level of oxidation of the central pyran ring of the main diphenylpropane structure; the

number and distribution of hydroxyl, carboxyl groups; and the degree of alkylation or

glycosilation. Flavones, flavanones, flavonols, flavanonols, flavan-3-ols and other related

compounds may be formed due to all these substitutions (Shahidi and Naczk 2003). From these

compounds, flavonols, flavones, flavanones, catechins, anthocyanidins and isoflavones are

commonly consumed (Le Marchand 2002).






AC@


Figure 2-1. Basic flavonoid structure (Pietta 2000).

Flavones and flavonols are the most abundant flavonoids in foods with approximately 100

flavones and 200 flavonols identified in plants. The most common flavonols are myricetin,

quercetin and kaempferol which are found in many important fruits and vegetables (Table 2-2).

Flavonols differ from flavones because of the presence of a hydroxyl group on the 3-position and

are also known as 3-hydroxyflavones (Shahidi and Naczk 2003; Le Marchand 2002).

Flavanones and flavanonols have a saturated C-ring. Flavanonols differ from flavanones due to

the presence of a hydroxyl group on the 3-position. Flavanones are mainly found in citrus fruits,

these compounds are frequently glycosylated in the 7-position with disaccharides (Tomas-

Barberan and Clifford 2000). Catechins and anthocyanins are also known as flavans and are an

important group of flavonoids. Catechins are primarily found in tea and red wine while

anthocyanins are generally found in many berries and flowers (Le Marchand 2002; Shahidi and

Naczk 2003).










Due to their bright colors, flavonoids can act as visual attractants for pollinating insects.

Some flavonoids might have a protective mechanism against predatory insects. UV protection

and growth and development are among other functions and processes associated with flavonoids

in plants (Winkel 2006; Pietta 2000). In addition to their physiological functions in plants,

flavonoids are also significant components of the human diet. Flavonoids are present in most

edible fruits and vegetables, and depending on their source, their bioavailability may vary

tremendously. Thus, dietary intake of flavonoids is variable, ranging from 3 to 800 mg/day

(Erlund 2004; Le Marchand 2002; Pietta 2000).

Table 2-2. Classes and dietary sources of flavonoids (Shahidi and Naczk 2003).
Class Dietary Source
Chalcone Miscellaneous
Flavone Fruit skins, parsley, celery, buckwheat, citrus, red pepper, red wine,
tomato skin
Flavanone Citrus
Flavonol Leek, broccoli, endives, grapefruit, tea, onion, lettuce, tomato, beeries,
apples, olive oil
Flavanonol White grape skins, soybean, fruits
Isoflavone Soybean
Flavanol Tea
Anthocyanidin Berries, dark fruits

2.2.1.3 Tannins

Tannins are a group of oligomeric and polymeric water-soluble polyphenols. Tannins are

divided in two main groups (Figure 2-2) due to their structure and susceptibility to acid

hydrolysis: condensed and hydrolyzable tannins (Meyers and others 2006). The name "tannins"

comes from the ability of these compounds to tan animal skin into leather by protein

precipitation (Lei 2002). These compounds are usually found in the bark of trees and can

precipitate proteins from aqueous solutions (Shahidi and Naczk 2003). Condensed tannins are

oligomers or polymers of flavan-3-ols. Approximately 50 proanthocyanidins have been

identified in the literature. Based on the monomer units, condensed tannins could be further









divided in procyanidins, propelargonidins, and prodelphinidins. These proanthocyanindins are

large molecules ranging from 2000 to 4000 Da. Hydrolyzable tannins are formed by the

glycosylation of gallic or ellagic acids, so, they are further divided in two groups: gallotannins

and ellagitannins. These tannins range from 500 to 2800 Da (Kraus and others 2003; Meyers

and others 2006; Shahidi and Naczk 2003).

Tannins have anti-nutritional properties due to the relation with proteins to form

complexes making them unavailable for digestion (Shahidi and Naczk 2003). In contrast,

tannins are associated with health benefits possessing antimutagenic and anticarcinogenic

properties, reduction of serum cholesterol, and many other biological functions in herbivore

animals (Meyers and others 2006; Kraus and others 2003).



HO
HOa4 HO O O

~"o c~- DHOH DH HO
HO 0 OC"~O OH H O



HOH OH


o~ OH

Figure 2-2. General structure of hydrolyzable tannins (left) and condensed tannins (right)
(Shahidi and Naczk 2003; Meyers and others 2006).

2.2.2 Polyphenolics as Antioxidants

Polyphenolics are naturally occurring antioxidants that prevent oxidation of substrates

containing these compounds. This prevention occurs not only in foods but also in humans,

relating phenolic compounds with the control of many degenerative diseases. Antioxidants

protect oxidative substrates by reducing the concentration of oxygen, intercepting singlet









oxygen, or scavenging initial radicals to prevent the activity of reactive oxygen, nitrogen and

chlorine species that are related with diseases such as arthritis, diabetes, atherosclerosis, among

others (Shahidi and Naczk 2003; Le Marchand 2002).

Even though the exact association of polyphenolics with some diseases is not fully

understood, flavonoids have proven not only to inhibit enzymes directly related in the generation

of reactive oxygen species, but also chelate metals which are important in the oxygen

metabolism (Pietta 2000). Moreover, since polyphenolics have a wide range of hydrophobicity,

both the hydrophilic and lipophilic environments could be protected by these compounds (Parr

and Bowell 2000).

The most predominant method of antioxidant activity seems to be the hydrogen donation,

also known as radical scavenging (Robbins 2003). Free radicals could cause extensive damage

to macromolecules in the body. Free radicals remove a proton from macromolecules, generating

highly reactive radicals of high molecular weight. This creates a chain degradation reaction

where radicals are trying to stabilize by removing a proton from a neighboring molecule.

Polyphenolics donate a hydrogen atom, breaking the degradation cycle. Furthermore, if phenolic

compounds react with the initial forms of free radicals, they donate their proton, thus quenching

the free radical and producing a less reactive radical that will be subsequently stabilized by

resonance delocalization (Parr and Bowell 2000; Shahidi and Naczk 2003).

Many studies have suggested that the antioxidant properties of phenolic compounds,

specifically flavonoids, are generally located in the B ring of the molecule, more specifically in

the number of hydroxyl groups present in that ring (Reviewed by Pietta 2000). However,

flavonoids that do not possess a hydroxyl group in the B ring still have antioxidant activity. In









the case of tannins, the antioxidant capacity is closely related to the degree of polymerization due

to the amount of radicals that can be quenched per molecule (De Bruyne and others 1999).

2.2.3 Polyphenolics in Mucscadine Grapes

Muscadine grapes are unique among grapes species due to the presence of anthocyanin

3,5-diglucosides, free ellagic acid and numerous derivatives of ellagic acid such as ellagic acid

glycosides and ellagitannins (Lee and Talcott 2004). As an isolated compound, anthocyanin

diglycosides are more resistant to oxidative and thermal processes than a respective

monoglycoside, however in vivo they exhibit less color, enhanced formation of polymers, and a

greater susceptibility to exhibit brown color than foods that contain anthocyanin monoglucosides

(Lee and Talcott 2004). Other unique compounds in muscadine grapes are ellagic acid and its

derivatives which are commonly associated with fruits such as blackberries, raspberries,

pomegranates, certain tree nuts and strawberries (Rommel and Wrosltad 1993; Lee 2004).

Characterization and quantification of phenolic compounds present in muscadine grapes

has been extensively studied. The polyphenolics, other than anthocyanins, found in muscadine

are flavanols (catechin and epicatechin), ellagic acid, flavonols (myricetin, quercetin and

kaempferol), gallic acid, and resveratrol ranging from 0.1 to 86.1 mg/100g of whole fruit in 10

different muscadine cultivars (Pastrana-Bonilla and others 2003). These compounds are mainly

located in the skins and the pulp. The skins contain ellagic acid, flavonols and resveratrol while

the seeds contain flavanols and gallic acid (Pastrana-Bonilla and others 2003). In another study

(Yi and others 2005) five anthocyanin aglycones were identified after hydrolysis. However, two

studies at the University of Florida (Talcott and Lee 2002; Talcott and others 2003) have

identified six main forms of anthocyanidins in muscadine grapes including pelargonidin.









2.3 Anthocyanins

Interest in natural food colorants and additives continues to increase in response to

consumer demands and the potential health benefits they impart (Del Pozo and others 2004).

Anthocyanins are the most important class of water-soluble pigments responsible for the red,

blue and violet colors in many fruits, vegetables, roots, tubers, bulbs, legumes, cereals, leaves

and flowers (Bridle and Timberlake 1997). Many fruits contain high concentrations of

anthocyanins and several studies have shown a relationship between fruit consumption and

reduction of certain diseases attributable to the presence of antioxidant polyphenolics (Rommel

and Wrosltad 1993; Parr and Bowell 2000; Aaby and others 2005). Polyphenolics such as

anthocyanins are effective radical scavengers and can break free radical reactions through their

electron donation, metal chelation, enzyme inhibition, and oxygen radical quenching capabilities

(Pastrana-Bonilla and others 2003).

2.3.1 Structure and Occurrence

Anthocyanins are flavonoids formed by condensation of phenylpropane compounds with

participation of three molecules of malonyl coenzyme A that form a chalcone that is cyclated

under acidic conditions (Shahidi and Naczk 2003). Currently, 17 naturally occurring

anthocyanidins have been identified, and only six of them (Figure 2-3) are common in higher

plants: pelargonidin (Pg), peonidin (Pn), cyanidin (Cy), malvidin (Mv), petunidin (Pt) and

delphinidin (Dp). From those six, Cy, Dp, and Pg are the most widespread in nature (Kong and

others 2003). The six different anthocyanin base forms vary based on their hydroxyl (OH) and

methoxyl (OCH3) Substitutions on the anthocyanidin base (B ring). Moreover, anthocyanins are

exclusively found as glycosides in undisturbed tissues of flowers and fruits, where they are

bound to one or more molecules of sugar yielding around 200 different anthocyanins that have

been identified (Shahidi and Naczk 2003).










The anthocyanidin form or flavylium cation (2-phenylbenzopyrilium) is the basic structure

of the anthocyanin molecule which has conjugated double bonds responsible for light absorption

around 500 nm, thus becoming visible with a red hue to the human eye (Rein 2005). Depending

on the presence of hydroxyl or carboxyl, the hue of the color will be either blue or red,

respectively (Shahidi and Naczk 2003).




HO, HO, OH2 HO. CH,0

O Hn O H I O

Pelargon din Cyanidin Peonidin



HO HOH OH
Dephnii MavdnPeuii
Figur 2-3. Chmia stutrsonhcaiisSaiiadNck20)






aroatcr liphaic dicabxl cd bounid t hntoynn Pthrugh stebndig h







most common acylating agents are: hydroxycinnamic acids such as p-coumaric, ferulic, caffeic

and sinapic acids, hydroxybenzoic acids like gallic acid, and aliphatic acids including malonic,

acetic, malic, succinic and oxalic acids (Francis 1989; Bruneton 1995; Cabrita and Andersen

1999).

The most important role of anthocyanins is their ability to impart color to the plants and

plant products in which they occur. This color establishment plays a crucial role in the attraction









of animals for pollination and seed dispersal. They also act as antioxidants, phytoalexins or

antibacterial agents, possess known pharmacological properties, and are used for therapeutic

purposes (Kong and others 2003).

2.3.2 Color Stability

Anthocyanins are unstable compounds and the color loss may depend on the hydrolysis of

the unstable aglycone form. The stability of anthocyanins is closely related to self-association,

concentration and structure, pH, organic chemicals, temperature, light, enzymes, oxygen,

copigments, metallic ions, ascorbic acid, sugars, and processing (Shahidi and Naczk 2003;

Stingzing and others 2002). Glycosylation, acylation, and hydroxylation contribute to the

anthocyanin stability. It has been shown that the acyl groups maintained color when bound to

anthocyanins. Polyacylated anthocyanins are more stable than monoacylated ones.

Hydroxylation in positions C-4 and C-5 prevents water addition to form colorless species (Saito

and others 1995; Rein 2005; Turker and others 2004; Shahidi and Naczk 2003). Copigmentation

enhances the color of anthocyanins by increasing the absorbance due to pigment concentration

and association with other compounds by hydrophobic interaction between aromatic bases of the

molecules involved (Shahidi and Naczk 2003). Copigmentation occurs through numerous

interactions, such as intermolecular and intramolecular complexes, self-association, and metal

complexation. Anthocyanin copigmentation results in a stronger and more stable color than a

singular anthocyanin molecule. Furthermore, overlapping association of copigmentation results

in a prevention of nucleophillic attack of water to the molecule (Rein 2005), thus providing the

new molecule with higher color intensity and more stablility. Copigments are colorless or

slightly yellowish natural molecules in plant cells that exist along with anthocyanins, the most

common copigments are flavonoids. Other copigments could be organic acids, amino acids, and

metal ions (Brouillard and others 1989).









Anthocyanins are very sensitive to pH shifts. In solutions they exist in four different

forms: blue neutral and ionized quinonoidial base, red flavylium cation or oxonium salt,

colorless pseudobase, and colorless chalcone (Figure 2-4). Each of the four species has a variety

of tautomeric forms and the chalcone could exist as cis or trains forms (Shahidi and Naczk 2003;

Clifford 2000).


OCHz
C :iOH
OH
HO -0 OCHJ
~O-Glucose
OH


-He


O OOCH, H,~O HO O H3


OH

Figure 2-4. Anthocyanin equilibria: quinonoidal base (A), flavylium cation (B), carbinol base or
pseudobase (C) and chalcone (D) (Clifford 2000; Shahidi and Naczk 2003).

Even though anthocyanins can exhibit a diversity of color tones in the pH range from 1-14,

they are more stable at acidic media showing an intense red coloration in the pH range of 1 to 3

(Rein 2005; Shahidi and Naczk 2003). The increase of pH decreases the concentration of the

flavylium cation, thus, decreasing the intense red color to form the carbinol base. This

compound does not have a conjugated double bond between the rings A and B so there is no

absorption of visible light. As the pH continues to increase (Figure 2-5), the colored


OCHz
B OH

HO OjOCH3

O--Glucose
OH










quinonoidial form is produced by the loss of a hydrogen atom. If pH continues to rise, the

cabinol base yields the colorless chalcone form (Rein 2005).


Human Blood pH approx7i
100 ~- Red Flavyllum Cation Small Intestine pH7T.5-.0
e i Colousr~les suobs




so Hurnan Stomach
as range from 2 to 5--
(post-prandial)




P I / IColourless Chalcone
Blue Quinoldal Base

0 1 2 3 4 5 ~6

pH value
Figure 2-5. Effect of pH value on anthocyanin equilibria (Clifford 2000).

Anthocyanin stability is also affected by temperature. This degradation process follows

first order kinetics (Kirca and others 2006). Elevated temperatures alter the anthocyanin

equilibria or hydrolyze the glycosidic bonding to form unstable chalcones or aglycone forms,

respectively, as the first step in thermal degradation. Ultimately, thermal degradation leads to

the formation of brown pigments (Rein 2005; Clifford 2000). On the other hand, extremely low

temperatures also affect the quality of anthocyanins. Quinonoidal formation is favored by low

temperatures, therefore if a product was frozen red, it might appear blue after thawing due to the

change of flavylium cation to quinonoidal form during that low temperature exposure (Bridle

and Timberlake 1997).

Oxygen intensifies the degradation of anthocyanins. Even though the formation of

unstable chalcones due to pH or thermal changes is reversible, the presence of oxygen during

these procedures impedes the normal reconversion of these compounds (Bridle and Timberlake









1997). The effect of oxygen on anthocyanins occurs as direct oxidative mechanisms or through

indirect oxidation, yielding colorless or brown end products (Rein 2005). Active forms of

oxygen are highly reactive, as described in the pecking order table (Buettner 1993), and can

degrade any type of molecule with a lower one-electron reduction potential (Eo)

Although light is needed in the biosynthesis of anthocyanins, once formed light damages

these compounds (Markakis 1982). Visible and UV light are harmful to anthocyanins, and a

study conducted on Berberis species illustrated the damaging effect of light on their anthocyanin

profile (Laleh and others 2006). Specifically, shorter wavelengths are more harmful than longer

ones (Skrede and Wrolstad 2000; Wang 2006). In a photochemical study, Furtado and others

(1993) found that aqueous solutions of anthocyanins submitted to irradiation help with the

disappearance of the flavylium cation due to the formation of the chalcone form.

Enzymes also have an important effect on anthocyanin stability, thus, inactivation of

enzymes is a key process in the production of a variety of fruit and vegetable products (Fang and

others 2006). The most common enzymes related to the degradation of anthocyanins are

glycosidases. Glycosidases are not specific in the structural requirements of the aglycone portion

of a molecule (Huang 1955), therefore they cleave the anthocyanins separating the sugar from

the unstable aglycone form. Peroxidases (POD) and polyphenol oxidases (PPO) are enzymes

naturally present in fruits that degrade phenolics compounds resulting in the formation of

precursors of brown pigments (Kader and others 1997). PPOs degrade anthocyanins indirectly

by the formation of quinones that subsequently will react with anthocyanins to form colorless

products (Kader and others 1998). Another investigation (Fang and others 2006) explained that

PPO oxidizes chlorogenic acid to form a quinone that will eventually react with anthocyanins to

form brown pigments.









Substances such as sugars and organic acids can react with other solutes like polyphenolics

and influence their stability as well. Sugars play a double role in anthocyanin stability. Sugars

and syrups could be used as cryoprotectants by associating with plant water by osmosis. The

process of sugar addition is also known as osmotic dehydration (Wang 2006). Syrups have

proven to work better than dry sugars because the sucrose, which is commonly the osmotic

agent, is dissolved (20-65%) and ready to migrate to the fruits or vegetables. Dry sugars are

used with fruits that contain a high percent of juice where sugars can be dissolved. An osmotic

step could protect color against degradation during drying (Torreggiani and Bertolo 2001).

However, once some browning derived products have been produced from sugar caramelization,

the degradation of anthocyanins is enhanced. The browning products responsible for the

degradation effect in anthocyanins are furfural and Maillard reaction products (Tsai and others

2005).

Ascorbic acid fortification has commonly been used in fruit juices as antioxidant

protection and to increase the nutritional value. Ascorbic acid has proven to retard enzymatic

browning by reducing the o-quinones to o-diphenols that no longer produce brown pigments or

degrade anthocyanins (Gregory 1996; Kader and others 1998). Another study (Rababah and

others 2005) showed that ascorbic acid addition in fruit products did not change the phenolic or

anthocyanin concentration, but dehydration together with ascorbic acid addition slightly reduced

the amount of anthocyanins. However, addition of ascorbic acid was shown to degrade

anthocyanins in pomegranate (Marti and others 2001) and Agai juice (Pacheco 2006).

Bisulfite and other sulfur compounds are also used to protect color and phenolic

compounds. These compounds are usually used in wine production. SO2 acts as an antioxidant

and bacteriostatic agent (Morata and others 2006). Bisulfite, like ascorbic acid, reacts with the o-









quinone to eliminate the basic compound to form brown pigments (Lindsay 1996). Nevertheless,

SO2 prevents the formation of visiting. Visitins are compounds formed by condensation of

anthocyanins and pyruvic acid or acetaldehyde released by certain strains of yeast. Visitins are

more stable than anthocyanins and do not affect the desired color of these compounds (Morata

and others 2006).

2.3.3 Anthocyanins in Muscadine Grape

Five main anthocyanin forms were reported showing the absence of pelargonidin aglycone

form (Yi and others 2005; Yi and others 2006). However, all six major anthocyanidins were

reported in muscadine grapes (Talcott and others 2003; Talcott and Lee 2002), and all of them

were present as diglucosides (Figure 2-6). These diglucosides are more resistant to oxidative and

thermal processes than a respective monoglycoside as isolated compounds. However, in vivo

they exhibit less color, enhanced formation of polymers, and a greater susceptibility to exhibit

brown color than foods that contain anthocyanin monoglucosides (Lee and Talcott 2004).




OHF-

HO~b~OH
HH


OH OH

OH-

Figure 2-6. Anthocyanin diglycoside structure (Cyanidin-3, 5-diglucoside).









2.4 Ellagic Acid


2.4.1 Structure and Occurrence

Ellagic acid (EA) is formed through the conjugation of two molecules of gallic acid which

is a derivative of hydroxyl benzoic acid (Figure 2-7). Ellagic acid is primarily found as

ellagitannins (ETs). The formation of free ellagic acid is a result of the spontaneous conversion

of both ester groups of hexahydroxydiphenoyl group (HHDP) into EA following its hydrolysis

from ETs (Rommel and others 1993). In the presence of tannase, tannins are hydrolyzed into

HHDP's and a sugar molecule. Subsequently, HHDP is converted to EA through coupled

oxidation and spontaneous lactonization (Shi and others 2005; Lei 2002).

EA is a polyphenolic located in the vacuole and represents the main phenolic compound in

the Rosaceae family (Atkinson and others 2005). EA is an important polyphenolic compound in

fruits such as raspberries, strawberries and blackberries. These fruits contain as much as three

times the concentration of EA of some nuts (Tomas-Barberan and Clifford 2000; Rommel and

others 1993). EA could also be found in pomegranate (Gil and others 2000), oak (Lei 2002) and

other woody plants (Lee 2004).


OO
O OH

HO
/ s OH

HO


Figure 2-7. Chemical structure of ellagic acid.

2.4.2 Ellagic Acid Derivatives

Ellagic acid derivatives could be divided in two main groups: Ellagic acid glycosides

(EAG) and ellagitannins (ETs). EAG are compounds that consist of a sugar moiety bound to an










EA molecule (Figure 2-8). Usually the sugars involved in the formation of EAG are pentoses

such as xylose, rhamnose, and arabinose. Glucose may also be present in the formation of

EAGs. The linkage between sugars and EA typically occurs in the 4-position since only ellagic

acid-4-glycosides have been reported (Lee 2004; Lee and others 2005; Zafrilla and others 2001;

Mullen and others 2003).


A 4
SO OH

OHO




C O C H z HO OH


OO


G OHO




Figure 2-8. Ellagic acid glycosides A) Ellagic acid-4-arabinoside, B) Ellagic acid-4-
acetylarabinoside, C) Ellagic acid-4-acetylxyloside (Mullen and others 2003).

ETs (Figure 2-9) are water soluble polyphenolics of molecular weights up to 4000 Da that

represent one of the largest groups of tannins. ETs are likely derived from a gallotannin

precursor (penta-O-galloyl-B-D-glucose) by oxidative coupling of at least two galloyl units

yielding a HHDP unit that is the base of an ET (Clifford and Scalbert 2000; Khanbabaee and Ree

2001). ETs are also categorized as hydrolysable conjugates, including one or more HHDP

groups esterified to a sugar molecule (Lee 2004). Currently, 500 different types of ETs were

reported in nature that differ from each other in the number of HHDP units, the conformation of










the glucose ring, and the location of the galloyl ester groups (Feldman and Sambandam 1995;

Helm and others 1999).

ETs can be divided in monomeric or oligomeric depending on the amount of glucose

molecules present. Monomeric ETs are HHDP ester groups bound to one molecule of glucose.

The coupling between HHDP groups and glucose generally occurs at the 4,6- carbon position

and/or 2,3- carbon position of the glucose molecule. 1,6-, 1,3-, 3,6-, and 2,4- arrangements can

also take place. These diverse combinations generate numerous monomeric ellagitanins. The

most prominent ellagitannins are 4,6-HHDP (tellimagrandin I and II), 2,3-HHDP (sanguin H4

anf H5 ) and 4,6-2,3-HHDP (pedunculagin and potentellin) (Lei 2002).

Oligomeric ETs are created by the polymerization of monomeric ETs. The most

predominant oligomeric ETs are dimeric and tetrameric ETs. The polymerization of monomeric

ETs occurs through oxidative C-O couplings between galloyl and HHDP moieties or the C-C

interaction s between glucose and HHDP. Examples of oligomeric ETs with C-O pairing,

coriariin and rugosin D, which are dimmers of tellimagradin I. Oligomeric ETs with C-C pairing,

roburin A and D, which are dimmers of vescagalin/castalagin (Lei 2002).


HQH





HO H o

HO HO OHHO /O \/OH
H H HO OH OH HO) OH
oH OH
Figure 2-9. Ellagitannins: Tellimagrandin II (monomeric ET) (left), Sanguiin H-6 (oligomeric
ET) (right) (Meyers and others 2006, Clifford 2000, Lei 2002).









2.4.3 Ellagic Acid and Its Derivatives in Muscadine Grape

EA was measured in muscadine grapes and it was found that it is one of the most abundant

non-colored polyphenolics in the skins along with myricetin (Pastrana-Bonilla and others 2003).

Another investigation (Lee and Talcott 2004) showed an average of 700.5mg/Kg and

1080.9mg/Kg of total EA in the skins of eight different cultivars of unripe and ripe muscadine

grapes respectively. Of this total EA, 3.4% was free EA and 5.4% was EAG in unripe fruit. In

ripe fruit, 9.2% constituted free EA and 8.7% was EAG of the total EA, proving that a large

portion of the ellagic acid was in the form of ETs.

Three EAGs were identified in muscadine grape (EA-rhamnoside, EA-xyloside, and EA-

glucoside), and in this study (Lee and others 2005), EAGs represented around 12.7% of the total

ellagic acid in Noble muscadine grapes.

Due to lack of chromatographic standards and the diversity of ETs, it is challenging to

identify these compounds. Commonly, the concentration of ellagic acid derivatives is measured

by the amount of free EA released after acid hydrolysis. ETs are measured by the difference

between total EA and EAG and free EA. Investigations have shown that ETs represent around

80%-90% of the ellagic acid present in muscadine grapes and it depends on the stage and

maturity of the fruit (Lee and Talcott 2004; Lee and others 2005). Four ETs were reported but

not completely identified in muscadine grapes. Moreover, two other ETs were identified as

Sanguiin H14 or H15 depending on the position of the galloyl group (Lee and others 2005).

2.5 Processing Effects on Polyphenolics

Numerous changes in properties, both physical and chemical, can occur during fruit and

vegetable processing. Some oxidative reactions may occur where electrons are removed from

molecules to form oxidized compounds. These oxidative reactions lead to browning, changes in

flavor and odor, changes in texture, and most importantly loss of nutritional value. The overall









concentration of nutrients could modify the antioxidant potential of fruit and vegetable products.

Therefore, fruit and vegetable processing is directly related to the changes in concentration and

functionality of phytochemicals (Wang 2006).

As a result of simple processes such as peeling, grating, cutting, and slicing, commodities

that usually had a shelf-life of weeks or months are perishable due to the disruption of plant cells,

thus liberating intracellular products and enzymes that will degrade polyphenolics and other

compounds of nutritional interest in fruits and vegetables (Laurila and Ahvenainen 2002;

Clifford 2000). Some commodities need peeling as part of their process. This step could be

accomplished in several ways, but in an industrial scale usually mechanical peeling, chemical

peeling, or high-pressure steam peeling is used. If this process is not gentle, the cell walls near

the peel may be disrupted and some compound may cause browning, and other degradation

procedures (Laurila and Ahvenainen 2002). Studies have shown that simple procedures should

be conducted with stainless steel materials that won't oxidize the compounds in fruits and

vegetables. Furthermore, these materials should be disinfected constantly during operation

(Laurila and Ahvenainen 2002).

2.5.1 Heat Procedures

Although the main obj ective of heat processing is safety, it is commonly known that this

procedure negatively affects the original properties of raw materials. Specifically, thermal

processing is responsible for the decrease of compounds of nutritional value such as ascorbate,

tocopherols and anthocyanins (Wang 2006). However, thermal process can be beneficial. Heat

treatments have proven to enhance the availability of some compounds due to thermal

destruction of cell walls and subcellular compartments with the release of nutritional compounds

and the denaturation of degrading enzymes such as polyphenol oxidases (PPO) and peroxidases

(PO) (Takamura and others 2002; Wang 2006).









Food canning is an important heat treatment widely used in the food industry to prevent the

presence of dangerous microorganisms such as Clostridium botulinunt, which is an anaerobic

organism that creates a lethal toxin. This microorganism grows at pH over 4.6 and is

thermolabile. Even though the possibility of C. botulinunt occurrence in high acid foods is

uncommon, an F-value of 5D must be accomplished to properly eliminate the risk of C.

botulinunt activity. To accomplish this task, canned products have to be exposed to a high

temperature retort that will have detrimental effects on its nutritional compounds (Pflug and

Esselen 1979).

Pasteurization is one of the most common heat procedures used in food products. The

most common pasteurization protocol, used to ensure proper heat transmission while reducing

the time of exposure, is "high temperature short time" system (HTST) used as a continuous

system for pasteurization in milk products and juices. A study (Klopotek and others 2005)

showed that pasteurization was highly influential in the total phenolic and anthocyanin

concentration of strawberry products.

2.5.2 Drying Procedures

Drying is one of the most ancient processes used to preserve foods. The purpose of this

procedure is to reduce the water activity of a fruit or vegetable to a level where growth of

spoilage microorganisms and occurrence of degrading chemical reactions are halted or slowed

down. Furthermore, drying was used to reduce the volume and weight of commodities for easier

distribution and extended shelf-life (Barbosa-Canovas and others 2005). However, drying can

diminish the nutritional content in foods. Due to thermal degradation, polyphenolics may be lost

or their bioactivity may be reduced. A study (Schmidt and others 2005) showed that different

processes did not decrease the phenolic content significantly but their bioactivity was reduced.

Another investigation (Rababah and others 2005) showed that even though concentration of










polyphenolics in strawberries, peaches and apples was not significantly changed after

dehydration, their antioxidant capacity decreased significantly.

Spray drying is one of the most well-known and widely used drying procedures. This

process take places at temperatures between 150-200oC (Orsat and Raghavan 2006). The

particles have a spherical shape. Due to the diminutive size of the particles, the drying procedure

is shorter compared to other drying protocols and is a suitable method for sensitive compounds

to heat deterioration. Powdered milk and whey concentrates are among the most common

products produced by spray drying. Other products include coffee, tea extracts, baby formula,

enzymes, and yeast (Barbosa-Canovas and others 2005).

It is important to control the feed rate, drying air temperature, and pressure of the air in the

nozzle in spray drying as these characteristics determine the final quality of the powder.

Moreover, carriers such as maltodextrins can be used to improve agglomeration and provide

stability (Orsat and Raghavan 2006). The best spray drying characteristics for higher yields of

roselle extract were analyzed (Andrade and Flores 2004) and results showed that the extract did

not differ in pH from the original liquid feed, but the flavor was lost. The best results for color,

pH, and yield recovery were detected at a temperature range of 178-190oC, and a pressure of 5-6

bar in the atomizer.

Also known as liophilization, freeze drying is another maj or drying procedure.

Liophilization was introduced to the food industry in 1954. Later, in 1964 coffee was subjected

to freeze drying. Freeze-dried products maintain most of their physico-chemical and sensory

characteristics due to the lack of heat exposure (Barbosa-Canovas and others 2005). Freeze

drying consists of two main steps: freezing the product and drying it under vacuum to cause

sublimation. For water, sublimation is accomplished when the temperature is lower or equal to










OoC and the pressure is below 672 Pa. This is called the triple point where ice could be

evaporated without melting (Figure 2-10). It is important to realize that the amount and nature of

solids in food play an important role in the sublimation temperature. Commercial freeze drying

is carried out at -10oC and absolute pressure of 2mm Hg or less (Barbosa-Canovas and others

2005).

p~e55Ufe






Edlid
liquid



672 Pa I--Triple p
Sub~liniatioli VapOUr

0" oCisnpearniue
Figure 2-10. Phase diagram of water (adapted from Barbosa-Canovas and others 2005).

Characteristics such as reconstitution, retention of volatile compounds, rehydration and

others are maintained by freeze drying. An investigation (van Golde and others 2004) showed

that the polyphenols of wine that were freeze dried had around a 70% recovery with the same

qualities as the polyphenols from the original wine. Freeze-drying appears to be a good process

for the conservation of large polyphenols like tannins whereas small polyphenols might not be

protected (Abascal and others 2005). Another investigation (Tambunan and others 2001)

demonstrated that the quality of freeze-dried herbal products was slightly decreased but the

overall quality was still higher than oven-dried samples. Freeze drying is considered the best

drying procedure because the initial material is frozen and the atmosphere around the sample has









a low concentration of oxygen. However, this procedure can be expensive and time consuming

(Barbosa-Canovas and others 2005).

Drum drying is a technique where heat is transferred to a material by conduction from a

rotating drum. After the product is dried, it is separated from the drum with a scraper (Orsat and

Raghavan 2006). This protocol is one of the cheapest drying methods. It is energy efficient,

saves space and is more economical than spray drying for small volumes. The disadvantages of

this procedure are: the product needs to be liquid; it has to adhere to a metal surface, resist an

oxygen exposure and high temperatures (Barbosa-Canovas and others 2005). Furthermore, a

study (Hsu and others 2003) showed that drum drying had the highest losses in antioxidant

capacity compared to freeze drying and hot air drying.

Other drying procedures, not as common as the ones previously discussed, can be found in

the industry. Convective drying is a procedure where a layer of product is exposed to heated air.

Vacuum drying is a procedure where steam heats the products under low pressure. Due to the

use of low pressure, vacuum drying improves the quality of a product by using lower

temperature (Orsat and Raghavan 2006).

2.5.3 Extraction Procedures and Storage

Pressing is a common procedure used for extracting juice from fruits. In this procedure a

significant concentration of polyphenolics may remain in the fruit solids. An investigation

(Klopotek and others 2005) showed step by step how the phenolic profie was changing during

strawberry processing. Total phenolics were reduced by 44% during mashing and pressing. On

the other hand, the anthocyanin content was maintained during initial steps of strawberry

processing.

Storage can also have severe effects on food quality if its temperature is not properly

controlled. Numerous studies have explored the detrimental effect of storage on polyphenolics.









Storage had more impact than thermal process in guava juice production (Fender 2005). Turker

and others (2004) showed a decrease in anthocyanin content and color density were decreased

during a 90 days storage study at 40oC. Another study (Kirca and others 2006) showed similar

results where the largest lost of compounds was at 37oC, followed by 20oC.

2.5.4 Enzymatic Procedures

The use of enzymes to achieve desirable changes in food products has been utilized for

centuries. Enzymes are catalysts that aid in the increase of yield, facilitate processes, and play an

important role on sensory characteristics. Pectinases are used to increase yield of pigments

extracted from grapes in wine production, Naringinases are used to reduce the bitter flavor in

citrus juices, pectin methyl esterase (PlVE) is use to increase the yield and clarify citrus juices,

and the list of enzymes and processes they are used in is vast (Whitaker 1994; Wang 2006).

Enzymes could be classified as endogenous and exogenous depending on whether the

enzyme was found in the substrate or was intentionally added to accomplish an activity.

Enzymes could be further divided into six main groups depending on the reaction they catalyze

(Whitaker 1994). However, not all enzymes are beneficial in the food industry. There are

enzymes that are found naturally in fruits and vegetables that need to be inactivated in order to

preserve the quality and prolong the shelf-life of a product. The main enzymes studied that are

closely related to fruit and vegetable deterioration are PPO and PO which are oxidoreductases

(Whitaker 1994; Kader and others 1997; Kader and others 1998).









CHAPTER 3
PHYTOCHEMICAL, ANTIOXIDANT AND PIGMENT STABILITY OF MUSCADINE
GRAPE POMACE AS AFFECTED BY CONCENTRATION AND DEHYDRATION

3.1 Introduction

Interest in phytochemicals has increased in recent years due to their association with

human health benefits as well as their role in foods as functional ingredients (Talcott and Lee

2002; Wang 2006). The major mechanism by which these compounds enhance food quality and

aid human health is radical scavenging (Robbins 2003), other mechanisms include enzymatic

inhibition, enzymatic co-factoring, growth selectivity and inhibition for deleterious

gastrointestinal bacteria, and essential nutrients absorption enhancement (Reviewed by Dillard

and German 2000). Radical scavenging stops a degradation chain reaction caused by free

radicals that are formed inside and outside the body. Around 100 radicals have been associated

with degenerative diseases such as cancer, atherosclerosis, arthritis, and cataracts, and

polyphenolics donate a hydrogen atom, obstructing the development of such diseases (Shahidi

and Naczk 2003; Parr and Bowell 2000).

Muscadine grapes (Vitis rotundifolia) are a significant fruit crop in the south and

southeastern U.S. and are unique among Vitis species not only for their increased tolerance to

Pierce's disease (Xylella fa;stidiosa), but also in their chemical composition. These grapes are

known to possess a diversity of polyphenolics, such as anthocyanin diglycosides, ellagic acid and

its derivatives, numerous phenolic acids and flavonoids (Pooling 1996; Mizell and others 2003;

Lee and others 2005). The severity of Pierce' s disease in the southeastern region of U.S. has

limited the production of Vitis species other than Vitis rotundifolia which has demonstrated to be

suitable for a commercial industry (Ruel and Walker 2006).

A growing concern for muscadine grape juice and wine producers is the handling of by-

products (skin and seeds) and the desire to produce a food-grade product with added value.









There are numerous phytochemical compounds in grape pomace that may have significance for

the food industry due to their association to human health, thus sustained efforts are currently

underway to produce value-added food ingredients from these otherwise waste products (Sort

2003). The unique properties of muscadine grapes that have presented technological challenges

include their thick pectin-laden skins, the selective recovery of polyphenolics, and, for juice

pomace, their high residual sugar content since wine by-product contains little or no sugar

concentration after fermentation. Recently, the literature on antioxidant compounds from

residual sources has been increasing steadily with investigations on grape pomace, leaves, and

skins (Monagas and others 2006; Bonilla and others 1999), olive mill waste (Visioli and others

1999), wine industry (Makris and others 2007) and several other fruits and vegetables (Peschel

and others 2006).

Therefore, the purpose of this study was to determine isolation techniques and subsequent

stability of polyphenolics recovered from muscadine grape skins using various extractions and

processing techniques to obtain a dry powder or a concentrate.

3.2 Materials and Methods

3.2.1 Materials and Processing

Grape pomace was obtained from Paulk Vineyards (Wray, GA) from deseeded muscadine

grapes grown in 2006. The pomace was obtained following a freeze-thaw cycle and a hydraulic

pressing for juice recovery in the absence of rice hulls as a pressing aid. The resulting pomace

was frozen and transported overnight to the Food Science and Human Nutrition Department at

the University of Florida, Gainesville, FL and held in frozen storage at -20oC until further

processing.

Upon thawing and removal of residual free-run juice grape skins were thawed and mixed

(1:1, 1:2, 1:5, and 1:10 w/w) with hot water (90-95oC). Extracts were manually stirred three or









four times a day to improve contact between the skins and water for a three-day assessment

period. Extracts were pressed and filtered through cheesecloth daily and mixed with new grape

skins to increase the phenolic concentration. For the first extraction grape skins were mixed with

hot water while the second and third extraction were not submitted to heat to minimize

degradation in compounds already extracted. After the third day of concentration the extract was

pressed and filtered to get rid of the skins. Extracts were analyzed for total anthocyanin content

as a marker for completion of the polyphenolic extraction (appendix A).

The disadvantage of a long concentration process was that the juice already started

undesired fermentation process and the color extraction in the second and third day was not as

important as the first one. Due to the long time spent in compound concentration, the mixture

process was reduced to only one day with hot water and likewise assessed for total anthocyanin

content as a marker for polyphenolic extraction (appendix B). At this point, the ratios of pomace

to water 1:1, 1:2, 1:5 were assessed. The high volume of water in dilution 1:10 would have

represented extensive drying in a future processing and was eliminated. After a one-day

extraction, dilution 1:5 was also eliminated due to a low concentration of compounds and high

volume of water. On the other hand, the most concentrated sample (1:1) did not have enough

water to facilitate handling of the product and minimal extraction of compounds, thus excluded

from further experimentation. Consequently, the dilution (1:2) was used for further processing

and investigation.

After the 24-hour process was conducted and the grape skin to water ratio was selected, the

mixture was assessed with samples every 30 minutes to determine the maximum compound

extraction time by total anthocyanin evaluation (appendix C). The extract was manually stirred

every 15 minutes to improve contact between the skins and water, and allowed to extract. The









maximum color extraction was accomplished at 3.5 hours and samples collection was conducted

for 90 more minutes to assure this maximum extraction. Therefore, the process was reduced

from 24 hours of water skin exposure to about 3.5 hours at which, a maximum concentration of

1,200 mg/kg total anthocyanins was accomplished. After this point the content of anthocyanins

was relatively stable.

Based on previous results, an experiment was run at a semi-industrial scale. Grape skins

were thawed and mixed (1:2 w/w) with 45.4 Kg of hot water (90-95oC) for 3.5 hours. The free

run extract was collected and skins pressed in a hydraulic press at (500 bar) to obtain an aqueous

extract. This extract was filtered through cheesecloth and a 1-cm bed of diatomaceous earth to

remove insoluble agents and to clarify the extract. This clarified extract was used as the starting

material for subsequent procedures to eliminate or reduce the presence of soluble sugars. Three

isolation methods were utilized including two solid phase extractions and a fermentation

procedure followed by three concentration protocols that included spray drying, freeze drying

and vacuum concentration. All handling and processing methods were compared to a control of

the starting clarified extract for calculation of phytochemical recovery of changes due to process

techniques. Upon completion of each isolation or processing protocol, samples were held at -

20oC until analysis.

3.2.2 Solid Phase Isolation

A batch of extract was separated as a control and for affinity column isolation treatments

(Amberlite XAD-4, reverse phase Cls). This batch was clarified through diatomaceous earth and

no further processing was done to a control sample of the extract that was stored frozen at -20oC

until analysis. Reversed phase C18 is a model system used commonly at a laboratory scale due

to its high cost. Moreover, particle size is also an impediment for its usage on a larger scale

(Kraemer-Schafhalter and others 1998). SEP-PAK Cls columns proved to efficiently separate









maj or phenolic compounds to improve analysis by HPLC and be very practical and easy to clean

(Jaworski and Lee 1987; Oszmianski and others 1988; Kraemer-Schafhalter and others 1998).

Amberlite copolymers, on the other hand, are used at laboratory scale but could also be used as

industrial alternatives for affinity column isolation due to their commercial availability and price

(Pietrzyk and Chu 1977b). Various types of Amberlite XAD copolymers have been industrially

utilized in the removal of impurities from waste and potable water as well as isolation of

carotenoids, steroids, and other biologically important compounds (Pietrzyk and Chu 1977a;

Fritz and Willis 1973). Amberlite copolymers, which vary in surface area, porosity, and activity,

where shown to vary in their mode of separation, quality of compound retention based upon

nature of the target compounds, pH of the environment, amount of Amberlite resin used as

adsorbent and the type of copolymer present in the resin (Kraemer-Schafhalter and others 1998;

Pietrzyk and Chu 1977a, Pietrzyk and Chu 1977b). Amberlite resin XAD-2 was partially

satisfactory in retaining polar solutes from aqueous extract and it was shown that Amberlite

XAD-7 could adsorb substances with both lipophillic and polar interaction even though

Amberlite XAD-2 showed greater affinity with aromatic compounds (McRae and others 1982).

Another work illustrated the efficiency and feasibility of Amberlite XAD-8 in the extraction of

polyphenolics because such resin was used for 2 years showing reproducible results. However,

when the Amberlite resin was overloaded with compounds, the solvent started to remove

material from the resin (Lalaguna 1993). Kraemer-Schafhalter and others (1998) explained that a

type of Amberlite XAD-7 showed poor separation and was a difficult column to clean, while

other Amberlite resins showed better separation but still showed cleaning complications.

Furthermore, Amberlite XAD-2 showed insufficient pigment retention. For present experiments,










reversed phase Cls column was used as a comparison to Amberlite XAD-4 as an industrial solid-

phase separation and isolation technique.

Amberlite XAD-4 resin (10g) previously washed with methanol and thoroughly cleaned

with deionized water was loaded into a small column whereby 2mL of extract was loaded and

allowed to adsorb for 1 hour. Following adsorption, unbound compounds were washed with

water (200mL) and desorbed with 100% methanol. Following evaporation to dryness,

compounds were re-dissolved in a known volume of 0. 1M citrate buffer at pH 3.0. Those

compounds not retained on the resin were subsequently adsorbed onto 1-gram of Sephedex LH-

20 (normal phase) placed in a mini-column, washed with water, eluted with 100% methanol,

evaporated, and likewise re-dissolved in citrate buffer.

Likewise, 5mL of extract was loaded onto a 5g reversed phase Cls mini-column and

allowed to adsorb by gravity feed. The column was then washed with water (200mL) and

phytochemicals desorbed with 100% methanol. Following evaporation, compounds were re-

dissolved to a known volume of the citrate buffer. Non-retained compounds were likewise

adsorbed onto a Sephedex LH-20 (normal phase) mini-column and re-dissolved in citrate buffer.

A third procedure to remove residual sugars involved fermentation of simple sugars by

inoculation with wine yeast (Saccharomyces cereviseae strain Premium Cuvee) at a rate of

2.5g/L and allowing aerobic fermentation to occur at 20-25oC until soluble solids content was

decreased to a constant amount by monitoring oBrix values every 12 hours. After fermentation,

the extract was clarified by passing through a 1-cm bed of diatomaceous earth with the aid of

vacuum .

Each of the three phytochemical isolation techniques that also served to remove residual

sugars was compared to a non-isolated control. To determine the effects of common processing









or concentration steps, three processing techniques were evaluated for the fermented isolate. The

isolate was sub-divided into equal portions for vacuum concentration, spray drying, and freeze

drying. A control sample was retained and frozen at -20oC until analysis. For spray drying, 2L

of the fermented extract was spray dried (Anhydro, Copenhagen, Denmark) at a temperature of

220-230oC and an exhaust temperature of 100-110oC over a 4 hour period. The resulting powder

obtained was re-dissolved in a known volume of 0. 1M citrate buffer at pH 3.0 for subsequent

analysis. A freeze dried sample of the same fermented extract (300mL) was accomplished in a

Freeze Drier 5 unit (Labconco, Kansas City, MO) at -100oC and 1 Torr inside the drying

chamber over an 8 hour period. The resulting powder was likewise re-dissolved in citrate buffer

for analysis. Lastly, 15mL of fermented extract was evaporated at 60oC using a rotary

evaporator over a 40 minute period and re-dissolved in a known volume of citrate buffer for

analy si s.

3.2.3 Chemical Analysis

3.2.3.1 Spectrophotometric determination of total anthocyanins

Total anthocyanin content was determined spectrophotometrically by the pH differential

method (Wrolstad 1976). Isolation and processing treatments were appropriately diluted with

buffer solutions at pH 1.0 and pH 4.5. Absorbance was read on a UV-Vis microplate reader

(Molecular Devices Spectra Max 190, Sunnyvale, CA) at a fixed wavelength of 520 nm and total

anthocyanin concentration calculated and reported in mg/kg equivalents of cyanidin-3 -glucoside

with an extinction coefficient of 29,600 (Jurd and Asen 1966).

3.2.3.2 Determination of polymeric anthocyanins

The percentage of polymeric anthocyanins was determined based on color retention in

presence of sodium sulfite (Rodriguez-Saona 1999). Treatments were diluted in pH 3.0 buffer

and each sample subdivided into two fractions. A solution containing 5% sodium sulfite was









added to one fraction while an equivalent volume of pH 3.0 buffer was added to the remaining

fraction. Absorbance at 520 nm was recorded for each on a UV-Vis microplate reader

(Molecular Devices Spectra Max 190, Sunnyvale, CA). Concentration of polymeric

anthocyanins was calculated and reported as the percentage of absorbance remaining after the

addition of sodium sulfite.

3.2.3.3 Determination of total soluble phenolics

Total soluble phenolics were determined by the Folin-Ciocalteu assay (Singleton and Rossi

1965). Samples were diluted in water and 100CLL of each were loaded into test tube for reaction

with 0.25N Folin-Ciocalteu reagent (Sigma Chemical Co. St. Louis, MO). After a 3 min reaction

of the reagent and the sample, IN sodium carbonate was added to form a blue chromophore that

was read after 30 minutes at 726 nm on a UV-Vis microplate reader (Molecular Devices Spectra

Max 190, Sunnyvale, CA). Total soluble phenolics were quantified in equivalents of a gallic

acid standard with data expressed in mg/kg of gallic acid equivalents.

3.2.3.4 Quantification of antioxidant capacity

Antioxidant capacity was determined by the oxygen radical absorbance capacity (ORAC)

method (Cao and others 1996), adapted to be performed with a 96-well Molecular Devices

fmax@ fluorescent microplate reader (485 nm excitation and 538 nm emission). The assay

measures the ability of an antioxidant to inhibit the decay of fluorescein induced by the peroxyl

radical generator 2,2-azobis (2-amidinopropane dihydrochloride) as compared to Trolox, a

synthetic, water-soluble vitamin E analog. For analysis, samples were diluted in pH 7.0

phosphate buffer and 50CIL of each sample was then transferred to a microplate along with a

Trolox standard curve (0, 6.25, 12.5, 25, 50CLM Trolox) and phosphate buffer blanks. 100CLL of

flourescein and 50CLL of peroxyl radical generator were added to all samples, standard curve, and

blanks. Readings were taken every 2 min over a 70 min period at 370C. Antioxidant capacity










was quantified by linear regression based on the Trolox standard curve and results were

expressed in Cpmol of Trolox equivalents per gram (Cpmol TE/g).

3.2.3.5 Half life determination

Samples were diluted with pH 3.0 citrate buffer and placed in a 96-cuvette microplate

subdivided into two fractions. A solution containing 3% hydrogen peroxide was added to one

fraction while an equivalent volume of pH 3.0 buffer was added to the remaining fraction.

Absorbance at 520nm was recorded using a UV-Vis microplate reader (Molecular Devices

Spectra Max 190, Sunnyvale, CA). The assay was carried out at 45oC for 60 minutes with

readings every 2 minutes to quantify color loss over time. Results were expressed as minutes of

half-life.

3.2.3.6 Analysis of polyphenolics by HPLC

Polyphenolic compounds were analyzed by reverse phase HPLC using modified

chromatographic conditions (Lee and others 2005) with a Dionex system equipped with an ASI-

100 Autosampler inj ector, a P-680 HPLC pump, and a PDA-100 Photodiode Array Detector.

Separations were performed on a 250 x 4.6 mm Acclaim 120-C18 column (Dionex, Sunnyvale,

CA) with a C18 guard column. Mobile phase A consisted of water acidified with o-phosphoric

acid (pH 2.4) and Mobile phase B consisted of 60:40 methanol and water acidified with o-

phosphoric acid (pH 2.4). Samples were hydrolyzed in 2N HCI (adjusted to contain 50%

methanol) for 90 min at 95oC before inj section. The gradient solvent program held Phase A for 3

min; then phase B from 0 to 30% in 3 min; 30 to 50% in 2 min, 50 to 70% in 5 min, 70 to

70.63% in 3 min, 70.63 to 70.7% in 1 min, 70.7 to 70.81% in 0.5 min, 70.81 to 71.2% in 2. 1 min,

71.2 to 71.3% in 2 min, 71.3 to 85% in 1.4 min and 85 to 100% in 10 min for a total run time of

30 minutes for both set of samples at a flow rate of 1mL/min. Polyphenolics were identified by

UV/VIS spectral interpretation, retention time and comparison to authentic standards (Sigma










Chemical Co., St. Louis, MO). All treatments were filtered through a 0.45CLM filter and directly

inj ected into the HPLC. Data was reported as mg/L of each compound. Anthocyanins were

compared to a cyanidin aglycone standard, flavonols were compared to a quercetin standard, and

ellagic acid was compared to an ellagic acid standard.

3.2.4 Statistical analysis

The study was designed as a completely randomized design (CRD) that included seven

treatments (Amberlite, Cls, spray drying, freeze drying, vacuum drying, and controls for both the

fermented and non-fermented extracts). Data for each treatment is the mean of three replicates.

Analysis of variance and means separations by LSD test (P < 0.05) were conducted using JMP

software (SAS Institute, Cary, NC).

3.3 Results and Discussion

The effects of processing were evaluated and results for all analyses were reported in units

from each assay mentioned earlier in the method section (Table 3-1).

Table 3-1. Quality analyses of muscadine pomace extract polyphenolss) as affected by various
processing protocols.
Total Total Polymeric Haflf4 5RC
Press Phenohics' Anthocyanins" Anthocyanins3
Es 1640 a6 1470 a 7.65 e 17.2 d 34.3 a
F 1580 a 1520 a 9.75 de 17.9 cd 28,7 b
BCis 1130 bc 996 c 25.8 a 24.7 a 16.7 d
UJBCis 4.55 e 0.25 e NA7 NA >0.1 f
BA 1030 c 873 d 18.6 b 21.4 b 22.6 c
UBA 186 d 1.65 e NA NA 1.91 e
SD 1240 b 1060 c 14.3 bcd 20.5 b 22.0 c
FD 1270 b 1230 b 11.3 cde 19.8 bc 18.1 d
VD 1620 a 1450 b 14.8 bc 20.5 b 28.8 b
IExpresed in gallic acid equivalents (mg/kg). 2Expressed in cyaniding-3-glucoside equivalents (mg/kg). 3Expressed
in percentage of polymeric anthocyanins (%/). 4Expressed in time (min). 5Expressed in Trolox equivalents (pLmol
TE/g). 6Similar letters within columns for each analysis are not significantly different (LSD test P < 0.05). 7NA
(not applicable) samples did not contain the analyte. "Treatment abbravietions: (E) Extract, (F) fermented extract,
(BC18) bound to reversed phase C18 column, (UBC18) unbound to reversed phase C18 column, (BA) bound to
Amberlite XAD-4, (UBA) unbound to Amberlite XAD-4, (SD) spray dried, (FD) freeze dried, and (VD) vacuum
dried.









3.3.1 Anthocyanin Color Stability

Anthocyanin stability was assessed spectrophotometrically and is shown in Figure 3-1.

Anthocyanin content was maintained during fermentation and vacuum drying (Table 3-1). Initial

anthocyanin concentration in extract (1470 & 102 mg/kg) was somewhat affected by freeze

drying (1230 & 114 mg/kg), but experienced a 28% decrease after spray drying (1060 & 26.3

mg/kg). Samples after affinity column isolation (reversed phase Cls column and Amberlite

resin) also showed significant decrease in their anthocyanin content (996 & 24.4 mg/kg and 873 &

78.9 mg/kg respectively). The extensive loss of color in both reversed Cls column and

Amberlite XAD-4 procedures (32 and 41% respectively) presumably occurred mainly while

anthocyanins were exposed to high pH environment since less than 0.03% of the initial

material's anthocyanins were found in the unbound fraction from reversed phase Cls and less

than 0.2% was found in the unbound fraction from the Amberlite resin. Another possible reason

for loss of anthocyanin by solid phase isolation might be column efficiency. Perhaps, a single

elution with methanol was not sufficient to dissociate polyphenolics from either column and,

after desorption, a fraction of these compounds remained in association with the column and was

washed away during cleaning of the resin and not collected for analysis. Thus, recovery from

reversed phase Cls bound anthocyanins were more efficient than those from Amberlite resin.

Another possible explanation is that Amberlite XAD-4 might have higher affinity with

polyphenolics than reversed phase Cls thus, making it difficult to recover compounds on a

simple desorption step. Furthermore, in both affinity column isolation techniques, anthocyanins

were subj ected to copious amounts of solvent to separate polyphenolics from sugars and other

compounds that were washed away. After adsorption, anthocyanins were desorbed with 100%

methanol followed by evaporation. Although evaporation uses mild temperatures, in the last

phase when most of the solvent has been evaporated, compounds could have been subj ected to










heat. Stability of anthocyanins is known to be jeopardized by lowering concentration of

anthocyanins in a medium (Giusti and Wrolstad 2003), decreasing acidity (Clifford 2000), and

applying heat (Klopotek and others 2005; Clifford 2000), explaining why both affinity column

isolation protocols significantly decreased anthocyanin concentration in muscadine pomace

extract.


2000

a a
S1500 h I b

I I Ibc b
> 1000 E-


S500



E F BC18 BA SD FD VD
Treatments

Figure 3-1. Total anthocyanin content of muscadine pomace extract as affected by various
processing methods. Error bars represent the standard error of each mean, n=3.

Reasons for color loss were oxygen exposure, low acidity levels in the environment and

heat. Vacuum drying maintained the concentration since some temperature was substituted by

low pressure and oxygen was removed from the medium to generate vacuum, thus making this

process mild and effective. On the other hand, freeze drying showed more than 16%

anthocyanin content loss despite the fact that no heat was applied to the extract. The exposure

time for the sample to freeze dry, and possibly errors at the recovery phase, might have had a

negative effect on anthocyanin concentration. Affinity column isolation techniques showed the

highest color losses due not only to heat and oxygen exposure, but also to higher pH










environment and low anthocyanin concentration due to massive amount of solvent used during

adsorption and desorption, thus illustrating the important role of pH in anthocyanin stability.

Polymerization of anthocyanins present in muscadine pomace extract was significantly

influenced by processing. The bound fraction of the reversed Cls column showed the highest

polymerization index (25.8 & 3.25%) followed by bound fraction of Amberlite resin, vacuum

dried, and spray dried that showed no significant difference between each other. At the same

time, the freeze dried, spray dried and the fermented extract samples showed no difference

between each other (Table 3-1). The extract showed the lowest polymerization index (7.65 &

0.52%) due to the minimum processing (concentration and clarification) it was subj ected to

(Figure 3-2).

When comparing the concentration of anthocyanins to polymeric anthocyanin content, it

was concluded that both affinity column isolation techniques had the lowest anthocyanin

concentration and yet had the highest polymerization index (Table 3-1). Therefore, anthocyanins

might be forming new high molecular weight compounds by association with other

polyphenolics such as condensed tannins and other anthocyanins. Processing was directly

related to the polymerization process since processed samples had higher polymerization index

while the starting material (extract) showed the lowest polymer formation, and as processing was

milder, polymer formation was decreased. A correlation between anthocyanin concentration and

polymeric index (r = -0.73) confirmed that as processes were conducted, polymers were forming

in the media. As compared to other studies (Weinert and others 1990), processing induced

polymerization which explained some of the losses in total anthocyanins attributed to this

process.











3596

S30% -1

25% r
20% --- bdbc

isi1% -1 de II cde

S10% -1 e
S5%-

086
E F BC18 BA SD FD VD
Treatments

Figure 3-2. Polymeric anthocyanins (%) in muscadine pomace extract as affected by various
processing methods. Error bars represent the standard error of each mean, n=3.

Half life determinations were made under accelerated conditions of storage (45oC) in the

presence of hydrogen peroxide, a strong oxidizing agent. Concentrations of peroxide and

holding temperatures were determined to create a slow decay curve, suitable to testing a wide-

range of anthocyanin concentrations with varying degrees of stability. Analogous to the

formation of the polymeric pigments, the effects of anthocyanin isolation and processing slightly

increased protection against hydrogen peroxide-induced oxidation. The bound fraction of Cls

showed the highest resistance (24.7 & 1.01 min) followed by all three drying protocols and the

bound fraction of Amberlite resin (Table 3-1). The initial extract showed shortest half life of all

treatments (17.2 & 0.55 min) and was due to the predominance of monomeric anthocyanins in

this fraction. Correlations between anthocyanin concentration and half life (r = -0.67) and half

life and polymeric index (r = 0.86) confirmed that as polymerization index increases,

anthocyanins become more stable, thus, increasing their durability (Figure 3-3). Stability of

anthocyanins has been enhanced due to polimerization (Weinert and others 1990; Rein and

Heinonen 2004), and copigmentation of anthocyanins showed reduction in pigment degradation










in grape anthocyanins (Brenes and others 2005), which explains the increase in anthocyanin half

life in processed muscadine extract samples.


30

25 b b b
~E 20 -1 d cd
15-
i= 10-



E F BC18 BA SD FD VD

Treatments

Figure 3-3. Half life (min) of muscadine pomace extract as affected by various processing
methods. Error bars represent the standard error of each mean, n=3.

Although processing reduced the concentration of anthocyanins, the anthocyanins

remaining in the matrix were more resistant. Polymeric anthocyanin proved to be more stable to

processing but that may affect the final quality of a food product since the color of the

anthocyanin might change due to this process. An investigation showed that some copigments

enhanced the a* value by retaining more red but also increased the b* value showing a yellowing

in the sample during storage (Rein and Heinonen 2004). Moreover, polymerization could

decrease the antioxidant capacity of phenolic compounds by variations in the hydroxyl groups

arrangement and availability which are related to their radical scavenging ability (Miller and

Ruiz-Larrea 2002), thus polymeric anthocyanins may not have as important benefit as compared

to monomeric anthocyanins. In addition, absorption of phenolic compounds might be conducted

by hydrolysis to obtain aglycone forms or simple phenols are likely to be absorbed more

efficiently in the human body (Reviewed by Miller and Ruiz-Larrea 2002).









3.3.2 Polyphenolic Concentration and Stability

Total soluble phenolics were assessed spectrophotometrically as affected by isolation and

food processes (Figure 3-4). Polyphenolic concentration in muscadine pomace extract and

processed samples experienced somewhat similar behavior as compared to the anthocyanin

content. Polyphenolic content was maintained during fermentation and vacuum dehydration

while affected by freeze drying and spray drying (Table 3-1 & Figure 3-4). Affinity column

isolation protocols (reversed phase Cls column and Amberlite resin) significantly decreased the

concentration of polyphenolics in the extract (1 130 & 43.8 and 1030 & 17.8 mg/kg respectively).

The unbound fraction of Cls did not show significant polyphenolic concentration (4.55 & 2.72

mg/kg) while the unbound fractions from Amberlite resin processing showed an important

concentration of polyphenolics (186 & 122 mg/kg). Amberlite was less effective in binding

polyphenolics than reversed phase Cls since the unbound fraction showed a much higher

concentration (15.3%) than the unbound fraction from Cl (<0.5%). The unbound fraction of

Amberlite resin contained mostly polyphenolics that did not absorb light at 520nm since

previous analysis showed a small concentration of anthocyanins in this fraction. Such

compounds might tentatively be ellagitannins since previous investigations have shown

occurrence of ellagitannins in unbound fractions of solid phase separation techniques (Lee 2004).

Polyphenolic losses in solid phase extraction techniques were possibly due to low acidity

medium, oxygen exposure, and heat exposure. Only vacuum drying maintained the

concentration due to temperature substitution and removal of oxygen by vacuum. On the other

hand, freeze drying showed almost 23% loss in polyphenolic content even though the product

was frozen during the procedure. Furthermore, there were no significant differences between

freeze drying, spray drying and reversed phase Cls column process.










Reversed phase Cls column effectively bound most polyphenolics since less than 0.5% was

detected in the unbound fraction. On the other hand, the Amberlite resin showed compound

losses due to the presence of polyphenolics that did not bind with the resin and were washed

away together with sugars and some other organic compounds. The unbound fraction had more

than 15% of the phenolics that were exposed to the Amberlite resin and more than 1 1% of the

polyphenolics from the starting material (extract).


2000
Sa a a

1500 -1 T I b


S1000-





E F BC18 UBC18 BA UBA SD FD VD
Treatments


Figure 3-4. Total phenolic content in muscadine pomace extract as affected by various
processing methods. Error bars represent the standard error of each mean, n=3.

3.3.3 Polyphenolics by HPLC

Polyphenolics present in muscadine pomace extract and various processing methods were

analyzed and monitored by HPLC at 360 and 520 nm. Analysis was focused on total ellagic

acid, anthocyanins, and flavonols. Compounds were identified and quantified in hydrolyzed

samples.

3.3.3.1 Anthocyanins by HPLC

HPLC analysis of muscadine pomace extracts confirmed the occurrence of six anthocyanin

aglycone forms (Figure 3-5). Previous investigations reported similar results (Talcott and Lee










2002; Talcott and others 2003), although some reports on the specific identity of anthocyanins in

muscadine grapes differed (Lee and Talcott 2004; Yi and others 2005; Yi and others 2006),

detecting only Hyve anthocyanins aglycones, excluding pelargonidin. Since samples from this

study were hydrolyzed and six peaks eluted, it was concluded that the six main forms of

anthocyanin aglycones were present and thus, anthocyanidins could be identified by their elution

order based on their structure and polarity.









00 20 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34
Time (mmn)


Figure 3-5. HPLC chromatogram of anthocyanidins present in muscadine pomace: delphinidin
(A), cyanidin (B), petunidin (C), pelargonidin (D), peonidin (E), malvidin (F).
Identification (520 nm) was done based on spectral characteristics and comparison to
cyanidin aglycone.

Addition of the corresponding peak areas for all six anthocyanidins yielded the total

anthocyanin content for each treatment which was used for general comparison between

treatments (Table 3-2). Delphinidin, cyanidin, petunidin, and peonidin constituted most of the

muscadine skin extract anthocyanin profile while pelargonidin accounted for only 1.1% and

malvidin for 8.2%. Low content of malvidin in the extract may be explained by its lower

polarity compared to the other five anthocyanidin bases. Possibly malvidin was not as well

extracted with hot water from the skin as other anthocyanidins. In muscadine grape juice, the

concentration of malvidin is somewhat similar peonidin (Del Pozo-Insfran 2006).

The unbound fraction from the Amberlite resin had small amounts of delphinidin and

cyanidin on its compound profile. The presence of these two anthocyanidins in the unbound









fraction from Amberlite can possibly be explained by their higher solubility in water as they are

the two most polar anthocyanindins. Due to such polarity, both anthocyanidins might not have

bound to the Amberlite resin as well as the others.

Data indicated that peonidin and malvidin were the most resistant anthocyanidins to

degradation under the processing conditions since only the spray dried sample indicated

significant differences with the starting material (extract). On the other hand, pelargonidin

showed the highest instability since only freeze drying and vacuum drying maintained the

concentration of this compound while other processing techniques significantly reduced it. The

Amberlite resin technique indicated the lowest concentrations of three of the four maj or

anthocyanidins (delphinidin, cyaniding, and petunidin), while spray drying affected pelargonidin,

peonidin, and malvidin the most. Fermentation preserved all anthocyanidins except pelargonidin

which had lower concentration compared to the extract. Previous investigations have illustrated

the unstable nature of pelargonidin during processing (Garz6n and Wrosltad 2001; Garz6n and

Wrosltad 2002; Kammerer and others 2007) while malvidin has been proven to resist thermal

processing (Del Pozo-Insfran 2006) and showed great stability in general due to the presence of

only one hydroxyl group in the B ring (Hradzina and others 1970, Talcott and others 2003; Lee

and Talcott 2004).

HPLC analysis results showed a slight discrepancy compared to the total anthocyanins

assay. Such discrepancy might be explained by the limitation of colorimetric assays to account

for copigmentation compared to single compounds examination (Talcott and Lee 2002; Talcott

and others 2003). Moreover, after HPLC results, data suggested that heat was not a maj or issue

in degradation since only Amberlite and spray dried samples indicated significantly lower

concentration of total anthocyanins compared to the starting material (Table 3-2). These results









illustrated the divergence of previous results compared to HPLC analysis data, opposing to early

conclusions mentioning vacuum drying being the only processing protocol that maintained

anthocyanin concentration. High variation on HPLC results and slight higher concentrations of

some anthocyanindins in processed samples were due to the difficulties of analyzing

anthocyanindins instead of anthocyanin glycosides, once the sugar molecule is separated from

the anthocyanidin, the aglycone form is very susceptible to degradation (Dao and others 1998).

Furthermore, consistency at the hydrolysis step prior to HPLC analysis and poor compound

separation might also explain these results.

Table 3-2. Anthocyanidin concentrations in the muscadine pomace extract as affected by various
processing protocols.
ProcessAnthocyanin concentration'
Dp CyPt Pg Pn My Toa
E4 316 ab' 333 bc 258 bc 15.7 a 367 ab 115 abc 1400 ab
F 401 a 411 a 329 ab 12.2 b 449 a 143 a 1750 a
BCis 243 bc 264 cd 219 cd 11.5 b 301 bc 95.2 bcd 1130 bc
UJBCls ND6 N D D
BA 165 c 186 e 169 d 9.27 b 280 bc 87.6 cd 897 c
UBA 12.7 d 7.74 f ND ND ND ND 20.4 d
SD 207 c 216 de 185 d 5.40 c 250 c 75.2 d 939 c
FD 313 ab 335 bc 283 abc 16.0 a 396 a 127 ab 1470 ab
VD 401 a 374 ab 336 a 16.2 a 439 a 135 a 1700 a
'Expresed in cyanidin aglycone equivalents (mg/kg). 2Anthocyanidin abbreviations: (Dp) Delphinidin, (Cy)
Cyanidin, (Pt) Petunidin, (Pg) Pelargonidin, (Pn) Peonidin, (My) Malvidin. 3Sum of all anthocyanidin bases.
4Treatment abbravietions: (E) Extract, (F) fermented extract, (BC18) bound to reversed phase C18 column11, (UBC18)
unbound to reversed phase C18 column, (BA) bound to Amberlite XAD-4, (UBA) unbound to Amberlite XAD-4,
(SD) spray dried, (FD) freeze dried, and (VD) vacuum dried. 5Values with similar letters within columns are not
significantly different (LSD P < 0.05). 6COmpounds were not detected.

3.3.3.2 Ellagic acid and flavonols by HPLC

HPLC analysis of non-anthocyanin polyphenolics in muscadine pomace extract confirmed

the presence of free ellagic acid, as previously characterized in other studies (Lee and Talcott

2002; Talcott and Lee 2002; Lee and Talcott 2004; Lee and others 2005; Pastrana-Bonilla and

others 2003; Yi and others 2006). However, only myricetin and quercetin could be detected after

hydrolysis, while previous investigations (Talcott and Lee 2002; Lee and Talcott 2004; Pastrana-










Bonilla and others 2003; Yi and others 2005; Yi and others 2006) have shown three flavonols in

muscadine grapes (myricetin, quercetin, and kaempferol). Identification was possible since the

retention time and spectroscopic attributes concurred with those of the authentic standards of

ellagic acid and quercetin aglycones (Figure 3-6).









00 20 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0
Time (mmn)
Figure 3-6. HPLC chromatogram of polyphenolics present in muscadine pomace: ellagic acid
(A), myricetin (B), and quercetin (C). Identification (360 nm) was done based on
spectral characteristics and comparison to authentic standards of ellagic acid and
quercetmn.

Total ellagic concentration was fairly stable and only affected by spray drying and

Amberlite treatments (Table 3-3). The content of ellagic acid was expected to be maintained in

most of the treatments since it has been proven to be quite stable to processing and increment its

concentration due to hydrolysis of ellagitannins which are unstable to heat and acidic conditions

(Amakura and others 2000; Zafrilla and others 2001; Tomas-Barberan and Clifford 2000).

However, another investigation showed that ellagic acid was decreased after cooking probably

due to oxidation reactions (Hakkinen and others 2000). The unbound fraction from the

Amberlite resin showed an important concentration of total ellagic acid (8.6%) that was not

bound to the resin and thus thought to be ellagitannins (Lee 2004). Similar to the total soluble

phenolics assay, reversed phase Cls was an efficient separation technique since negligible

concentrations of free ellagic acid was detected in the unbound fraction from this technique.

In the case of flavonols, myricetin and quercetin were observed in the muscadine [omace

extract (10.2 mg/kg and 5.44 mg/kg respectively). No significant differences were detected in










flavonols due to processing (Table 3-3). Remaining isolation protocols showed higher

concentrations of flavonols, thus, the only conclusion that could be derived from this data is that

processes had no effect on the flavonol concentration of the muscadine pomace product. Such

results differed with some investigations that illustrated the loss of flavonols due to heat

processing (Amakura and others 2000; Hakkinen and others 2000). However, flavonols have

indicated better stability than anthocyanindins in storage at 20 and 37oC (Talcott and Lee 2002).

Such results could be explained by the high variation detected in HPLC analysis due to the use of

aglycone forms of flavonoids that are know to be very unstable making this assay difficult.

Neither myricetin or quercetin were detected in the unbound fractions (Cls and Amberlite), thus

flavonols might bind more effectively to both columns compared to other polyphenolics.

Table 3-3. Ellagic acid and flavonol concentrations in the muscadine pomace extract as affected
by various processing protocols.
Process Polyphenolic Concentration
Ellagic acidly Myricetin2 Quercetin2
E3 168 ab4 10.2 c 5.44 bc
F 176 ab 22.2 ab 7.26 a
BCis 123 cd 18.6 b 6.54 ab
UJBCls ND5 ND ND
BA 113 d 11.3 c 5.16 bc
UBA 9.77 e ND ND
SD 122 cd 13.8 c 4.94 c
FD 155 bc 20.7 ab 7.13 a
VD 202 a 22.8 a 7.67 a
'Expresed in ellagic acid equivalents (mg/kg). 2Expressed in quercetin equivalents (mg/kg). 3 Treatment
abbravietions: (E) Extract, (F) fermented extract, (BC18) bound to reversed phase C18 column, (UBC18) unbound to
reversed phase C18 column, (BA) bound to Amberlite XAD-4, (UBA) unbound to Amberlite XAD-4, (SD) spray
dried, (FD) freeze dried, and (VD) vacuum dried. 4Values with similar letters within columns are not significantly
different (LSD P < 0.05). 5Compounds were not detected.

3.3.4 Antioxidant Capacity

Antioxidant capacity of muscadine pomace extract (34.3 & 0.57 Cpmol Trolox equivalents

(TE)/g) was found to be relatively high and comparable to other fruits such as cherries (33.4 &

3.4 Cpmol TE/g) and strawberries (35.4 & 4.2 Cpmol TE/g), and vegetables such as red cabbage









(31.5 & 6 Cpmol TE/g) (Wu and others 2004). Processing in general had a negative effect on the

antioxidant capacity of the extract (Figure 3-7). Fermentation, which maintained the

concentration of polyphenolics from the starting extract, had significant differences in

antioxidant capacity (28.7 & 2.53Cpmol TE/g). Following the fermentation procedure only

vacuum drying maintained antioxidant capacity (28.8 & 1.23 Cmol TE/g) while spray and freeze

drying significantly reduced it (22.0 + 0.75Cpmol TE/g and 18.1 & 3.13Cpmol TE/g respectively).

Solid phase isolation process also had a detrimental effect on the antioxidant capacity of the

starting material since both affinity column techniques decrease the antioxidant capacity by more

than 34% (Amberlite) and 51% (reversed phase Cls). The unbound fraction of Amberlite

showed a comparatively low antioxidant capacity probably due to the presence of ellagitannins

in the fraction (1.91 & 3.64Cpmol TE/g) since more than 15% of polyphenolics that were subjected

to the Amberlite were found in the unbound fraction and were thought to contain comparable

content of antioxidant capacity that was washed away. Data of the unbound fraction only

indicated 8% of antioxidant capacity which confirmed the previous assumption of ellagitannins

occurrence in the fraction since its antioxidant capacity is considered to be lower than ellagic

acid aglycone as explained by Lee (2004) where antioxidant capacity was higher in aglycone

forms and gradually reduced by polymerization of ellagic acid. Data from the unbound fraction

of reversed phase Cls did not indicate presence of antioxidant compounds. Furthermore, HPLC,

total anthocyanins, and total phenolics analyses confirmed a negligible concentration of

anthocyanins and polyphenolics in this fraction (Table 3-1).

All treatments affected the initial antioxidant capacity from the extract which agrees with

Schmidt and others (2005), who suggested that even if the polyphenolic concentration of any

sample is maintained after processing, its bioactive characteristics are modified. Vacuum










concentration resulted in a greater retention of antioxidant capacity compared to spray drying

and freeze drying which resulted in losses of 23% and 37% respectively. Also, vacuum

concentration was the only isolation procedure that preserved the antioxidant capacity following

fermentation. Consequently, the vacuum drying process did not have a negative effect on the

quality of the final extract.


10
35-bb
S30-
v,25- c cd
a 20- d
15 -

~ e
5-f
E- 0
-5

E F BC18 UBC18 BA UBA SD FD VD
Treatments

Figure 3-7. Antioxidant capacity of muscadine pomace extract as affected by various processing
protocols. Error bars represent the standard error of the mean, n=3.

3.4 Conclusions

Target compounds extracted from muscadine grape pomace showed fairly high antioxidant

activity (34.3 + 0.57 Cpmol TE/g) that is comparable to some fruits and vegetables. Processing

positively influenced polymerization and stability of anthocyanins. In the first section of this

work, high temperature together with low concentration of polyphenolics, oxygen exposure, and

high pH environment were thought to be the most harmful factors that affected polyphenolic

content and bioactive characteristics. However, later information from HPLC analysis illustrated

the impact of polymerization was not accounted by colorimetric analysis and heat was not a

major problem, thus showing biased results. Although processing did not show significant









compound losses in all treatments except Amberlite and spray drying (by HPLC analysis),

antioxidant capacity was significantly affected by processing. Vacuum drying proved to be the

best treatment of all since it best maintained both anthocyanins and other polyphenolics

concentration and also preserved the antioxidant capacity following fermentation. Fermentation

showed better results than solid phase isolation to get rid of sugars without j eopardizing the

quality of the Einal product. Moreover, solid phase isolation, specifically the Amberlite resin

technique, could not bind all polyphenolics efficiently. Drying procedures following an aerobic

fermentation were more practical than solid phase isolation followed by methanol evaporation,

thus drying processes were not only statistically different but also showed practicality to be

considered for implementation by the industry. Furthermore, the use of methanol, a non edible

alcohol, in both affinity column isolation techniques could represent an environmental downside

and a potential operative cost compared to use of water in the fermentation procedure.









CHAPTER 4
ECONOMIC ANALYSIS OF AN ISOLATED PRODUCT OBTAINED FROM MUSCADINE
GRAPE POMACE

Functional foods and beverages consumption is growing due to maj or consumer trends

toward health consciousness (Milo 2005). As a result of consumer awareness, natural products

such as food colorants and antioxidants have gained substantial attention in the market. In 1996,

26% of the food antioxidant market was occupied by natural antioxidants with a growth rate of

6-7% annually. The sources of natural antioxidants such as vitamin C, tocopherols,

polyphenolics, and organic acids include fruits, vegetables, spices, and herbs (Meyer and others

2002). In berries and fruits, the most abundant antioxidants are vitamin C and polyphenolics.

Companies such as Optiture (USA), Chr. Hansen (Denmark), Overseal Natural Ingredients (GB),

Quim Dis (France), Inheda (France), and Folexco (USA) share the market for extracts and

concentrates from fruits and berries (Meyer and others 2002).

A growing demand for natural products has created an opportunity to substitute synthetic

antioxidants by natural compounds. Among the traditional antioxidants used are butylated

hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) which have been associated with

potential toxicity. Furthermore, the manufacturing costs of these compounds are more expensive

than natural antioxidants from fruits and vegetables (Moure and others 2001).

Polyphenolics can be widely found in pomace or wastes of numerous fruits and vegetables

after processing (Koplotek and others 2005; Pastrana-Bonilla and others 2003; Visioli and others

1999; Bonilla and others 1999) and these sources represent an inexpensive material to potentially

create food ingredients. As a result, the interest in natural antioxidants and the occurrence of

these compounds in by-products have driven fruit and vegetable processors to attempt to extract

polyphenolics from their wastes and increase the profitability of their operations. In the case of

muscadine grapes, pomace constitutes around 40% of the total fruit and it is an important source










of polyphenolics (Pastrana-Bonilla and others 2003; Morris and Brady 2004) that has not been

utilized, but could be an important source to generate food ingredients. Research has focused on

converting muscadine and blueberry pomace into nutraceutical products that could be sold for as

much as $100/1b. In Georgia alone, around 1.5 million pounds of dry muscadine skins are

produced yearly and could be transformed into a very profitable product (Phillips 2006).

Therefore, the aim of this part of the study was to analyze the profitability of proposed

technologies to extract and concentrate polyphenolics from muscadine grape pomace as an

incremental operation to a typical grape juice production facility.

4.2 Materials and Methods

4.2.1 Data Collection

Primary data, regarding the nature of the muscadine industry and its operations, was

collected by interviewing muscadine processors of South Georgia. Other primary information,

regarding equipment specification, capacity and pricing, was collected by interviewing some

companies responsible for selling used equipment for food industries. Assumptions used in this

study were drawn from interview comments and educated estimations.

4.2.2 Economic Analysis

An economic analysis was conducted based on profitability, sensitivity, and economic

return of three alternatives of polyphenolic isolation following fermentation. Potential

profitability was evaluated by comparing total revenue and total costs by a breakeven analysis of

both prices and volumes of production. Results were reported as minimum price per pound

($/lb) and minimum volume (lb) needed to cover total annual costs respectively. A ratio between

production volume of extract and the volume of dried residue from the skins following

fermentation was used for relevant calculations. A ratio between prices of the extract and the

dried skins was also applied assuming that prices would fluctuate proportionally. Variations in









the output of the model were assessed by a sensitivity analysis conducted by modifying the

production and price of both the extract and the dried skins (+40%). The gross revenue and the

profit were displayed in the analysis for better illustration of the outcome. Results were reported

in US dollars ($) per season. Finally, the economic return to the business was assessed over a

period of ten years by a cash flow analysis. Net Present Value (NPV) was calculated based on a

rate of 12% which was compared to a calculated Internal Rate of Return (IRR). Inflation was

estimated as 3.43%. Depreciation of equipment was conducted by the single line method with a

10% salvage value. A loan is calculated based on a fixed interest rate with higher payments

towards interest at the beginning, paying the loan in 10 years. Results were expressed as US

currency per season.

4.2.2.1 Description of the operation

The intended operation is an extension of a juice process facility illustrated in Figure 4-1.

After grapes have been pressed for juice manufacture, the waste is collected and mixed with hot

water (1:2) to allow soluble compounds to migrate to water creating a "liquid extract". A

pressing procedure following the initial extraction is necessary to remove skins to facilitate

fermentation. Furthermore, skins can be dried and sold as animal feed. Next, sugars which have

migrated into the liquid extract together with other compounds must be removed to facilitate

further processing. Sugars are removed by an aerobic fermentation followed by a series of

filtrations that allow separation of insoluble compounds. Lastly, a drying protocol is necessary

to obtain an isolated product rich in polyphenolics. Three concentration protocols have been

considered for the process: spray drying, freeze drying, and vacuum evaporation.

4.2.2.2 Economic assumptions

The analyses in this study are based on the following assumptions:

*Land is previously owned and is not included in the capital investment.











































Figure 4-1. Operation flow for a typical grape juice processor planning to process its byproduct.

* The company already has some equipment needed for the operation and it is not included
in the capital investment.

* Muscadine pomace has no cost since it is a by-product of their own operation and no costs
were considered for elimination of this material.

* 90 tons of fruit will go through juice processing and the pomace will be the by-product
operation which will be used as the starting material for polyphenolic extraction.

* This operation lasts two months due to the harvesting season of muscadine grapes (61
days).

* The final consumer is a processor that will use the product as a food ingredient.

* Equipment specifications (Table 4-1) are approximates drawn from interviews with
equipment vendors.










* The process needs 4 employees working part time (4h/day) on the process with a wage of
$8/h. Employees already work in the company.

* Proposed price for yeast required for fermentation is $5.45/1b, and intended to be used at a
ratio of 0.251b yeast/1001b of extract.

* Price for diatomaceous earth (DE) for clarification is quoted at $0.80/1b and planned to be
used at a ratio of 11b of DE/1001b of extract.

* Most of the working time will be spent in the extract manufacturing (70%) while 30% is
spent on skin drying.

* Currently no costs in hauling or marketing of the final product are taken into account.

* The final product is intended to be sold to formulator of dietary supplements and/or
functional foods.

* The estimated price used for the extract in the analysis is $70/1b.

* The dried skins after this process are going to be sold as animal feed.

* The estimated price of the dried skins in the analysis is $1.5/1b.

* Depreciation is calculated at 5 years for equipment, 20 years for land, and 10 years for
other materials.

* The rate used for taxes is 20% for a self employed operation according to IRS.

* There is a loan for the building construction of $70,000 to be paid in ten years at a 7.5%
interest rate (Table 4-2).

* For other activities such us cleaning and maintenance needed in the operation, 1% of the
total revenue was assigned in the cash flow.

* Installation cost for dryers are 40% of the total cost of the machinery.

* Installation cost for filters are 70% of the total cost of the machinery.

* Pumps have an electrical consumption of 17KW each.

* Operation time was calculated based on the volume produced per day, for electrical
calculations, extra time (1 hour) was added for basic maintenance and warming up of
equipment.





Evaporator $ 150,000 600 39.0
Spray dryer $ 84,000 200 25.0
Freeze Dryer $ 77,000 150 18.4
Residue dryer $ 100,000 500 16.0


Table 4-2. Loan payment plan for ten years at a fixed rate of 7.5%.
Intial debt (ID) Rate' Payment2 Interest Amortization (A) Debt3 (ID -A)
$ 70,000 7.50% $ 7,525 $ 998 $ 6,528 $ 63,473
$ 63,473 7.50% $ 7,525 $ 893 $ 6,633 $ 56,840
$ 56,840 7.50% $ 7,525 $ 788 $ 6,738 $ 50,103
$ 50,103 7.50% $ 7,525 $ 683 $ 6,843 $ 43,260
$ 43,260 7.50% $ 7,525 $ 578 $ 6,948 $ 36,313
$ 36,313 7.50% $ 7,525 $ 473 $ 7,053 $ 29,260
$ 29,260 7.50% $ 7,525 $ 368 $ 7,158 $ 22,103
$ 22, 103 7.50% $ 7,525 $ 263 $ 7,263 $ 14,840
$ 14,840 7.50% $ 7,525 $ 158 $ 7,368 $ 7,473
$ 7,473 7.50% $ 7,525 $ 53 $ 7,473 $
1Rate estimated from a normal bank for a fixed rate loan. 2Payment is constituted by amortization and interest.
3Debt is the result of subtracting annual amortization from previous period debt.

4.3 Results and Discussion

Proposed technologies were economically evaluated and results were reported in US

currency ($US).

4.3.1 Economic Analysis

For all three operations (spray, freeze and vacuum drying), the process was identical until

the final step where isolation of polyphenolics took place via three different methods. According

to results from Chapter 3, the extract has a concentration of polyphenolics (1640 + 18. 1 mg/kg)

that is intended to be isolated as a final product (extract). From this number we consider that the

final product will be at least 0.16% concentrated and that all calculations are based on this

proportion of the liquid extracted from the process. With 90 tons of grapes expected to be used

in this process, 40% represent skins (Pastrana-Bonilla and others 2002) that are directly intended


Table 4-1. Generalities of drying equipment.


Estimated cost
($US)


Estimated capacity
(lb/h)


Estimated electrical
demand (KW)


Equipment









for the secondary process to obtain the desired extract. This results in a season skin production

of 72,000 lb of fresh skin that is intended to be mixed with hot water for a production of 144,000

lb of liquid extract and 72,000 lb of residual skins. If 0. 16% of the liquid extract represents

polyphenolics and we are trying to obtain a product with 5% moisture, the final concentration of

extract in a season would be 249 lb. From the remaining skins after fermentation, 30% is

considered solids (Phillips, 2006) and is proposed to be sold as animal feed as part of the by-

product operation, which will also contain 5% moisture as a final product. From the remaining

skins obtained, a total of 22,619 lb of dry skin can be produced in a season. Currently, efforts to

process muscadine pomace are still emerging and the amount of time and capital intended for

such an operation is limited. Therefore, proposed processes are basic and economically feasible

for a typical muscadine producer.

4.3.1.1 Spray drying operation

A used spray dryer with a capacity to remove 200 lb of water per hour was considered for

the process. An estimated price ($60,000) was quoted for the equipment which needed to

operate 13 hours daily to remove the amount of water required to obtain a 5% moisture isolated

product. The initial investment was $120,640 which included the spray dryer, its installation fee

($24,000), and other required materials (Table 4-3). Additional equipment necessary for this

specific operation would typically already be owned by a juice processor. As mentioned earlier

in the assumptions, the new building construction is intended to be built with a bank loan.

From the seasonal production of extract and dried skins (2491b and 22,6191b respectively)

and the medium price estimated and expected by an average muscadine grape producer for such

extract and dried skins production ($70/1b and $1.5/1b respectively ), total income for the

incremental operation was calculated. Furthermore, with knowledge of the equipment and

material required by this operation, total costs were also calculated and used for further









economic analysis. Once income and cost sources were determined, a production volume

breakeven point established the minimum amount required to sustain the intended by-product

operation. From the break even equation [1], where I represents income from the extract (IE) and

the residue skins (IR), C TepreSents cost both fixed (FC) and variable from the two products (VCE

and VCR) and the ratio between extract (QE) and dried skins (QR) prOductions (lb per season) [2],

161 lb of extract and 14,616 lb of dried skins must be produced to cover both annual fixed costs

and variable costs of an operation using a spray dryer with similar technical characteristics as the

method for polyphenolic isolation. Figure 4-2 illustrates that at prices $70/1b for the extract (pE)

and $1.50/1b for the dried skins (pR), the total cost to cover for a season is $33,168 which is

covered by 161 lb of extracts and proportionally by 14,616 lb of dried skins. The production of

dried skins was not shown in the x-axis of this figure to avoid confusion.

Table 4-3. Capital expenditure to initiate a marginal process obtaining extract and dried skins
from muscadine grape skins using spray drying as the isolation technique.
Description Unit Unit cost Quantity Total estimated cost
1. EQUIPMENT COSTS'
Filter Each $ 25,000 1 $ 25,000
Spray Dryer Each $ 84,000 1 $ 84,000
Pumps Each $ 1,200 2 $ 2,400
Containers (bins) Each $ 220 42 $ 9,240
Sub-total $ 120,640
2. CONSTRUCTION COSTS
New Building3 Each $ 70,000 1 $ 70,000
Sub-total $ 70,000

GRAND TOTAL $ 190,640
'Equipment listed in the table is applied directly to the expenditure of the operation since the rest of equipment
needed for the operation is already owned. 2The total construction cost for the new building includes wiring,
electrical and tubing installations. 3NOW building will be constructed with a bank loan.

IE R, = FC +VyCE VCR [1E = 0.011QR [ 2]

PE X E + R XR = FC +VyCE x E +VyCR x R

70QE +1.5QR = 25,162 +39.82QE +0. 11QR










70(0.011QR)+1.5QR, = 25,162 + 39.82(0.011QR,)+ 0. 11QR

0.77QR +1.5QR = 25,162 +0.44QR + 0. 11QR

2.27QR 0.55QR = 25,162

1.72QR = 25,162


QR _25,616 72


QR=14,615.56 lb QE=160.63 lb


$70,000
$60,00 *FC-m-VC --TC -x-1

S$50,000-
S$40,000-

~j$30,000 -
$20,000-
$10,0 -

0 50 100 150 200 250 300
Volume of extract production (Ib)

Figure 4-2. Volume break-even point for a facility using a spray dryer as a final step for product
isolation. Abbreviations: FC Fixed costs, VC variable costs, TC total costs, I income.

Another useful analysis is to keep production volume steady and to vary prices to

determine the minimum price needed to cover total costs. For a price break even point

calculation, the break even equation [1] and the ratio between extract and dried skins productions

[2] were also employed. Furthermore, a ratio between prices ($/lb) [3] was used under the

assumption that prices of extract and dried skins would maintain a difference between each other

and that such difference would remain proportional. With such assumptions in mind, the prices

where the actual production of extract (249 lb) and dried skins (22,619 lb) cover the annual total

cost are $51.21/1b and $1.10/1b, respectively. Figure 4-3 illustrates five diverse scenarios with









different incomes (I) illustrating the significant impact price alterations can have on the

operation. Prices ranged from $20/1b (I-1) to $100/1b (I-5) with increments of $20 for the extract

and proportionally from $0.43/1b to $2. 14/1b with increments of 43 cents for the dried skins.

When prices were below $40/1b (extract) and $0.86/1b (dried skins), the minimum production to

cover annual costs exceeded 300 lb of extract and more than 27,000 lb of dried skins. On the

other hand, when prices reached $100/1b (extract) and $2. 14/1b (dried skins), net earnings can be

reached after only 100 lb of extract and 9,000 lb of dried skins produced. With such prices and

the intended season production volume, gross income of almost $90,000 would be generated and

profit would increase up to $48,379 in a single season.

IE R, = FC +VyCE VCR 1

PE X E + R XR = FC +VyCE x E V CR x R

pE Rx L 0.011) + pR X R = FC +V~CE R x 0.011)+ +VCR LR



Q 0.011pE RIn~~+ RliC FC+.01CE C



pE = 46.67 pR [3]


0.011(46.67pR R _= 25,16 2,619+0.011(39.82)+0.11


pR = $1.10/1b pE =$51.21/1b

A sensitivity analysis of the circumstances when a spray dryer (with characteristics

previously described) is used as the isolation method was conducted (Table 4-4). Data suggested

that a loss of more than $1 1,000 could be anticipated if production and prices dropped 40%.

Losses were still registered when one characteristic, either price or volume, was reduced 40%.













































Low Sales $ 6, 188 $ 36,664 $ 51,685
17816,56 Profit" $ (11,363)7 $ (887) $ 14,133

0 Sales $ 36,664 $ 51,329, $ 72,358
eMedium 249 /22,619
Profit $ (887) $ 13,778 $ 34,807

a2 Sales $ 51,329 $ 71,861 $ 101,302
High 348 /31,666
Profit $ 13,778 $ 34,310 $ 63,750


Under initial circumstances (medium price and volume), a profit of almost $14,000 in a season

was registered. The remaining situations also showed positive numbers. Therefore, this

operation is viable when the combination of production volume and prices surpasses low-low

and low-medium combinations.


$100,000
$90, 000 -( +TC -m- 1-1 t 1-2 ~ 1-3 + 1-4 -0 1-5
$80,000-
$70,000-
S$60,000-
$50,000-
$40,000-
$30,000-
$20,000-
$10,000-
$0
0 50 100 150 200 250 300
Volume (Ib)

Figure 4-3. Break-even point as affected by price for a facility using a spray dryer as a Einal step
for product isolation. Abbreviations: I income, TC total costs.

Table 4-4. Sensitivity analysis for a marginal process obtaining extract and dried skins from
muscadine grape skins using spray drying as the isolation technique.
Prices ($US)
Low' Medium High2
Ext DS p~t/pDS4 $50 /$1.07 $70 /$1.50 $100 /$2.10


'Low represent 40% less of both medium prices and volumes. 2High represent 40% more of both medium prices
and volumes. 3Extract and dried skins productions are displayed vertically in the table. 4Prices for extract and dried
skins are displayed horizontally in the table. 5Sales are shown for all 9 different supposed scenarios. 6Profits are
shown for all 9 different supposed scenarios. 7Number in parenthesis indicate profit loss.

The economic return was assessed after 10 years of operation by a cash flow analysis

(Table 4-5). With an investment of $120,640 and a bank loan of $70,000, the operation could









return the investment and generate almost $60,000 of net earnings in ten years. With average

annual income of more than $30,000, the internal rate of return (IRR) was 10% higher than the

rate used for the net present value (NPV) calculation, thus showing thriving results for this

additional operation for the muscadine industry. It is important to denote that more compounds

could be extracted from the skins if processes are to be optimized resulting in a higher yield,

extract production and thus, generating profit for selling more product at $70/1b than $1.5/1b.

4.3.1.2 Freeze drying operation

A used freeze dryer with a capacity to remove 150 lb of water per hour was evaluated for

this isolation technique and according to its specifications, it needed 15.7 hours to remove water

from the daily liquid extract produced. An estimated price ($55,000) was quoted for the

equipment. The initial investment ($113,640) included the freeze dryer, its installation fee

($4,400), and other required materials (Table 4-6). Additional equipment necessary for this

specific operation was assumed to be already owned by a typical juice processor. The facilities

required to conduct a by-product isolation process were intended to be built with a bank loan as

described in the assumption section of this chapter.

Table 4-6. Capital expenditure to initiate a marginal process obtaining extract and dried skins
from muscadine grape skins using freeze drying as the isolation technique.
Description Unit Unit cost Quantity Total estimated cost
1. EQUIPMENT COSTS'
Filter Each $ 25,000 1 $ 25,000
Freeze Dryer Each $ 77,000 1 $ 77,000
Pumps Each $ 1,200 2 $ 2,400
Containers (bins) Each $ 220 42 $ 9,240
Sub-total $ 113,640
2. CONSTRUCTION COSTS
New Building3 Each $ 70,000 1 $ 70,000
Sub-total $ 70,000

GRAND TOTAL $ 183,640
'Equipment listed in the table is applied directly to the expenditure of the operation since the rest of equipment
needed for the operation is already owned. 2The total construction cost for the new building includes wiring,
electrical and tubing installations. 3NOW building will be constructed with a bank loan.











Table 4-5. Cash flow analysis for a marginal process obtaining extract and dried skins from muscadine grape skins using spray drying
as the isolation technique.
Periods (years)
01 23 45 67 8 910


Investment $120,640
Income
Extract
Residue
Expenses
Labor
Materials
General costs
Depreciation
Interest (7.5%)
Gross Income
Tax (20%)
Utilities after Taxes
S(+) Depreciation
" Amortization
Salvage Value
Net Income

Net Present Value (NPV)
Intern Rate of Return (IRR)


$ 17,401 $ 17,998 $ 18,615 $ 19,254 $ 19,914 $ 20,597 $ 21,304 $ 22,035 $ 22,790 $ 23,572
$ 33,928 $ 35,092 $ 36,296 $ 37,540 $ 38,828 $ 40,160 $ 41,537 $ 42,962 $ 44,436 $ 45,960

$ 7,168 $ 7,412 $ 7,664 $ 7,924 $ 8,194 $ 8,472 $ 8,760 $ 9,058 $ 9,366 $ 9,685
$ 3,341 $ 3,455 $ 3,572 $ 3,694 $ 3,819 $ 3,949 $ 4,083 $ 4,222 $ 4,366 $ 4,514
$ 1,753 $ 1,813 $ 1,874 $ 1,938 $ 2,004 $ 2,072 $ 2,142 $ 2,215 $ 2,291 $ 2,369
$ 18,303 $ 18,303 $ 18,303 $ 18,303 $ 18,303 $ 4,424 $ 4,424 $ 4,424 $ 4,424 $ 4,424
$ 998 $ 998 $ 893 $ 788 $ 683 $ 578 $ 473 $ 368 $ 263 $ 158
$ 19,767 $ 21,215 $ 22,710 $ 24,253 $ 25,845 $ 41,367 $ 43,064 $ 44,815 $ 46,622 $ 48,488
$ 3,953 $ 4,243 $ 4,542 $ 4,851 $ 5,169 $ 8,273 $ 8,613 $ 8,963 $ 9,324 $ 9,698
$ 15,813 $ 16,972 $ 18,168 $ 19,402 $ 20,676 $ 33,094 $ 34,451 $ 35,852 $ 37,298 $ 38,791
$ 18,303 $ 18,303 $ 18,303 $ 18,303 $ 18,303 $ 4,424 $ 4,424 $ 4,424 $ 4,424 $ 4,424
$ 6,528 $ 6,633 $ 6,738 $ 6,843 $ 6,948 $ 7,053 $ 7,158 $ 7,263 $ 7,368 $ 7,473
$ 7,711
$ 27,589 $ 28,643 $ 29,734 $ 30,863 $ 39,742 $ 30,465 $ 31,717 $ 33,013 $ 34,354 $ 35,742


$177,808
22.2%


rate 12.00%









When income and cost sources were determined, a production volume breakeven point was

conducted to determine the minimum amount of extract and dried skins required for the by-

product operation to be feasible. Analogous to the previous technology (spray drying), the break

even equation (US dollars) [1] and the proportion between extract and dried skins productions (lb

per season) [2] were used to conduct a break even analysis. Data suggested that 152 lb of extract

and 13,847 lb of dried skins were needed to cover total annual costs ($3 1,424) of the operation

when prices for the extract and the dried skins were $70/1b and $1.50/1b respectively (Figure 4-

4).

IE+ R = FC +VyCE VCR [1E = 0.011QR [ 2]

PE X E +R X R = FC +VyCE x E +VyCR x R

70QE +1.5QR = 23,862 +39.68QE + 0. 11QR

70(0.011QR)+1.5QR = 23,862 + 39.68(0.011QR)+ 0.11QR

0.77QR +1.5QR = 23,862 +0.44QR +0. 11QR

2.27QR 0.55QR = 23,862

1.72QR = 23,862


QR 23,86 72

QR 13,847.41 lb QE=152.18 lb











$70,000
$60,00 *FC-m-VC --TC -x-1

$50,000-

S$40,000-

-0 $30,000-
$20,000-
$10,0 -


0 50 100 150 200 250 300
Volume of extract production (Ib)

Figure 4-4. Volume break-even point for a facility using a freeze dryer as a final step for product
isolation. Abbreviations: FC Fixed costs, VC variable costs, TC total costs, I income.

For the break even price analysis, the break even equation US currency [1] and the ratio

between extract and dried skins productions in pounds per season [2] were used for estimations.

A ratio between prices ($/lb) [3] was calculated and also used under the assumption that prices

would maintain a proportional difference between each other. To cover total annual costs when

medium production volumes of extract (249 lb) and dried skins (22,619 lb) are produced, the

prices should be at least $39/1b and $1.06/1b, respectively. Five scenarios in which prices varied

from $20/1b (I-1) to $/100/1b (I-5) with increments of $20 for the extract and $0.43/1b (I-1) and

$2. 14/1b (I-5) with increments of 43 cents for the dried skins were analyzed (Figure 4-5). Similar

to an operation with spray drying as the isolation method, when prices are below $40/1b (extract)

and $0.86/1b (dried skins), the minimum production to cover annual costs would have to be

greater than 300 lb of extract and more than 27,000 lb of dried skins. In contrast, when prices

just surpass $40/1b (extract) and $0.86/1b (dried skins) profit is reached under such production

volumes. This slight difference in the calculation between spray drying and freeze drying

operations is attributable to the reduction in the investment. The freeze dryer suggested in this

chapter is 10% cheaper.










IE R, = FC +VyCE VCR 1

PE X E + R XR = FC +VyCE x E V CR x R

pEK Rx ~ 0.011 ) + pRK R = FC +V~CEi R Kx 0.011 )+ +VCR R






0.011p Rl, --, FC +0.011VCEC +VCR1


pE = 46.67 pR


0.011(46.67 )pR R _23,86 2,619+0.011(39.68)+0.11


pR=$1.06/1b pE=$49.39/1b


$100,000
$90, 000 -( +TC -m- 1-1 -A- 1-2 -x- 1-3 -a- 1-4 -e 1-5
$80,000-
$70,000-
S$60,000-
=$50,000-
$40,000-
$30,000-
$20,000-
$10,000-
$0
0 50 100 150 200 250 300
Volume (Ib)

Figure 4-5. Break-even point as affected by price for a facility using a freeze dryer as a final step
for product isolation. Abbreviations: I income, TC total costs.

A sensitivity analysis of the circumstances where freeze drying is proposed as the isolation

method (Table 4-7) indicated loss of $10,025 when production and prices dropped 40%. The

low-medium combinations showed a small profit ($450) in a single year. The rest of the

sensitivity analysis indicated positive numbers (profit) for the eight remaining situations.









Therefore, the only circumstance under which this operation could not be viable was when both

production and prices were 40% lower than the expected values (medium).

Table 4-7. Sensitivity analysis for a marginal process obtaining extract and dried skins from
muscadine grape skins using freeze drying as the isolation technique.
Prices ($US)
Lowl Medium High2
Ext /DS3 pExt / pDS4 $50 /$1.07 $70 /$1.50 $100 /$2.10
1 Sales $ 26, 188 $ 36,664 $ 51,685
Low' 178 16156 Profit6 $ (10,025) $ 450 $ 15,471

0 Sales $ 36,664 $ .51,329 $ 72,358
eMedium 249 /22,619
Profit $ 450 $ 15,116 $ 36,145

a2 Sales $ 51,329 $ 71,861 $ 101,302
High 348 /31,666
Profit $ 15,116 $ 35,648 $ 65,088
ILow represent 40% less of both medium prices and volumes. 2High represent 40% more of both medium prices
and volumes. 3Extract and dried skins productions are displayed vertically in the table. 4Prices for extract and dried
skins are displayed horizontally in the table. 5Sales are shown for all 9 different supposed scenarios. 6PTOfits are
shown for all 9 different supposed scenarios. 7Number in parenthesis indicate profit loss.

Economic return of this operation assessed through a ten-year period suggested that the

operation was profitable and could return the investment in less than 6 years with and NPV of

more than 177,000 with a IRR of almost 24% (Table 4-8). This increase in IRR compared to the

spray drying operation is consequence of a reduction in the investment.

4.3.1.3 Vacuum drying operation

The evaporator cited in this situation had a capacity of 600 lb of water removed per hour

and water produced from the daily operation could be evaporated in less than 4 hours. An

estimated price ($107, 143) was quoted for the equipment, and initial investment of $186,640

included the evaporator, its installation fee ($42,857), and other required materials (Table 4-9).

Additional equipment necessary for the isolation operation was thought to be previously owned

by a typical juice processor and thus not included in the current analysis.











Table 4-8. Cash flow analysis for a marginal process obtaining extract and dried skins from muscadine grape skins using freeze
drying as the isolation technique.
Periods (years)
0 1 2 3 4 5 6 7 8 9 1


Investment $113,640
Income
Extract
Residue
Expenses
Labor
Materials
General costs
Depreciation
Interest (7.5%)
Gross Income
Tax (20%)
Utilities after Taxes
(+) Depreciation
SAmortization
Salvage Value
Net Income

Net Present Value (NPV)
Intern Rate of Return (IRR)


$ 17,401 $ 17,998 $ 18,615 $ 19,254 $ 19,914 $ 20,597 $ 21,304 $ 22,035 $ 22,790 $ 23,572
$ 33,928 $ 35,092 $ 36,296 $ 37,540 $ 38,828 $ 40,160 $ 41,537 $ 42,962 $ 44,436 $ 45,960

$ 7,168 $ 7,412 $ 7,664 $ 7,924 $ 8,194 $ 8,472 $ 8,760 $ 9,058 $ 9,366 $ 9,685
$ 3,341 $ 3,455 $ 3,572 $ 3,694 $ 3,819 $ 3,949 $ 4,083 $ 4,222 $ 4,366 $ 4,514
$ 1,716 $ 1,774 $ 1,835 $ 1,897 $ 1,961 $ 2,028 $ 2,097 $ 2,168 $ 2,242 $ 2,318
$ 17,403 $ 17,403 $ 17,403 $ 17,403 $ 17,403 $ 4,424 $ 4,424 $ 4,424 $ 4,424 $ 4,424
$ 998 $ 998 $ 893 $ 788 $ 683 $ 578 $ 473 $ 368 $ 263 $ 158
$ 20,704 $ 22,154 $ 23,650 $ 25,194 $ 26,788 $ 41,411 $ 43,109 $ 44,862 $ 46,671 $ 48,538
$ 4,141 $ 4,431 $ 4,730 $ 5,039 $ 5,358 $ 8,282 $ 8,622 $ 8,972 $ 9,334 $ 9,708
$ 16,563 $ 17,723 $ 18,920 $ 20,155 $ 21,430 $ 33,129 $ 34,487 $ 35,889 $ 37,337 $ 38,831
$ 17,403 $ 17,403 $ 17,403 $ 17,403 $ 17,403 $ 4,424 $ 4,424 $ 4,424 $ 4,424 $ 4,424
$ 6,528 $ 6,633 $ 6,738 $ 6,843 $ 6,948 $ 7,053 $ 7,158 $ 7,263 $ 7,368 $ 7,473
$ 7,211
$ 27,439 $ 28,494 $ 29,586 $ 30,716 $ 39,096 $ 30,501 $ 31,754 $ 33,051 $ 34,393 $ 35,782


$177,065
23.8%


rate 12.00%









Table 4-9. Capital expenditure to initiate a marginal process obtaining extract and dried skins
from muscadine grape skins using vacuum drying as the isolation technique.
Description Unit Unit cost Quantity Total estimated cost
1. EQUIPMENT COSTS
Filter Each $ 25,000 1 $ 25,000
Vaccum Dryer Each $ 150,000 1 $ 150,000
Pumps Each $ 1,200 2 $ 2,400
Containers (bins) Each $ 220 42 $ 9,240
Sub-total $ 186,640
2. CONSTRUCTION COSTS
New Building3 Each $ 70,000 1 $ 70,000
Sub-total $ 70,000

GRAND TOTAL $ 256,640
1Equipment listed in the table is applied directly to the expenditure of the operation since the rest of equipment
needed for the operation is already owned. 2The total construction cost for the new building includes wiring,
electrical and tubing installations. 3NOW building will be constructed with a bank loan.

Once revenue and cost sources have been determined, a production volume breakeven

analysis was used to determine the minimum amount required to sustain the intended by-product

operation. The break even equation [1] and the proportion between extract and dried skins

productions [2] were used as part of the analysis. Calculations indicated that in order to cover

both annual fixed and variable costs of the operation, 236 lb of extract and 21,5 16 lb of dried

skins should be produced at $70/1b and $1.50/1b respectively. Figure 4-6 illustrates these

calculations by showing the point at which the gross income generated covers season costs of

$48,828. When using an evaporator of such specifications, the demand of volume of both

products is high due to the elevated cost of the equipment (investment). According to these

calculations and the medium production, only 13 lb of extract and 103 lb of dried skins represent

earnings in the season.

IE+ R = FC +VyCE VCR [1E = 0.011QR [ 2]

PE X E + R XR = FC +VyCE x E V CR x R

70QE +1.5QR = 37,419 +37.75QE +0. 11QR










70(0.011QR)+ 1.5QR, = 37,419 +38.23(0.011QR,)+0. 11QR

0.77QR +1.5QR = 37,419 + 0.42QR + 0. 11QR

2.27QR 0.53QR = 37,419

1.74QR = 37,419


QR 37,41 74,


QR = 21,516.44 lb QE =236.47hlb


$70,000
$60,00 *FC-m-VC --TC -x-1

S$50,000-

S$40,000 -1
~j$30,000-
$20,000-
$10,0 -


0 50 100 150 200 250 300
Volume of extract production (Ib)

Figure 4-6. Volume break-even point for a facility using a vacuum dryer as a final step for
product isolation. Abbreviations: FC Fixed costs, VC variable costs, TC total costs, I
income.

A price break even analysis for an operation with an evaporator was also conducted.

Similar to previous break even analysis, the break even equation [1], the ratio between extract

and dried skins productions [2], and a ratio between prices [3] were used for calculations. Total

annual costs could be covered if prices of extract (249 lb) and dried skins (22,619 lb) surpassed

$67/1b and $1.44/1b respectively. Figure 4-7 illustrates five scenarios in which prices varied

from $20/1b (I-1) to $/100/1b (I-5) with increments of $20 for the extract and $0.43/1b (I-1) and

$2. 14/1b (I-5) with increments of 43 cents for the dried skins. Unlike the two previous analysis









(spray drying and freeze drying), profit with a production under 300 lb of extract and over

27,000 lb of dried skins requires a price of at least $60/1b (extract) and $1.29/1b (dried skins). At

such prices a profit of only $1,203 could be accomplished. In contrast, net season earnings of

$36,600 can be reached when 300 lb of extract and 27,000 lb of dried skins are produced at the

highest prices analyzed ($100/1b and $2.14/1b respectively). Although price of the evaporator

was high, profit can be reached in the first year and cover season costs. Moreover, if the process

is successful and proj ected to grow, the evaporator analyzed in this scenario has enough capacity

to support such growth while the other two equipment (spray and freeze dryers) operate more

than 10 hours daily while the evaporator works only 4. Thus, while this isolation technique is

capital demanding, if implemented it has room for growth without severe changes in the process

line.

IE R, = FC +VyCE VCR

PE X E + R XR = FC +VyCE x E V CR x R

pE Rx L 0.011) + pR X R = FC + VCE R x 0.011) +VCR LR




Q 0.011pE RIn~~+ RliC FC+.01CE C



pE = 46.67 pR


0.011(46.67pR Rp 37,41 2,6+00193C+0.1(3.5)01

pR=$1.44/1b pE=$67.22/1b




































Low Sales $ 6, 188 $ 36,664 $ 51,685
178/1,156Profit" $ (23,105)7 $ (12,630) $ 2,391

0 Sales $ 36,664 $ 51,329, $ 72,358
eMedium 249 /22,619
Profit $ (12,630) $ 2,036 $ 23,065

a2 Sales $ 51,329 $ 71,861 $ 101,302
High 348 /31,666
Profit $ 2,036 $ 22,568 $ 52,008


$100,000
$90, 000 -( TC -m- 1-1 1--2 ~ 1-3 1-4 -0 1-5
$80,000-
$70,000-
S$60,000-
$50,000-
$40,000-
$30,000-
$20,000-
$10,000-
$0
0 50 100 150 200 250 300
Volume of extract production (Ib)

Figure 4-7. Break-even point as affected by price for a facility using a vacuum dryer as a final
step for product isolation. Abbreviations: I income, TC total costs.

Table 4-10. Sensitivity analysis for a marginal process obtaining extract and dried skins from
muscadine grape skins using vaccum drying as the isolation technique.
Prices ($US)
Low' Medium High2
ExtIDS p~t ipDS4 $50 / $1.07 $70 / $1.50 $100 / $2.10


'Low represent 40% less of both medium prices and volumes. 2High represent 40% more of both medium prices
and volumes. 3Extract and dried skins productions are displayed vertically in the table. 4Prices for extract and dried
skins are displayed horizontally in the table. 5Sales are shown for all 9 different supposed scenarios. 6Profits are
shown for all 9 different supposed scenarios. 7Number in parenthesis indicate profit loss.

When an evaporator is proposed as the isolation method (Table 4-10), there were losses in

the low-low and medium-low combinations of production and prices. The remaining situations

showed positive numbers but only the medium-high and high-high combinations showed five-

digit figures of profit. At the original circumstances (medium price and volume), resulted in

only $2,000 are registered in profit per season. As mentioned before, extract production is likely










to increase if process is optimized and therefore this isolation method is also profitable and

convenient.

Vacuum drying, due to the characteristics of the equipment quoted, resulted in a low IRR

(12.3%) and NPV of 188,627 (Table 4-11). Although the NPV calculated for this operation was

higher than the last two, the investment demanded most of the money earned in a 10 year-

operation period. However as mentioned earlier, this process did generate profit and had room

for expansion if the process was successfully implemented by a muscadine processor. The

machine at the early stages of the operation is going to be sub-utilized while spray dryer and

freeze dryer quoted for previous operations are going to be working at almost full capacity. With

the actual liquid extract volume, the vacuum evaporator worked less than 4 hours while spray

dryer and freeze dryer were working 12 hours and 16 hours respectively.











Table 4-11. Cash flow analysis for a marginal process obtaining extract and dried skins from muscadine grape skins using vacuum
drying as the isolation technique.
Periods (years)
01 23 45 67 8 910


Investment $186,640
Income
Extract
Residue
Expenses
Labor
Materials
General costs
Depreciation
Interest (7.5%)
Gross Income
Tax (20%)
Utilities after Taxes
(+) Depreciation
O Amortization
Salvage Value
Net Income

Net Present Value (NPV)
Intern Rate of Return (IRR)


$ 17,401 $ 17,998 $ 18,615 $ 19,254 $ 19,914 $ 20,597 $ 21,304 $ 22,035 $ 22,790 $ 23,572
$ 33,928 $ 35,092 $ 36,296 $ 37,540 $ 38,828 $ 40,160 $ 41,537 $ 42,962 $ 44,436 $ 45,960

$ 7,168 $ 7,412 $ 7,664 $ 7,924 $ 8,194 $ 8,472 $ 8,760 $ 9,058 $ 9,366 $ 9,685
$ 3,341 $ 3,455 $ 3,572 $ 3,694 $ 3,819 $ 3,949 $ 4,083 $ 4,222 $ 4,366 $ 4,514
$ 1,357 $ 1,403 $ 1,451 $ 1,500 $ 1,551 $ 1,604 $ 1,659 $ 1,715 $ 1,773 $ 1,834
$ 26,789 $ 26,789 $ 26,789 $ 26,789 $ 26,789 $ 4,424 $ 4,424 $ 4,424 $ 4,424 $ 4,424
$ 998 $ 998 $ 893 $ 788 $ 683 $ 578 $ 473 $ 368 $ 263 $ 158
$ 11,677 $ 13,139 $ 14,648 $ 16,205 $ 17,812 $ 41,836 $ 43,548 $ 45,315 $ 47,140 $ 49,023
$ 2,335 $ 2,628 $ 2,930 $ 3,241 $ 3,562 $ 8,367 $ 8,710 $ 9,063 $ 9,428 $ 9,805
$ 9,342 $ 10,511 $ 11,718 $ 12,964 $ 14,250 $ 33,468 $ 34,838 $ 36,252 $ 37,712 $ 39,219
$ 26,789 $ 26,789 $ 26,789 $ 26,789 $ 26,789 $ 4,424 $ 4,424 $ 4,424 $ 4,424 $ 4,424
$ 6,528 $ 6,633 $ 6,738 $ 6,843 $ 6,948 $ 7,053 $ 7,158 $ 7,263 $ 7,368 $ 7,473
$ 12,425
$ 29,603 $ 30,668 $ 31,770 $ 32,910 $ 46,516 $ 30,840 $ 32,105 $ 33,414 $ 34,768 $ 36,170


$188,627
12.3%


rate 12.00%









4.4 Conclusions

Aerobic fermentation following a simple concentration step was an inexpensive way to

obtain polyphenolics from muscadine grape skins since most of the investment was focused in

the latter procedure (isolation). Results from three isolation techniques of polyphenolic from

muscadine grape skins suggested that this additional operation can be suitable and profitable for

a typical muscadine producer. Further investigation is needed to optimize the polyphenolic

concentration procedure prior to fermentation to increment the extract produced and,

consequently increase profit. Using initial production conditions (medium price and volume),

the profit generated for each of the techniques (in order from most to least) was freeze drying >

spray drying > vacuum drying. In contrast, the vacuum evaporator used as an example for this

chapter had three times the capacity of the spray dryer and four times the capacity of the spray

dryer, thus only the evaporator could support a growth in extract production if the volume

processed is increased significantly. Another advantage of vacuum evaporation for a muscadine

grape processor is the versatility to obtain both powder and/or concentrate product depending on

the purchaser requirements whereas spray drying and freeze drying will produce only a powder

limiting the market for the isolated extract. Moreover, to implement a freeze drying line,

refrigeration is needed to freeze the liquid extract before it is submitted to drying. This chapter

illustrated various assumptions that could be changed to recalculate and to adjust the investment

and costs of any processor. Due to limited information regarding equipment, this chapter only

contained general information for engineering and equipment specifications, thus, many

specifications can be added to predict economic information more accurately. Furthermore, this

process could be adjusted and implemented for by-products from various fruits and vegetables

sources to estimate the increase in profit of their industries.









CHAPTER 5
SUMMARY AND CONCLUSIONS

Interest in by-product utilization has encouraged fruit and vegetable processors to

investigate ways to efficiently and economically add value to otherwise low-benefit processing

residues. For muscadine grapes, the potential exists to extract valuable compounds that remain

in the by-product and bring these novel products to a revenue-generating market. In parallel,

interest in natural antioxidants has driven the production of nutraceuticals from fruit and

vegetable sources. Prior studies have focused on extracting polyphenolics from diverse fruit and

vegetable sources, but the present studies are the first to explore the potential economic and

processing environment impact on polyphenolic content and stability. The studies illustrate the

ability to extract polyphenolics from muscadine grape pomace and measuring the effect of

various processes on chemical composition, chemical reactivity, and oxidative and thermal

stability. Furthermore, these studies tried to illustrate the feasibility of obtaining by-product

isolates rich in polyphenolics from muscadine grape pomace. Such feasibility was determined

by simple economic indicators, using logical assumptions and the best economic predictors for

success of a by-product extraction venue.

Polyphenolics extracted from muscadine grape pomace showed antioxidant activity

comparable to fruits and vegetables such as cherries, strawberries, and red cabbage.

Fermentation was better than solid phase isolation in getting rid of residual sugars with minimal

damage to the chemical profile and antioxidant activity of the final product, as well as showing

more practicality on a larger scale. Vacuum drying proved to be the best treatment following

fermentation since it best maintained polyphenolic content and preserved the antioxidant

capacity. Initial concentration followed by aerobic fermentation was an inexpensive way to

obtain polyphenolics from muscadine grape skins. Isolation of polyphenolics from muscadine










grape pomace can be suitable and profitable for a typical muscadine juice producer if their

circumstances are somewhat similar with the ones indicted in these studies. Assumptions can be

adjusted to any fruit or vegetable processor situation for more accurate calculations. Further

research is needed to support the work shown in these studies. Investigations need to be focused

on the optimization of polyphenolic concentration prior to fermentation, the impact of processing

on phytochemical characteristics at an industrial scale, and more detailed economic evaluation.













APPENDIX A
PRELIMINARY STUDY I


.


-


]


2500

2000


1500

1000


500

0


a,
o~s,
cn~E
o~s,


Ca,
um

Oa,


Days

-o-1/10x -m- 1/5x -A-1/2x -K 1/1 x


Figure A-1. Total anthocyanins during a 3-day extraction procedure.


12

10

8

6

4

2

0 ]


Days

-*- 1/1x -m- 1/5x 1/2x -K- 1/ Ix


Figure A-2. oBrix values during a 3-day concentration procedure.











APPENDIX B
PRELIMINARY STUDY II


1,600

1,400

1,200

1,000
800

600

400

200

0


1/2x

Concentration ratio (skin/w ater)


Figure B-1. Total anthocyanin content in a 1-day extraction procedure.


10
9
8
7
6


4
3
2
1
-


Concentration ratio (skin/w ater)


Figure B-2. oBrix values in a 1-day extraction procedure.










APPENDIX C
PRELIMINARY STUDY III


1100

.0 1200-


1 00 -


E 6000

400-

200-


0 30 60 90 120 150 180 210 240 270 300 330
Minutes


Figure C-1. Total anthocyanin content after a 5-hour concentration procedure.









LIST OF REFERENCES

Aaby K, Skrede G, Wrolstad R. 2005. Phenolic composition and antioxidant activities in flesh
achenes of strawberries (Fragaria anana~ssa). J Agric Food Chem 53:4032-40.

Abascal K, Ganora L, Yarnell E. 2005. The effect of freeze-drying and its implications for
botanical medicine: a review. Phytother Res 19:655-60.

Amakura Y, Umino, Y, Tsuji S, Tonogai Y. 2000. Influence of jam processing on the radical
scavenging activity and phenolic content in berries. J Aric Food Chem 48:6292-97.

Andrade I, Flores H. 2004. Optimization of spray drying of roselle extract (Hibiscus sabdarrifa~ddd~~~ddd~~~dd
L.). Proceedings of the 14th International Drying Symposium A: 597-604. Sho Paulo,
Brazil.

Atkinson CJ, Nestby R, Ford YY, Dodds PAA. 2005. Enhancing the beneficial antioxidants in
fruits: a plant physiological perspective. Biofactors 23:229-34.

Barbosa-Canovas GV, Ortega-Rivas E, Juliano P, Yan H. 2005. Drying. In: Food Powders,
Physical Properties, Processing, and Functionality. New York, NY. Kluwer
Academic/Plenum Publishers. p 271-304.

Bonilla F, Mayen M, Merida J, Medina M. 1999. Extraction of phenolic compounds from red
grape marc for use as food lipid antioxidants. Food Chem 66:209-15.

Brenes CH, Del Pozo-Insfran, D, Talcott, ST. 2005. Stability of copigmented anthocyanins and
ascorbic in a grape juice model system. J Agric Food Chem 53:49-56.

Bridle P, Timberlake C. 1997. Anthocyanins as food colors, selected aspects. Food Chem 58(1-
2):103-9.

Brouillard R, Mazza G, Saad Z, Albrecht-Gary AM, Cheminat A. 1989. The copigmentation
reaction of anthocyanins: a microprobe for the structural study of aqueous solutions. J
Am Chem Soc 111:2604-10.

Bruneton J. 1995. Pharmacognosy, Phytochemistry, Medical Plants. Tec & Doc-Lavoisier, Paris.

Buettner GR. 1993. The pecking order of free radicals and antioxidants: lipid peroxidation,
alpha-tocopherol, and ascorbate. Archives of biochemistry and biophysics 3 00(2):53 5-43.

Cabrita L, Andersen OM. 1999. Anthocyanins in blue berries of Vaccinium padifolium.
Phytochem 52:1693-96.

Cao G, Wang G, Prior R. 1996. Total antioxidant capacity of fruits. J Agric Food Chem 44:701-


Clifford MN. 2000. Anthocyanins: nature, occurrence and dietary burden. J Sci Food Agric
80:1063-72.









Clifford MN, Scalbert A. 2000. Ellagitannins-nature, occurrence and dietary burden. J. Sci Food
Agric 80: 1118-25.

Cline B, Fisk C. 2006. Overview of muscadine acreage, cultivars and production areas in
southeastern US. Plant Pathology and Horticultural Science. North Carolina State
University .

Crocker TE, Mortensen JA. 2001 The Muscadine Grape. Extension. Institute of Food and
Agricultural Sciences. University of Florida.

Croft KD. 1999. Antioxidant effects of plant phenolic compounds. In: Basu TK, Temple NJ,
Garg ML editors. Antioxidant in Human Health and Disease. New York NY. CABi Pub.
p 109-22.

Dao LT, Takeoka GR, Edwards RH, Berrios JDJ. 1998. Improved method for the stabilization of
anthocyanidins. J Agric Food Chem 46:3564-9.

De Bruyne T, Pieters L, Deelstra H, Vlietinck A. 1999. Condensed vegetable tannins:
biodiversity in structure and biological activities. Biochem Syst Ecol 27:445-59.

Degner RL, Rodan LW, Mathis K. 1981. Farmer to Consumer, direct marketing of grapes in
Florida: producer and consumer benefits. Florida Agricultural Market Research Center.
Institute of Food and Agricultural Sciences. University of Florida.

Del Pozo-Insfran D. 2006. Emerging technologies and strategies to enhance anthocyanin
stability. Dissertation. University of Florida

Del Pozo-Insfran D, Brenes C, Talcott S. 2004. Phytochemical composition and pigment stability
of agai (Euterpe oleracea Mart.). J Agric Food Chem 52: 1539-45.

Dillard CJ, German JB. 2000. Review: Phytochemicals: nutraceuticals and human health. J Sci
Food Agric 80 (12):1744-56.

Ector BJ. 2001. Compositional and nutritional characteristics. In: Basiouny FM Himelrick DG,
editors. Muscadine Grapes. Alexandria, VA.: ASHS ASHS Press. p 341-67.

Erlund I. 2004. Review of the flavonoids quercetin, hesperetin, and naringenin. Dietary sources,
bioactivities, bioavailability, and epidemiology. Nutrition Research 24:851-74.

Fang Z, Zhang M, Sun Y, Sun J. 2006. How to improve bayberry (M~yrica rubra Sieb. et Zucc.)
juice color quality: effect of juice processing on bay berry anthocyanins and
polyphenolics. J Agric Food Chem 54:99-106.

Feldman KS, Sambandam A. 1995. Ellagitannin Chemistry. The first total chemical synthesis of
an O(2),O(3)-galloyl-coupled ellagitannin, Sanguiin H-5. J Prg Chem 60:8171-8.

Fender L. 2005. Phytochemical, antioxidant, and storage stability of thermally processed guava
(Psidium guajva) and guava juice blends. Thesis. University of Florida.










Francis FJ. 1989. Food colorants: anthocyanins. Crit Rev Food Sci Nutr 28:273-314.

Fritz JS, Willis RB. 1973. Chromatography separation of phenols using an acrylic resin. J
Chromatography 79:107-19.

Furtado P, Figueiredo P, Chaves das Neves H, Pina F. 1993. Photochemical and thermal
degradation of anthocyanidins. J Photochem Photobiol A 75:1 13-8.

Garz6n GA, Wrolstad RE. 2001. The stability of pelargonidin-based anthocyanins at varying
water activity. Food Chem 75 185-96.

Garz6n GA, Wrolstad RE. 2002. Comparison of the stability of pelargonidin-based anthocyanins
in strawberry juice and concentrate. Journal of Food Science 67:(4) 1289-99.

Gil MI, Tomas-Barberan FA, Hess-Pierce B, Holcroft DM, Kader AA. 2000. Antioxidant
activity of pomegranate juice and its relationship with phenolic composition and
processing. J Agric Food Chem 48:4581-9.

Giusti MM, Wrolstad RE. 2003. Acylated anthocyanins from edible sources and their
applications in food systems. Biochem Eng J 14:217-25.

Gregory III JF. 1996. Vitamins. Chapter 8 in Food Chemistry 3rd Edition, Marcel Dekker, Inc.
New York NY.

Hakkinen SH, Karenlampi SO, Mykkanen HM, Heinonen IM, Toirroinen AR. 2000. Ellagic acid
content in berries: Influence of domestic processing and storage. Eur Food Res Technol
212:75-80.

Halbrooks MC. 1998. Alternative opportunities for small farms: muscadine grape production
review. Cooperative Extension Service. Institute of Food and Agricultural Sciences.
University of Florida.

Helm RF, Zhentian L, Ranatunga T, Jervis J, Elder T. 1999. Toward understanding monomeric
ellagitannin byosynthesis. In Plant Polyphenolics 2: chemistry, biology, pharmacology,
ecology. Academic/Plenum Publishers, New York, NY.

Himelrick DG. 2003. Handling, storage, and postharvest physiology of muscadine grapes: a
review. Small Fruits Review 2(4):45.

Hradzina G, Borzell AJ, Robinson WB. 1970. Studies on the stability of the anthocyanin-3,5-
diglucosides. Am J Enol Vitic 21:201

Hsu CL, Chen W, Weng YM, Tseng CY. 2003. Chemical composition, physical properties, and
antioxidant activities of yam flours as affected by different drying methods. Food Chem
83:85-92.

Huang HT. 1955. Decolorization of anthocyanins by fungal enzymes. J Agric Food Chem
3:141-6.










Jaworski AW, Lee CY. 1987. Fractionation and HPLC determination of grape phenolics. J Agric
Food Chem 35:257-9.

Jurd L, Asen S. 1966. The formation of metal and co-pigment complexes of cyanidin 3-
glucoside. Phytochem 5:1263-71.

Kader F, Haluk JP, Nicolas JP, Metche M. 1998. Degradation of cyanidin-3 -glucoside by
blueberry polyphenol oxidase: kinetic studies and mechanisms. J Agric food Chem
46:3060-5.

Kader F, Rovel B, Girardin M, Metche M. 1997. Mechanism of browning in fresh highbush
blueberry fruit (Vaccinium corymbosum L). Partial purification and characterization of
blueberry polyphenol oxidase. J Sci Food Agric 73:513-6.

Kammerer DR, Schillmoiller S, Maier O, Schieber A, Carle R. 2007. Colour stability of canned
strawberries using black carrot and elderberry juice concentrates as natural colourants.
Eur Food Res Technol 224:667-9.

Khanbabaee K, Ree TV. 2001. Tannins: classification and definition. Nat Prod Rep 18:641-9.

Kirca A, Ozkan M, Cemeroglu B. 2006. Stability of black carrot anthocyanins in various fruit
juices and nectars. Food Chem 97:598-605.

Klopotek Y, Otto K, Bohm V. 2005. Processing strawberries to different products alters contents
of vitamin C, total phenolics, total anthocyanins and antioxidant capacity. J Agric Food
Chem 53:5640-6.

Kong J, Chia L, Goh N, Chia T, Brouillard R. 2003. Analysis and biological activities of
anthocyanins. Phytochem 64:923-33.

Kraemer-Schafhalter A, Fuchs H, Pfannhauser W. 1998. Solid-phase extraction (SPE) a
comparison of 16 materials for the purification of anthocyanins from Aronia
melan2ocarpa var Nero. J Sci Food Agric 78:435-40.

Kraus TEC, Dahlgren RA, Zasoski RJ. 2003. Tannins in nutrient dynamics of forest ecosystems-
a review. Plant and Soil 256:41-66.

Lalaguna F. 1993 Purifieation of fresh cassava root polyphenolics by solid-phase extraction with
Amberlite XAD-8 resin. J Chromatography 657:445-9.

Laleh GH, Frydoonfar H, Heidary R, Jameei R, Zare S. 2006. The effect of light, temperature,
pH and species on stability of anthocyanin pigments in four Berberis species. Pakistan
Journal of Nutrition. 5(1):90-2.

Laurila E, Ahvenainen R. 2002. Minimal processing in practice. In: Ohlsson T, Bengtsson N,
editors. Minimal processing technologies in the food industry. Boca Raton, FL. CRC
Press. p 219-44.










Lee JH, Talcott ST. 2004. Fruit maturity and juice extraction influences ellagic acid derivatives
and other antioxidant polyphenolics in muscadine grapes. J Agric Food Chem 52:361-6.

Lee JH. 2004. Hydrolytic and antioxidant properties of ellagic acid and its precursors present in
muscadine grape. Dissertation. University of Florida.

Lee JH, Johnson JV, Talcott ST. 2005. Identification of ellagic acid conjugates and other
polyphenolics in muscadine grapes by HPLC-ESI-MS. J Agric Food Chem 53:6003-10.

Lei Z. 2002. Monomeric ellagitannins in oaks and sweetgum. Dissertation. Virginia Polytechnic
Institute and State University.

Le Marchand L. 2002. Cancer preventive effects of flavonoids- a review. Biomed Pharmacother
56:296-301.

Lindsay RC. 1996. Food Additives. In: Fenemma, OR editor. Food Chemistry 3rd Edition. New
York NY. Marcel Dekker, Inc. p 767-824.

Makris DP, Boskou G, Andrikopoulos NK. 2007. Polyphenolic content and in vitro antioxidant
characteristics of wine industry and other agri-food solid waste extracts. Journal of Food
Composition and Analysis 20:125-32.

Marais JPJ, Deavours B, Dixon RA, Ferreira D. 2006. The stereochemistry of flavonoids. In:
Grotewold E, editor. The Science of Flavonoids. New York, NY. Springer. p 1-46.

Markakis P. 1982. Stability of anthocyanins in foods. In: Anthocyanins as Food Colors. New
York, NY. Academic Press, Inc. p 163-80.

Marti N, Perez-Vicente A, Garcia-Viguera C. 2001. Influence of storage temperature and
ascorbic acid addition on pomegranate juice. J Sci Food Agric 82:217-21.

McRae TG, Gregson RP, Quinn RJ. 1982. Amberlite XAD-7 as a chromatographic absorbent.
Journal Chromatographic Science 20:475-9.

Meyer AS, Suhr KI, Nielsen P, Lyngby, Holm F. 2002. Natural food preservatives. In: Ohlsson
T, Bengtsson N, editors. Minimal processing technologies in the food industry. Boca
Raton, FL. CRC Press. p 124-76.

Meyers KJ, Swiecki TJ, Mitchell AE. 2006. Understanding the native californian diet:
identification of condensed hydrolazable tannins in Tanoak Acorns (Lithocarpus
densiflorus). J Agric Food Chem 54:7686-91.

Miller NJ, Ruiz-Larrea MB. 2002. Flavonoids and other plant phenols in the diet: their
significance as antioxidants. Journal of Nutritional & Enviromental Medicine 12:39-5 1.

Milo L. 2005. Nutraceuticals & Functional Foods. Food Technology 59(5):65-7.










Mizell R, Andersen P, Tipping C, Brodbeck B. 2003. Xylella fastidiosa diseases and their
leafhopper vectors. Institute of Food and Agricultural Sciences (IFAS). University of
Florida.

Monagas M, Hernandez-Ledesma B, G6mez-Cordoves C, Bartolome B. 2006. Commercial dietary
ingredients from Vitis vinifera L. leaves and grape skins: Antioxidant and Chemical
Characterization. J Agric Food Chem 54:319-27.

Morata A, G6mez-Cordoves MC, Calder6n F, Suarez JA. 2006. Effects of temperature, pH and
SO2 On the formation of pyranoanthocyanins during red wine fermentation with two
species of Saccharomyces. Int J Food Micro 106: 123-9.

Morris JR, Brady PL. 2004. The muscadine experience: Adding value to enhance profits.
Arkansas Agricultural Experiment Station. Institute of Food Science and Engineering.
University of Arkansas.

Moure A, Cruz JM, Franco D, Dominguez JM, Sineiro J, Dominguez H, Nufiez MJ, Paraj6, JC.
2001. Natural antioxidants from residual sources. Food Chem 72:145-71.

Mullen W, Stewart AJ, Lean MEJ, Gardner P, Duthie GG, Crozier A. 2002. Effect of freezing
and storage on the phenolics, ellagitannins, flavonoids, and antioxidant capacity of red
raspberries. J Agric Food Chem 50:5197-201.

Mullen W, Yokota T, Lean ME, Crozier A. 2003. Analysis of ellagitannins and conjugates of
ellagic acid and quercetin in raspberry fruits by LC-MSn. Phytochemistry 64:617-24.

Orsat V, Raghavan GSV. 2006. Dehydration technologies to retain bioactive components. In: Shi
J, editor. Functional Food Ingredients and Nutraceuticals: Processing and Technologies.
Boca Raton, FL. CRC Taylor & Francis Group. p 173-192.

Oszmianski J, Ramos T, Bourzeix M. 1988. Fractionation of phenolic compounds in red wine.
Am J Enol Vitic 39(3):260-2.

Pacheco LA. 2006. Phytochemical, antioxidant and color stability of Agai (Euterpe oleracea
Mart.) as affected by processing and storage in juice model systems. Thesis. University of
Florida.

Parr A, Bowell G. 2000. Review: Phenols in the plant and in man. The potential for possible
nutritional enhancement of the diet by modifying the phenol content or profile. J Sci
Food Agric 80:985-1012.

Pastrana-Bonilla E, Akoh C, Sellapan S, Krewer G. 2003. Phenolic content and antioxidant
capacity of muscadine grapes. J Agric Food Chem 51:5497-503.

Peschel W, Sanchez-Rabaneda F, Diekmann W, Plescher A, Gartzia I, Jimenez D, Lamuela-
Raventos R, Buxaderas S, Codina C. 2006. An industrial approach in the search of
natural antioxidants from vegetable and fruit wastes. Food Chem 97(1):137-50.










Pflug IJ, Esselen WB. 1979. Heat sterilization of canned food. In: Jackson JM, Shinn BM,
editors. Fundamentals of Food Canning Technology. Westport, CT. AVI Publishing
Company, Inc. p 10-94.

Phillips RD. 2006. Pilot-scale, pre-commercial production of nutraceuticals from Georgia
commodities. In Fiscal Year 2005-2006 Report to industry. Georgia's Traditional
Industries Program for Food Processing. Food Processing Advisory Council. p 10.

Pietrzyk DJ, Chu CH. 1977a. Amberlite XAD copolymers in reversed phase gravity flow and
high pressure liquid chromatography. Anal Chem 49(6):757-64.

Pietrzyk DJ, Chu CH. 1977b. Separation of organic acids on amberlite XAD copolymers by
reversed phase high pressure liquid chromatography. Anal Chem 49(6):860-7.

Pietta PG. 2000. Flavonoids as antioxidants. J Nat Prod 63:1035-42.

Poling EB. 1996. Muscadine grapes in the home garden. Department of Horticultural Science.
North Carolina Cooperative Extension Service. North Carolina State University.

Rababah TM, Ereifej KI, Howard L. 2005. Effect of ascorbic acid and dehydration on
concentration of total phenolics, antioxidant capacity, anthocyanins, and color in fruits. J
Agric Food Chem 53:4444-7.

Rein MJ, Heinonen M. 2004. Stability and enhancement of berry juice color. J Agric Food Chem
52:3106-14.

Rein MJ. 2005. Copigmentation reactions and color stability of berry anthocyanins. Dissertation.
University of Helsinki.

Robbins R. 2003. Phenolic acids in foods: An overview of analytical methodology. J Agric Food
Chem 51:2666-87.

Rodriguez-Saona LE, Guisti MM, Wrolstad RE. 1999. Color and pigment stability of red radish
and red fleshed potato anthocyanins in juice model systems. J Food Sci 64:451-6.

Rommel A, Wrolstad RE. 1993. Ellagic acid content of raspberry juice as influenced by cultivar
processing, and environmental factors. J Agric Food Chem 41:1951-60.

Ruel J, Walker A. 2006. Resistance to Pierce's disease in M~uscadinia rotundifolia and other
native grape species. Am J Enol Vitic 57:158-66.

Saito N, Tatsuzawa F, Yoda K, Yokoi M, Kasahara K, lida S, Shigihara A, Honda T. 1995.
Acylated cyanidin glycosides in the violet-blue flowers oflpomoea purpurea. Phytochem
40(4):1283-9.

Schmidt BM, Erdman JW, Lila MA. 2005. Effects of food processing on blueberry
antiproliferation and antioxidant capacity. Journal of Food Science 70:389-94.










Shahidi F, Naczk M. 2003. Phenolics in Food and Nutraceuticals. Boca Raton, FL. CRC Press p
558.

Shi B, He Q, Yao K, Huang W, Li Q. 2005. Production of ellagic acid from degradation of
valonea tannins by Aspergillus niger and Can2dida utilis. J Chem Technol Biotechnol
80:1154-9.

Sims CA. Morris JR. 1985. pH effects on the color of wine from two grape species. Ark Farm
Res 34(2):9.

Singleton VL, Rossi J. 1965. Colorimetry of total phenolics with phosphomolybdic-
phosphotungstic acid reagents. Am J Enol Vit 16: 144-53.

Skrede G, Wrolstad RE. 2000. Flavonoids from berries and grapes. In: Mazza G, editor.
Functional Foods: Biochemical and Processing Aspects. Lancaster, PA. Technomic Pub.
p 71-134.

Sort X. 2003. Environmental problem of agroindustrial spills. Environmental Management.
Electron. J Environ Agric Food Chem 2:205-7.

Stintzing F, Stintzing A, Carle R, Frei B, Wrolstad R. 2002. Color and antioxidant properties of
cyanidin-based anthocyanin pigments. J Agric Food Chem, 50: 6172-81.

Takamura H, Yamaguchi T, Terao J, Matoba T. 2002. Change in radical-scavenging activity of
spices and vegetables during cooking. In: Lee TC, Ho CT, editors. Bioactive Compounds
in Foods: Effects of Processing and Storage. Washington, DC. American Chemical
Society. p 34-43.

Takeda F, Saunders MS, Saunders JA. 1983. Physical and chemical changes in muscadine grape
during postharvest storage Vitis rotundifolia. Am J Enol Vit 34(3): 180-5.

Talcott ST, Lee JH. 2002. Ellagic acid and flavonoid antioxidant content of muscadine wine and
juice. J Agric Food Chem 50:3186-92.

Talcott ST, Brenes CH, Pires DM, Del Pozo-Insfran D. 2003. Phytochemical stability and color
retention of copigmented and processed muscadine grape juice. J Agric Food Chem
51:957-63.

Tambunan AH, Yudistira, Kisdiyani, Hernani. 2001. Freeze drying characteristics of medical
herbs. Drying Tech 19(2):325-31.

Tomas-Barberan FA, Clifford MN. 2000. Dietary hydroxybenzoic acid derivatives-nature,
occurrence and dietary burden. J Sci Food Agric 80:1024-32.

Torreggiani D, Bertolo G. 2001. Osmotic pre-treatments in fruit processing: chemical, physical
and structural effects. J Food Eng 49:247-53.










Tsai PJ, Delva L, Yu TY, Huang YT, Dufosse L. 2005. Effect of sucrose on the anthocyanin
capacity of mulberry extract during high temperature heating. Food Res Int 3 8:1059-65.

Turker N, Aksay S, Ekiz I. 2004. Effect of storage temperature on the stability of anthocyanins
of a fermented black carrot (Daucus carota. beverage: Shalgam. J Agric Food Chem 52:
3807-13.

Van Golde PH, Van der Westelaken M, Bouma BN, Van de Wiel A. 2004. Characteristics of
piraltin, a polyphenols concentrate, produced by freeze-drying of red wine. Life Sci
74:1159-66.

Visioli F, Romani A, Mulinacci N, Zarini S, Conte D, Vincieri FF, Galli C. 1999. Antioxidant
and other biological activities of olive mill waste waters. J Agric Food Chem 47:3397-
401.

Wang SY. 2006. Fruits with high antioxidant activity as functional foods. In: Shi J, editor.
Functional Food Ingredients and Nutraceuticals: Processing and Technologies. Boca
Raton, FL. CRC Taylor & Francis Group. p 371-414.

Weinert IAG, Solms J, Escher F. 1990. Polymerization of anthocyanins during processing and
storage of canned plums. Lebensm-Wiss u Technol 23:445-50.

Winkel BSJ. 2006. The biosynthesis of flavonoids. In: Grotewold E, editor. The Science of
Flavonoids. New York, NY. Springer. p 71-96.

Whitaker JR. 1994. Principles of enzymology for the food sciences 2nd edition. Ney York, NY.
Marcel Dekker. p 625.

Wrolstad RE. 1976. Color and pigment analysis in fruit products. Oregon Agricultural
Experimental Station Corvallis. Bulletin 624.

Wu H, Haig T, Prately J, Lemerle D, An M. 2000. Allelochemicals in wheat (Triticum aesvestum
L.): Variation of phenolic acids in root tissues. J Agric Food Chem 48:5321-5.

Wu X, Beecher GR, Holden JM, Haytowitz DB, Gebhardt SE, Prior RL. 2004. Lipophillic and
Hydrophillic Antioxidant Capacities of Common Food in the United States. J Agric Food
Chem 52:4026-37.

Yi W, Fischer J, Akoh CC. 2005. Study of anticancer activities of muscadine grape phenolics in
vitro. J Agric Food Chem 53:8804-12.

Yi W, Akoh, CC, Fischer J, Krewer G. 2006. Effect of phenolic compounds in blueberries and
muscadine grapes on HepG2 cell viability and apoptosis. Food Res Int 39:628-38.

Zafrilla P, Ferreres F, Tomas-Barberan FA. 2001. Effects of processing and storage on the
antioxidant ellagic acid derivatives and flavonoids of red raspberry (Rubus idaeus) jams.
J Agric Food Chem 49:3651-5.









BIOGRAPHICAL SKETCH

Jorge Cardona was born on October 13, 1983, in Cochabamba, Bolivia. Before graduating

from high school in 2000, he traveled to Devon, PA as an exchange student for 11 months with

AFS exchange program. He went back to Bolivia to graduate from high school in 2001. After

high school, he entered Zamorano University (Escuale Agricola Panamericana) in Honduras,

Central America to obtain his bachelor' s degree in agroindustry. After his graduation in 2005, he

was offered an assistantship to pursue his graduate education at the Food Science and Human

Nutrition Department at University of Florida, under the supervision of Dr. Stephen Talcott and

Dr. Charles Sims. In August 2007, he earned a Master of Science in Food Science and Human

Nutrition and will continue his studies towards a doctoral degree.





PAGE 1

1 CHEMICAL AND ECONOMIC ANALYSIS OF A VALUE-ADDED PRODUCT FROM MUSCADINE GRAPE POMACE By JORGE ALFREDO CARDONA PONCE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007

PAGE 2

2 2007 Jorge Alfredo Cardona Ponce

PAGE 3

3 To my family and all the peopl e that have believed in me

PAGE 4

4 ACKNOWLEDGMENTS I want to thank my first advisor Dr. Stephen Talcott to guide me th rough this experience, make this work possible, and for giving me the opportunity to be in graduate school. Also, I extend special gratitude to Dr. Charles A. Sims who guided me through the second part of this research and accepted me as his student to fi nish my graduate student experience at the University of Florida. I also thank Dr. Mura t Balaban and Dr. Allen Wysocki for their countless advices and time to help in the production of this work. And last but no t least, I thank all my friends and lab mates who have graduated with Dr. Talcott: Flor, Y oungmok, Chris, Lisbeth, Joon, David, Lanier, and Kristine for sharing good times, helping me out in every way possible, and sharing their knowledge and time. I also want to thank my family (Fernando, Susana, and Diego) for supporting me in my decisions that have guided me all the way from Bolivia to Florida. I ow e them all the knowledge and respect I have for others. I truly appreciate thei r schooling and philosophy of life. Finally, my most sincere and deepest appreciation to Th elma, for all the patience and support during my experience in college, graduate school, and for all the happiness we share.

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ............10 CHAPTER 1 INTRODUCTION..................................................................................................................12 2 LITERATURE REVIEW.......................................................................................................15 2.1 Muscadine Grape............................................................................................................ ..15 2.2 Polyphenols................................................................................................................ .......17 2.2.1 Structure and Classification....................................................................................17 2.2.1.1 Phenolic acids and simple phenols...............................................................18 2.2.1.2 Flavonoids....................................................................................................18 2.2.1.3 Tannins.........................................................................................................20 2.2.2 Polyphenolics as Antioxidants...............................................................................21 2.2.3 Polyphenolics in Mucscadine Grapes.....................................................................23 2.3 Anthocyanins............................................................................................................... .....24 2.3.1 Structure and Occurrence.......................................................................................24 2.3.2 Color Stability........................................................................................................26 2.3.3 Anthocyanins in Muscadine Grape........................................................................31 2.4 Ellagic Acid............................................................................................................... .......32 2.4.1 Structure and Occurrence.......................................................................................32 2.4.2 Ellagic Acid Derivatives.........................................................................................32 2.4.3 Ellagic Acid and Its Derivatives in Muscadine Grape...........................................35 2.5 Processing Effects on Polyphenolics................................................................................35 2.5.1 Heat Procedures......................................................................................................36 2.5.2 Drying Procedures..................................................................................................37 2.5.3 Extraction Procedures and Storage.........................................................................40 2.5.4 Enzymatic Procedures............................................................................................41 3 PHYTOCHEMICAL, ANTIOXIDANT AND PIGMENT STABILITY OF MUSCADINE GRAPE POMACE AS AFFECTED BY CONCENTRATION and DEHYDRATION...................................................................................................................42 3.1 Introduction............................................................................................................... ........42 3.2 Materials and Methods.....................................................................................................43 3.2.1 Materials and Processing........................................................................................43 3.2.2 Solid Phase Isolation..............................................................................................45

PAGE 6

6 3.2.3 Chemical Analysis..................................................................................................48 3.2.3.1 Spectrophotometric determination of total anthocyanins.............................48 3.2.3.2 Determination of polymeric anthocyanins...................................................48 3.2.3.3 Determination of total soluble phenolics.....................................................49 3.2.3.4 Quantification of antioxidant capacity.........................................................49 3.2.3.5 Half life determination.................................................................................50 3.2.3.6 Analysis of polyphenolics by HPLC............................................................50 3.2.4 Statistical analysis..................................................................................................51 3.3 Results and Discussion.....................................................................................................51 3.3.1 Anthocyanin Color Stability...................................................................................52 3.3.2 Polyphenolic Concentration and Stability..............................................................57 3.3.3 Polyphenolics by HPLC.........................................................................................58 3.3.3.1 Anthocyanins by HPLC...............................................................................58 3.3.3.2 Ellagic acid and flavonols by HPLC............................................................61 3.3.4 Antioxidant Capacity..............................................................................................63 3.4 Conclusions................................................................................................................ .......65 4 ECONOMIC ANALYSIS OF AN ISOL ATED PRODUCT OBTAINED FROM MUSCADINE GRAPE POMACE.........................................................................................67 4.2 Materials and Methods.....................................................................................................68 4.2.1 Data Collection.......................................................................................................68 4.2.2 Economic Analysis.................................................................................................68 4.2.2.1 Description of the operation.........................................................................69 4.2.2.2 Economic assumptions.................................................................................69 4.3 Results and Discussion.....................................................................................................72 4.3.1 Economic Analysis.................................................................................................72 4.3.1.1 Spray drying operation.................................................................................73 4.3.1.2 Freeze drying operation................................................................................78 4.3.1.3 Vacuum drying operation.............................................................................83 4.4 Conclusions................................................................................................................ .......91 5 SUMMARY AND CONCLUSIONS.....................................................................................92 APPENDIX A PRELIMINARY STUDY I....................................................................................................94 B PRELIMINARY STUDY II...................................................................................................95 C PRELIMINARY STUDY III..................................................................................................96 LIST OF REFERENCES............................................................................................................. ..97 BIOGRAPHICAL SKETCH.......................................................................................................106

PAGE 7

7 LIST OF TABLES Table page 2-1 Estimated muscadine grape acreage in the southeastern United States.............................16 2-2 Classes and dietary sources of flavonoids.........................................................................20 3-1 Quality analyses of muscadine pomace extr act (polyphenols) as affected by various processing protocols...........................................................................................................51 3-2 Anthocyanidin concentrations in the muscadine pomace extract as affected by various processing protocols..............................................................................................61 3-3 Ellagic acid and flavonol concentrations in the muscadine pomace extract as affected by various processing protocols.........................................................................................63 4-1 Generalities of drying equipments.....................................................................................72 4-2 Loan payment plan for ten y ears at a fixed rate of 7.5%...................................................72 4-3 Capital expenditure to initiate a margin al process obtaining extract and dried skins from muscadine grape skins using spra y drying as the isolation technique......................74 4-4 Sensitivity analysis for a marginal process obtaining extract and dried skins from muscadine grape skins using spray dr ying as the isolation technique...............................77 4-6 Capital expenditure to initiate a margin al process obtaining extract and dried skins from muscadine grape skins using freeze drying as the isolation technique.....................78 4-5 Cash flow analysis for a marginal pr ocess obtaining extract and dried skins from muscadine grape skins using spray dr ying as the isolation technique...............................79 4-7 Sensitivity analysis for a marginal process obtaining extract and dried skins from muscadine grape skins using freeze dr ying as the isolation technique..............................83 4-8 Cash flow analysis for a marginal pr ocess obtaining extract and dried skins from muscadine grape skins using freeze dr ying as the isolation technique..............................84 4-9 Capital expenditure to initiate a margin al process obtaining extract and dried skins from muscadine grape skins using vacuum drying as the isolation technique..................85 4-10 Sensitivity analysis for a marginal process obtaining extract and dried skins from muscadine grape skins using vaccum drying as the isolation technique...........................88 4-11 Cash flow analysis for a marginal pr ocess obtaining extract and dried skins from muscadine grape skins using vacuum drying as the isolation technique...........................90

PAGE 8

8 LIST OF FIGURES Figure page 2-1 Basic flavonoid structure.................................................................................................. .19 2-2 General structure of hydro lyzable tannins (left) and condensed tannins (right)................21 2-3 Chemical structures of anthocyanidins..............................................................................25 2-4 Anthocyanin equilibria: qui nonoidal base (A), flavylium ca tion (B), carbinol base or pseudobase (C) and chalcone (D)......................................................................................27 2-5 Effect of pH value on anthocyanin equilibria....................................................................28 2-6 Anthocyanin diglycoside struct ure (Cyanidin-3,5-diglucoside)........................................31 2-7 Chemical structure of ellagic acid......................................................................................32 2-8 Ellagic acid glycosides A) Ellagic acid-4-arabino side, B) Ellagic acid-4acetylarabinoside, C) Ellagic acid-4-acetylxyloside..........................................................33 2-9 Ellagitannins: Tellimagrandin II (monomer ic ET) (left), Sangu iin H-6 (oligomeric ET) (right).................................................................................................................... ......34 2-10 Phase diagram of water.................................................................................................... ..39 3-1 Total anthocyanin content of muscad ine pomace extract as affected by various processing methods. Error bars represent the standard error of each mean, n=3..............53 3-2 Polymeric anthocyanins (%) in muscad ine pomace extract as affected by various processing methods. Error bars represent the standard error of each mean, n=3..............55 3-3 Half life (min) of muscadine pomace ex tract as affected by various processing methods. Error bars represent the st andard error of each mean, n=3................................56 3-4 Total phenolic content in muscadine pomace extract as affected by various processing methods. Error bars represent the standard error of each mean, n=3..............58 3-5 HPLC chromatogram of anthocyanidins present in muscadine pomace: delphinidin (A), cyanidin (B), petunidi n (C), pelargonidin (D), pe onidin (E), malvidin (F). Identification (520 nm) was done based on sp ectral characteristics and comparison to cyanidin aglycone..............................................................................................................59 3-6 HPLC chromatogram of polyphenolics pres ent in muscadine pomace: ellagic acid (A), myricetin (B), and quercetin (C). Identification (360 nm) was done based on spectral characteristics and comparison to au thentic standards of ellagic acid and quercetin...................................................................................................................... .......62

PAGE 9

9 3-7 Antioxidant capacity of muscadine pomace extract as affected by various processing protocols. Error bars represent the standard error of the mean, n=3..................................65 4-1 Operation flow for a typical grape jui ce processor planning to process its byproduct......70 4-2 Volume break-even point for a facility us ing a spray dryer as a final step for product isolation. Abbreviations: FC Fixed costs, VC variable costs, TC total costs, I income......................................................................................................................... .......75 4-3 Break-even point as affected by price for a facility using a spray dryer as a final step for product isolation. Abbreviati ons: I income, TC total costs.........................................77 4-4 Volume break-even point for a facility us ing a freeze dryer as a final step for product isolation. Abbreviations: FC Fixed costs, VC variable costs, TC total costs, I income......................................................................................................................... .......81 4-5 Break-even point as affected by price for a facility using a freeze dryer as a final step for product isolation. Abbreviati ons: I income, TC total costs.........................................82 4-6 Volume break-even point for a facility using a vacuum dryer as a final step for product isolation. Abbreviations: FC Fixed cost s, VC variable costs, TC total costs, I income......................................................................................................................... .......86 4-7 Break-even point as affected by price for a facility using a vacuum dryer as a final step for product isolation. Abbrev iations: I income, TC total costs.................................88 A-1 Total anthocyanins during a 3-day extraction procedure...................................................94 A-2 oBrix values during a 3-day concentration procedure........................................................94 B-1 Total anthocyanin content in a 1-day extraction procedure...............................................95 B-2 oBrix values in a 1-da y extraction procedure.....................................................................95 C-1 Total anthocyanin content after a 5-hour concentration procedure...................................96

PAGE 10

10 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Master of Science CHEMICAL AND ECONOMIC ANALYSIS OF VALUE-ADDED PRODUCT FROM MUSCADINE GRAPE POMACE By Jorge Alfredo Cardona Ponce December 2007 Chair: Charles A. Sims Major: Food Science & Human Nutrition Polyphenolics are recognized for their antioxidant capacity and their contribution to flavor and color of several fruits and vegetables. H ealth benefits of these compounds are still under investigation and their use is current ly based on observed experiments both in vitro and in vivo The sources of natural antioxidants such as polyph enolics include fruits, vegetables, spices, and herbs. Muscadine grapes ( Vitis rotundifolia ) are an important fruit crop in the southeastern region of U.S. due to their tolerance to Pier ces disease. These grapes have a unique polyphenolic profile compared to other Vitis species that has sustained efforts to develop a valueadded product from them. By-products (skin an d seeds) from muscadine grape processing have presented processors with an inexpensive s ource of polyphenolics to produce valuable food ingredients. This research study evaluated isol ation techniques and s ubsequent stability of compounds recovered from muscadine grape pomace. Methods to reduce or eliminate sugars were also explored by the us e of yeast fermentation and solid-phase isolation. Following fermentation, methods to remove residual water were explored using freeze drying, spray drying and vacuum evaporation. Additi onally, processes were assessed to determine the efficiency and profitability for the muscadin e grape industry. Target compounds extracted from muscadine grape pomace showed high antioxidant activity (34.3 0.57 mol Trolox Equivalents/g).

PAGE 11

11 Although processing positively influenced polym erization and stability of polyphenolics, antioxidant capacity was significantly decreased. Initially, high temperature, low concentration of polyphenolics, oxygen exposure, and high pH envi ronment were considered major factors that affected polyphenolic content and bioactive charact eristics. Latter information indicated that such factors had a significant effect on the pol yphenolics antioxidant capacity but only a small change on their concentration. Vacuum drying showed the be st results for maintaining polyphenolics concentration and pr eserving their antioxidant capaci ty following fermentation. Fermentation proved to be a more practical proce dure than solid phase isolation to eliminate or reduce sugars without putting the va luable nutritional characteristics of the final product at risk. Fermentation following a simple concentrati on step was an economical way to obtain polyphenolics from muscadine grape skins. Resu lts from three isolation techniques suggested that a simple muscadine grape by-product concen tration followed by a drying operation can be suitable and profitable for a typical muscadine pr oducer. This process could be adjusted and implemented by any fruit or vegetable processor to estimate the potential increase in profit of their additional byproduct operation.

PAGE 12

12 CHAPTER 1 INTRODUCTION Polyphenlics are plant metabolites that are r ecognized for their antioxidant capacity and their contribution to flavor and color of several fruits and vegeta bles (Croft 1999). In recent years, polyphenolic characteristics such as enzyme inhibition and radical scanvenging have captured consumers attention because of the as sociation of these compounds, and their activity, with long-term human hea lth (Parr and Bowell 2000). Since the knowledge of how polyphenolics be have in the body is still limited, the efficiency of natural antioxidant products is not easy to estimate. Furthermore, phenolic compounds could act syne rgistically or antagon istically which complicates the antioxidant product assessment. Therefore, th e use of most natural antioxid ant products is currently based on empirical knowledge from research conducted in model systems and some existing products (Meyer and others 2002). The sources of natural antioxidants such as vitamin C, tocopherols, polyphenolics, and organic acids include fruits, ve getables, spices, and herbs. The share of polyphenolics in the market of antioxidants has increased as the de mand for antioxidants from natural sources grows steadily. In 1996, 26% of the food antioxidant mark et was occupied by natural antioxidants with a yearly growing rate of 6-7% (Meyer and others 2002). In berri es and fruits, the most abundant antioxidants are vitamin C and polyphenolics. Companies such as Optiture (USA), Chr. Hansen (Denmark), Overseal Natural Ingredients (GB) Quim Dis (France), Inheda (France), and Folexco (USA) share the market of extracts a nd concentrates from these sources (Meyer and others 2002). Muscadine grapes ( Vitis rotundifolia ) are unique among grape speci es due to the presence of polyphenolics such as anthocya nin diglucosides and ellagic aci d and its deriva tives (Lee and

PAGE 13

13 Talcott 2004). Moreover, muscad ine grapes are an important na tive fruit crop to the south and southeastern U.S. due to their remarkable toleran ce to Pierces disease. Pierces disease is caused by a bacterium ( Xyllela fastidiosa ) that invades the vascular sy stem of grape vines causing decay, and this microorganism is commonly transported by glassy-winged sharpshooters (Pooling, 1996; Mizell and others 2003). Due to the resistance to Pierces disease, muscadine grapes have sustained a commer cial industry in the southeast region of the U.S. (Ruel and Walker 2006). A growing concern for grape juice and wine pr oducers is the handli ng of by-products (skin and seeds) and the production of food-grade pro ducts with added value. The importance of compounds in grape pomace that may have sign ificance to the food industry due to their association to human health ha s sustained efforts to produce va luable food ingredients (Sort 2003). In the case of muscadine grapes, almost half of the fresh fruit weight constitutes skin that is a very rich source of phytochemicals such as resveratrol, ellagic acid and some flavonols (Pastrana-Bonilla and others 2003; Morris and Brady 2004). Howeve r, unique characteristics of muscadine grapes have presented technological challenges to extract polyphenolics from the pomace. This research study assessed isolation techniqu es and subsequent st ability of polyphenolics recovered from muscadine grape pomace (target compounds from the grape skins only) using various extraction and processing techniques to obtain a dry powder or a low water concentrate. Methods to reduce or eliminate sugars were ex plored by the use of aerobic yeast fermentation and partitioning from solid-phase supports with specific affinities to the compounds of interest. Processing methods to reduce or remove residual water were explored using freeze drying, spray drying and vacuum evaporation techniques. It was hypothesized that extract processing would

PAGE 14

14 affect polyphenolic stability. Processes were sou ght to optimize the concentration and stability of target polyphenolics in an effort to determin e the most efficient and profitable process for the industry. The specific object ives of this study were: To evaluate the antioxidant capacity, polyphe nolic composition and pigment stability of muscadine grape pomace extract as affected by concentration, sugar elimination, and dehydration processes. To propose protocols suitable for the Muscadine grape industry to deve lop an extract rich in polyphenolics. To evaluate the profitability of best proposed protocols to manufacture an extract from the muscadine grape pomace.

PAGE 15

15 CHAPTER 2 LITERATURE REVIEW 2.1 Muscadine Grape Muscadine grapes ( Vitis rotundifolia ) are an important native fruit crop to the south and southeastern U.S and have been cultivated and utilized by people in these regions before European colonization (Poling 1996). Due to the high pressu re of insect vectors and environmental routes for plant diseases, it is not possible to commercially cultivate most Vitis species, other than Vitis rotundifolia Because of their adaptation, many muscadine grape cultivars have shown remarkable tolerance to pests and diseases (Poling 1996). More specifically, muscadine grapes are re sistant to Pierces disease caused by Xylella fastidiosa a bacteria that is commonly spread by glassy-winged sharpshooters ( Homalodisca coagulate ) that invade the vascular system of grape vines and cause a significant vine decline over time (Mizell and others 2003). Due to the severity of Pier ces disease in the southeastern region of U.S., Vitis rotundifolia and Vitis arizonica both native to these regions, ha ve demonstrated resistance to Pierces disease sufficient to create a commercial industry (Ruel and Walker 2006). Muscadine grapes are found in tight small clus ters of 3 to 10 berries that may not ripen uniformly, thus, they are harvested as single berr ies instead of bunches (Himelrick 2003; Takeda and others 1983). The fruit possess a much thicker skin than other grape species, has large seeds, is very turgid, and has musky-flavored pulp. The fruit is found in black to bronze colo rs (Croker and Mortensen 2001; Himelrick 2003). The pro duction area of muscadine grapes is around 5,000 acres. Georgia and North Carolina encompass more than half of the total acreage, and, Arkansas and Florida also hold an important portion of the cultivated area (Table 2-1). Commercial production of muscadin e grapes is divided mainly into fresh fruit and wine. According to Halbrooks (1998), muscadine juice manufacture has been positively evaluated, but

PAGE 16

16 its production has still not been exploited. Fresh fruit is marketed as pick-your-own and packaged berries. In 1979, 95% of the grape production of Florida wa s sold directly to customers (Degner and others 1981). A recent re port divided the muscadine market into juice, wine, vinegar, sweet spreads, dry products, and by-products and nutraceuticals (Morris and Brady 2004). The most common product manufactured with muscadine grapes is wine, which is attractive due to its fruity flavor. The shelf lif e of these wines may be shorter than other wines due to changes in their pigments during aging. If there is an oversupply of muscadine wine, high quality vinegar might be produced to create an extra product of this industry (Sims and Morris 1985; Morris and Brady 2004). Table 2-1. Estimated muscadine grape acreage in th e southeastern United States (Cline and Fisk 2006). State Acreage Alabama < 75 Arkansas 400 500 Florida 600 1,000 Georgia 1,400 Lousiana 70 Missisipi 300 North Carolina 1,300 Oklahoma < 50 South Carolina 300 Tennessee 160 Texas < 50 Virginia < 50 Since 40% of the fruit is skin (Pastrana-Boni lla and others 2003), onl y about half of the fruit is used in conventional pr oducts such as juice and wine. After pressing, processors must decide the best way to handle th eir waste and such a substantial volume of residue can only mean significant costs to the overall procedure. Furt her processing of pomaces rich in polyphenolics, such as muscadine grapes, can lead to an incr ease in economic value pe r ton of fruit and the decrease of waste material (Ector 2001). Pigments could be extracted from the skins and be used

PAGE 17

17 as food ingredients; these co mpounds could then contribute to the overall product color and increase its nutraceutical c ontent (Morris and Brady 2004). 2.2 Polyphenols There is substantial interest in polypheno lic compounds in foods due to their effects on food quality and their association with human health benefits against coronary heart disease and cancer (Parr and Bowell 2000). Polyphenolic compounds are not only recognized for their bioactive properties but also for their contributio n to flavor and color of several fruits and vegetables (Croft 1999). Approximately 8000 ph enolic compounds have been identified that possess a common aromaticring st ructure with at least one hydroxyl group (Robbins 2003). These phenolic compounds originate as secondary plant metabolites, from phenylalanine and tyrosine precursors and the phenylpropanoid pa thway, and are essential for plant reproduction, stability, and growth processes in plan ts (Croft 1999; Shahidi and Naczk 2003). 2.2.1 Structure and Classification According to the number of phenol subunits phenolic compounds can be divided into simple phenols and polyphenols and are further divided into other categories depending on their structure and activity (Robbins 2003; Shahidi an d Naczk 2003). The term polyphenolics is commonly used to describe compounds of this nature. Phenolic compounds are formed by the release of ammonia from phenylal anine and tyrosine due to the action of phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) to form trans -cinnamic acid and p -coumaric acid, respectively. Subsequentl y, these two compounds serve as pr ecursors in the formation of several phenolic compounds (Shahidi and Naczk 2003). De Bruyne and others (1999) explained that phenolic compounds are product s of a plant aromatic pathway: the shikimate section that generates the aromatic amino acids phenylalanin e, tyrosine and tryptophan; the phenylpropanoid pathway that produces the cinnamic acid derivati ves; and the flavonoid ro ute that produces a

PAGE 18

18 diversity of flavonoid compounds. Phenolic compounds are divided into the following groups: hydroxylated derivatives of benzoi c or cinnamic acids (phenolic acids); coumarins; flavonoids and stilbenes; lignans and lignins; suberins a nd cutins; and tannins (S hahidi and Naczk 2003). 2.2.1.1 Phenolic acids and simple phenols Phenylpropanoids are typically known as phenolic acids. These compounds are a group of aromatic secondary plant metabolites that ha ve one carboxylic acid functional group. While the basic skeleton in all these compounds is simi lar, the number and positions of the hydroxyl or carboxyl groups on the aromatic ring generate a diversity of compounds (Shahidi and Naczk 2003; Robbins, 2003). Current research relates pheno lic acids with various plan t functions, including nutrient uptake, protein synthesis, enzyme activity, phot osynthesis, among others (Wu and others 2000). In foods, phenolic acids are related to sensor y characteristics and nut ritional properties. Moreover, these compounds are intimately relate d with enzymatic browning thus affecting food quality and shelf life (Robbins 2003). 2.2.1.2 Flavonoids Flavonoids are widely known for their red, pu rple, and blue color and their association with health in diets rich in fruits and vegeta bles. Currently more than 6,400 flavonoids have been identified from diverse plant species. Fla vonoids include a broad gr oup of compounds that share a similar diphenylpropane (C6-C3-C6) ba sic structure (Figure 2-1) and depending on the position of the association of the aromatic ring and the benzopyrano, flav onoids could be divided in three main groups: flavonoids, isoflavonoids, and neoflavonoids (Marais and others 2006; Winkel 2006). Flavonoids are formed from the condensation of phenylpropane with coenzyme A to form chalcones that will then form other end products. The differences within flavonoids are dictated

PAGE 19

19 by the level of oxidation of the central pyran ri ng of the main diphenylpr opane structure; the number and distribution of hydroxyl, carboxyl groups; and th e degree of alkylation or glycosilation. Flavones, flava nones, flavonols, flavanonols, fl avan-3-ols and other related compounds may be formed due to all these substi tutions (Shahidi and Naczk 2003). From these compounds, flavonols, flavones, flavanones, cate chins, anthocyanidins and isoflavones are commonly consumed (Le Marchand 2002). Figure 2-1. Basic flavonoi d structure (Pietta 2000). Flavones and flavonols are the most abundant flavonoids in foods with approximately 100 flavones and 200 flavonols identified in plants The most common flavonols are myricetin, quercetin and kaempferol which ar e found in many important fruits and vegetables (Table 2-2). Flavonols differ from flavones because of the pr esence of a hydroxyl group on the 3-position and are also known as 3-hydroxyflavones (Shahi di and Naczk 2003; Le Marchand 2002). Flavanones and flavanonols have a saturated Cring. Flavanonols differ from flavanones due to the presence of a hydroxyl group on the 3-position. Flavanones are mainly found in citrus fruits, these compounds are frequently glycosylated in the 7-position with disaccharides (TomsBarbern and Clifford 2000). Catechins and ant hocyanins are also known as flavans and are an important group of flavonoids. Catechins are primarily found in tea and red wine while anthocyanins are generally found in many berri es and flowers (Le Marchand 2002; Shahidi and Naczk 2003).

PAGE 20

20 Due to their bright colors, fla vonoids can act as visual attract ants for pollinating insects. Some flavonoids might have a protective mechan ism against predatory insects. UV protection and growth and development are among other functi ons and processes associ ated with flavonoids in plants (Winkel 2006; Pietta 2000). In addition to their physio logical functions in plants, flavonoids are also significant components of th e human diet. Flavonoids are present in most edible fruits and vegetables, and depending on their source, their bioavailability may vary tremendously. Thus, dietary intake of flavonoi ds is variable, ranging from 3 to 800 mg/day (Erlund 2004; Le Marchand 2002; Pietta 2000). Table 2-2. Classes and di etary sources of flavonoids (Shahidi and Naczk 2003). Class Dietary Source Chalcone Miscellaneous Flavone Fruit skins, parsley, celery, buckwheat, citrus, red pepper, red wine, tomato skin Flavanone Citrus Flavonol Leek, broccoli, endives, grapefru it, tea, onion, lettuce, tomato, beeries, apples, olive oil Flavanonol White grape skins, soybean, fruits Isoflavone Soybean Flavanol Tea Anthocyanidin Berries, dark fruits 2.2.1.3 Tannins Tannins are a group of oligomeric and polym eric water-soluble polyphenols. Tannins are divided in two main groups (Fi gure 2-2) due to their structur e and susceptibility to acid hydrolysis: condensed and hydrolyzab le tannins (Meyers and others 2006). The name tannins comes from the ability of these compounds to tan animal skin into leather by protein precipitation (Lei 2002). These compounds are us ually found in the bark of trees and can precipitate proteins from aqueous solutions (S hahidi and Naczk 2003). Condensed tannins are oligomers or polymers of flavan-3-ols. A pproximately 50 proanthoc yanidins have been identified in the literature. Based on the m onomer units, condensed tannins could be further

PAGE 21

21 divided in procyanidins, propela rgonidins, and prodelphinidins. These proanthocyanindins are large molecules ranging from 2000 to 4000 Da. Hydrolyzable tannins are formed by the glycosylation of gallic or ellagic acids, so, they are further divided in two groups: gallotannins and ellagitannins. These tannins range from 500 to 2800 Da (Kraus and others 2003; Meyers and others 2006; Shahidi and Naczk 2003). Tannins have anti-nutritional properties due to the relation with proteins to form complexes making them unavailable for digesti on (Shahidi and Naczk 2003). In contrast, tannins are associated with h ealth benefits possessing anti mutagenic and anticarcinogenic properties, reduction of serum cholesterol, a nd many other biological fu nctions in herbivore animals (Meyers and others 2006; Kraus and others 2003). Figure 2-2. General structure of hydrolyzable tannins (left) and condensed tannins (right) (Shahidi and Naczk 2003; Meyers and others 2006). 2.2.2 Polyphenolics as Antioxidants Polyphenolics are naturally occurring antioxida nts that prevent oxida tion of substrates containing these compounds. This prevention occurs not only in foods but also in humans, relating phenolic compounds with the control of many degenerati ve diseases. Antioxidants protect oxidative substrates by reducing the concentration of oxygen, intercepting singlet

PAGE 22

22 oxygen, or scavenging initial radicals to preven t the activity of reactive oxygen, nitrogen and chlorine species that are related with diseases such as arthritis, diabet es, atherosclerosis, among others (Shahidi and Nacz k 2003; Le Marchand 2002). Even though the exact associ ation of polyphenolics with some diseases is not fully understood, flavonoids have proven not only to inhibit enzymes direc tly related in the generation of reactive oxygen species, but also chelat e metals which are important in the oxygen metabolism (Pietta 2000). More over, since polyphenolics have a wide range of hydrophobicity, both the hydrophilic and lipophilic environments could be protected by these compounds (Parr and Bowell 2000). The most predominant method of antioxidant activity seems to be the hydrogen donation, also known as radical scavenging (Robbins 2003). Free radicals could cause extensive damage to macromolecules in the body. Free radicals rem ove a proton from macromolecules, generating highly reactive radicals of high molecular weight This creates a chain degradation reaction where radicals are trying to stabilize by removing a proton from a neighboring molecule. Polyphenolics donate a hydrogen atom, breaking the de gradation cycle. Furt hermore, if phenolic compounds react with the initial forms of free ra dicals, they donate thei r proton, thus quenching the free radical and producing a less reactive radi cal that will be subs equently stabilized by resonance delocalization (Parr and Bowell 2000; Shahidi and Naczk 2003). Many studies have suggested that the antioxidant prope rties of phenolic compounds, specifically flavonoids, are generally located in the B ring of the molecule, more specifically in the number of hydroxyl groups present in that ring (Reviewed by Pietta 2000). However, flavonoids that do not possess a hydroxyl group in the B ring still have anti oxidant activity. In

PAGE 23

23 the case of tannins, the antioxidant capacity is cl osely related to the degree of polymerization due to the amount of radicals that can be quenc hed per molecule (De Bruyne and others 1999). 2.2.3 Polyphenolics in Mucscadine Grapes Muscadine grapes are unique among grapes sp ecies due to the pres ence of anthocyanin 3,5-diglucosides, free ellagic acid and numerous derivatives of ella gic acid such as ellagic acid glycosides and ellagitannins (Lee and Talcott 2004). As an isolated compound, anthocyanin diglycosides are more resist ant to oxidative and thermal processes than a respective monoglycoside, however in vivo they exhibit less color, enhanced formation of polymers, and a greater susceptibility to exhibit brown color than foods that contain anthocyanin monoglucosides (Lee and Talcott 2004). Other unique compounds in muscadine grapes are ellagic acid and its derivatives which are commonly associated with fruits such as blackberries, raspberries, pomegranates, certain tree nuts and strawber ries (Rommel and Wr osltad 1993; Lee 2004). Characterization and quantification of pheno lic compounds present in muscadine grapes has been extensively studied. The polyphenolics, other than anthocyanins, found in muscadine are flavanols (catechin and ep icatechin), ellagic acid, fla vonols (myricetin, quercetin and kaempferol), gallic acid, and resveratrol rang ing from 0.1 to 86.1 mg/100g of whole fruit in 10 different muscadine cultivars (Pastrana-Bonilla and others 2003). These compounds are mainly located in the skins and the pulp. The skins cont ain ellagic acid, flavonols and resveratrol while the seeds contain flavanols and gallic acid (Pastrana-Bonilla and others 2003). In another study (Yi and others 2005) five anthocyanin aglycones were identified after hydrolysis. However, two studies at the University of Florida (Talcott and Lee 2002; Talcott and others 2003) have identified six main forms of anthocyanidins in muscadine grapes including pelargonidin.

PAGE 24

24 2.3 Anthocyanins Interest in natural food colorants and addi tives continues to in crease in response to consumer demands and the potential health benef its they impart (Del Pozo and others 2004). Anthocyanins are the most important class of water-soluble pigments responsible for the red, blue and violet colors in many fruits, vegetables, roots, tubers bulbs, legumes, cereals, leaves and flowers (Bridle and Timberlake 1997). Ma ny fruits contain high concentrations of anthocyanins and several studies have shown a relationship between fruit consumption and reduction of certain diseases attributable to the presence of antioxida nt polyphenolics (Rommel and Wrosltad 1993; Parr and Bowell 2000; Aaby a nd others 2005). Polyphenolics such as anthocyanins are effective radical scavengers and can break free radical reactions through their electron donation, metal chelati on, enzyme inhibition, and oxygen radical quenching capabilities (Pastrana-Bonilla and others 2003). 2.3.1 Structure and Occurrence Anthocyanins are flavonoids formed by c ondensation of phenylpropane compounds with participation of three molecules of malonyl coenzy me A that form a chalcone that is cyclated under acidic conditions (Shahidi and Naczk 2003). Currently, 17 naturally occurring anthocyanidins have been iden tified, and only six of them (F igure 2-3) are common in higher plants: pelargonidin (Pg), peonidin (Pn), cyanid in (Cy), malvidin (Mv), petunidin (Pt) and delphinidin (Dp). From those si x, Cy, Dp, and Pg are the most widespread in nature (Kong and others 2003). The six different anthocyanin ba se forms vary based on their hydroxyl (OH) and methoxyl (OCH3) substitutions on the anthocyanidin base (B ring). Moreover, anthocyanins are exclusively found as glycosides in undisturbed tissues of flow ers and fruits, where they are bound to one or more molecules of sugar yielding around 200 different ant hocyanins that have been identified (Shahidi and Naczk 2003).

PAGE 25

25 The anthocyanidin form or flavylium cation (2 -phenylbenzopyrilium) is the basic structure of the anthocyanin molecule which has conjuga ted double bonds responsible for light absorption around 500 nm, thus becoming visible with a red hue to the human eye (Rein 2005). Depending on the presence of hydroxyl or carboxyl, the hue of the color will be e ither blue or red, respectively (Shahidi and Naczk 2003). Figure 2-3. Chemical structures of an thocyanidins (Shahidi and Naczk 2003). Anthocyanins are usually glycosylated with glucose, galactose, ar abinose, xylose, or rhamnose as 3-glycosides (monoglycosides), 3,3, 3,5 or 3,7-diglycosides (diglycosides) or triglycosides (Rein 2005). Anthocyanins can also be acylated with organic acids that are usually aromatic or aliphatic dicar boxyl acids bound to the anthocya nin through ester bonding. The most common acylating agents are: hydroxycinnamic acids such as p -coumaric, ferulic, caffeic and sinapic acids, hydroxybenzoic ac ids like gallic acid, and ali phatic acids including malonic, acetic, malic, succinic and oxalic acids (Francis 1989; Bruneton 1995; Cabrita and Andersen 1999). The most important role of ant hocyanins is their ability to im part color to the plants and plant products in which they occur. This color es tablishment plays a crucia l role in the attraction

PAGE 26

26 of animals for pollination and seed dispersal. They also act as antioxidants, phytoalexins or antibacterial agents, possess known pharmacological properties, and are used for therapeutic purposes (Kong and others 2003). 2.3.2 Color Stability Anthocyanins are unstable compounds and the color loss may depend on the hydrolysis of the unstable aglycone form. The stability of anth ocyanins is closely rela ted to self-association, concentration and structure, pH, organic chem icals, temperature, light, enzymes, oxygen, copigments, metallic ions, ascorbic acid, suga rs, and processing (Shahidi and Naczk 2003; Stingzing and others 2002). Glycosylation, acylation, and hy droxylation contribute to the anthocyanin stability. It has been shown that the acyl gr oups maintained color when bound to anthocyanins. Polyacylated anthocyanins ar e more stable than monoacylated ones. Hydroxylation in positions C-4 and C-5 prevents wa ter addition to form colorless species (Saito and others 1995; Rein 2005; Turker and others 2004; Shahidi and Naczk 2003). Copigmentation enhances the color of anthocyanins by increa sing the absorbance due to pigment concentration and association with other compounds by hydrophobi c interaction between aromatic bases of the molecules involved (Shahidi and Naczk 2003) Copigmentation occurs through numerous interactions, such as intermol ecular and intramolecular complexe s, self-association, and metal complexation. Anthocyanin copigmentation results in a stronger and more stable color than a singular anthocyanin molecule. Furthermore, ove rlapping association of copigmentation results in a prevention of nucleophillic a ttack of water to the molecule (Rein 2005), thus providing the new molecule with higher color intensity and mo re stablility. Copigm ents are colorless or slightly yellowish natural molecules in plant ce lls that exist along with anthocyanins, the most common copigments are flavonoids. Other copigm ents could be organic acids, amino acids, and metal ions (Brouillard and others 1989).

PAGE 27

27 Anthocyanins are very sensitive to pH shifts. In solutions they exist in four different forms: blue neutral and ioni zed quinonoidial base, red fla vylium cation or oxonium salt, colorless pseudobase, and colorless chalcone (Figure 2-4). Each of the four species has a variety of tautomeric forms and the chalcone could exist as cis or trans forms (Shahidi and Naczk 2003; Clifford 2000). Figure 2-4. Anthocyanin e quilibria: quinonoidal base (A), flavy lium cation (B), carbinol base or pseudobase (C) and chalcone (D) (C lifford 2000; Shahidi and Naczk 2003). Even though anthocyanins can exhibit a diversity of color tones in the pH range from 1-14, they are more stable at acidic media showing an intense red coloration in the pH range of 1 to 3 (Rein 2005; Shahidi and Naczk 2003). The increase of pH decreases the concentration of the flavylium cation, thus, decreasing the intense re d color to form the carbinol base. This compound does not have a conjugated double bond between the rings A and B so there is no absorption of visible light. As the pH con tinues to increase (Fi gure 2-5), the colored

PAGE 28

28 quinonoidial form is produced by the loss of a hydr ogen atom. If pH continues to rise, the cabinol base yields the colorl ess chalcone form (Rein 2005). Figure 2-5. Effect of pH value on anthocyanin equilibria (Clifford 2000). Anthocyanin stability is also affected by temp erature. This degrad ation process follows first order kinetics (Kirca and others 2006). Elevated temper atures alter the anthocyanin equilibria or hydrolyze the glyc osidic bonding to form unstable chalcones or aglycone forms, respectively, as the first step in thermal degr adation. Ultimately, therma l degradation leads to the formation of brown pigments (Rein 2005; Cl ifford 2000). On the other hand, extremely low temperatures also affect the quality of anthoc yanins. Quinonoidal formation is favored by low temperatures, therefore if a product was frozen re d, it might appear blue after thawing due to the change of flavylium cation to quinonoidal form during that low temper ature exposure (Bridle and Timberlake 1997). Oxygen intensifies the degrad ation of anthocyanins. Even though the formation of unstable chalcones due to pH or thermal change s is reversible, the pr esence of oxygen during these procedures impedes the normal reconversio n of these compounds (Bridle and Timberlake

PAGE 29

29 1997). The effect of oxygen on anthocyanins occu rs as direct oxidative mechanisms or through indirect oxidation, yielding co lorless or brown end products (Rein 2005). Active forms of oxygen are highly reactive, as described in th e pecking order table (Buettner 1993), and can degrade any type of molecule with a lower oneelectron reduction potential (Eo). Although light is needed in the biosynthesis of anthocyanins, once formed light damages these compounds (Markakis 1982). Visible and UV light are harm ful to anthocyanins, and a study conducted on Berberis species illustrated the damaging effect of light on their anthocyanin profile (Laleh and others 2006). Specifically, sh orter wavelengths are more harmful than longer ones (Skrede and Wrolstad 2000; Wang 2006). In a photochemical study, Furtado and others (1993) found that aqueous solutions of anthocyani ns submitted to irradiation help with the disappearance of the flavylium cation due to the formation of the chalcone form. Enzymes also have an important effect on anthocyanin stability, thus, inactivation of enzymes is a key process in the production of a variety of fruit and vege table products (Fang and others 2006). The most common enzymes relate d to the degradation of anthocyanins are glycosidases. Glycosidases are not specific in th e structural requirements of the aglycone portion of a molecule (Huang 1955), theref ore they cleave the anthocyani ns separating the sugar from the unstable aglycone form. Peroxidases (P OD) and polyphenol oxidases (PPO) are enzymes naturally present in fruits that degrade phe nolics compounds resulting in the formation of precursors of brown pigments (Kader and others 1997). PPOs degrade ant hocyanins indirectly by the formation of quinones that subsequently w ill react with anthocyanins to form colorless products (Kader and others 1998). Another investigation (Fang a nd others 2006) explained that PPO oxidizes chlorogenic acid to form a quinone that will eventually react with anthocyanins to form brown pigments.

PAGE 30

30 Substances such as sugars and organic acids can react with other solutes like polyphenolics and influence their stability as we ll. Sugars play a double role in anthocyanin stability. Sugars and syrups could be used as cryoprotectants by associating with plant water by osmosis. The process of sugar addition is al so known as osmotic dehydration (Wang 2006). Syrups have proven to work better than dry sugars because the sucrose, which is commonly the osmotic agent, is dissolved (20-65%) and ready to migrate to the fruits or vegetables. Dry sugars are used with fruits that contain a high percent of ju ice where sugars can be dissolved. An osmotic step could protect color agai nst degradation during drying (T orreggiani and Bertolo 2001). However, once some browning derived products ha ve been produced from sugar caramelization, the degradation of anthocyanins is enhance d. The browning products responsible for the degradation effect in anthocyani ns are furfural and Maillard r eaction products (Tsai and others 2005). Ascorbic acid fortification has commonly b een used in fruit juices as antioxidant protection and to increase the nut ritional value. Ascorbic acid has proven to retard enzymatic browning by reducing the o -quinones to o -diphenols that no longer produce brown pigments or degrade anthocyanins (Gregory 1996; Kader an d others 1998). Another study (Rababah and others 2005) showed that ascorbic acid addition in fruit products did not change the phenolic or anthocyanin concentration, but dehydration together with ascorbic acid addition slightly reduced the amount of anthocyanins. However, addi tion of ascorbic acid was shown to degrade anthocyanins in pomegranate (Marti and others 2001) and Aa juice (Pacheco 2006). Bisulfite and other sulfur compounds are al so used to protect color and phenolic compounds. These compounds are usua lly used in wine production. SO2 acts as an antioxidant and bacteriostatic agent (Morata and others 2006). Bisulfite, like ascorb ic acid, reacts with the o -

PAGE 31

31 quinone to eliminate the basic compound to form brown pigments (Lindsay 1996). Nevertheless, SO2 prevents the formation of visitins. Visi tins are compounds formed by condensation of anthocyanins and pyruvic acid or acetaldehyde releas ed by certain strains of yeast. Visitins are more stable than anthocyanins and do not aff ect the desired color of these compounds (Morata and others 2006). 2.3.3 Anthocyanins in Muscadine Grape Five main anthocyanin forms were reported sh owing the absence of pelargonidin aglycone form (Yi and others 2005; Yi and others 2006). However, all six major anthocyanidins were reported in muscadine grapes (Talcott and othe rs 2003; Talcott and Lee 2002), and all of them were present as diglucosides (Fi gure 2-6). These diglucosides are more resistant to oxidative and thermal processes than a resp ective monoglycoside as isolated compounds. However, in vivo they exhibit less color, enhanced formation of polymers, and a gr eater susceptibility to exhibit brown color than foods that contain anthoc yanin monoglucosides (L ee and Talcott 2004). Figure 2-6. Anthocyanin diglycoside st ructure (Cyanidin3,5-diglucoside).

PAGE 32

32 2.4 Ellagic Acid 2.4.1 Structure and Occurrence Ellagic acid (EA) is formed through the conjugation of two mole cules of gallic acid which is a derivative of hydroxyl benzoic acid (Figur e 2-7). Ellagic acid is primarily found as ellagitannins (ETs). The formation of free ella gic acid is a result of th e spontaneous conversion of both ester groups of hexahydroxydiphenoyl group (HHDP) into EA following its hydrolysis from ETs (Rommel and others 1993). In the pr esence of tannase, tann ins are hydrolyzed into HHDPs and a sugar molecule. Subsequentl y, HHDP is converted to EA through coupled oxidation and spontaneous lactonizati on (Shi and others 2005; Lei 2002). EA is a polyphenolic located in the vacuole and represents the main phenolic compound in the Rosaceae family (Atkinson and others 2005). EA is an important polyphenolic compound in fruits such as raspberries, strawberries and black berries. These fruits contain as much as three times the concentration of EA of some nuts (Toms-Barbern and Clifford 2000; Rommel and others 1993). EA could also be found in pomeg ranate (Gil and others 2000), oak (Lei 2002) and other woody plants (Lee 2004). Figure 2-7. Chemical stru cture of ellagic acid. 2.4.2 Ellagic Acid Derivatives Ellagic acid derivatives could be divided in two main groups : Ellagic acid glycosides (EAG) and ellagitannins (ETs). EAG are compounds that consist of a sugar moiety bound to an

PAGE 33

33 EA molecule (Figure 2-8). Usually the sugars involved in the formati on of EAG are pentoses such as xylose, rhamnose, and arabinose. Gluc ose may also be present in the formation of EAGs. The linkage between sugars and EA typica lly occurs in the 4-position since only ellagic acid-4-glycosides have been reported (Lee 2004; Lee and others 2005; Zafrilla and others 2001; Mullen and others 2003). Figure 2-8. Ellagic acid glyc osides A) Ellagic acid-4-ara binoside, B) Ellagic acid-4acetylarabinoside, C) Ellagic acid-4-acetylxyloside (Mullen and others 2003). ETs (Figure 2-9) are water soluble polyphenolic s of molecular weights up to 4000 Da that represent one of the largest gr oups of tannins. ETs are likely derived from a gallotannin precursor (pentaO -galloyl-D-glucose) by oxidative coupling of at least two galloyl units yielding a HHDP unit that is the base of an ET (Clifford and Scalbert 2000; Khanbabaee and Ree 2001). ETs are also categorized as hydrolysab le conjugates, includi ng one or more HHDP groups esterified to a sugar molecule (Lee 2004) Currently, 500 different types of ETs were reported in nature that differ from each other in the number of HHDP units, the conformation of

PAGE 34

34 the glucose ring, and the location of the gallo yl ester groups (Feldman and Sambandam 1995; Helm and others 1999). ETs can be divided in mono meric or oligomeric depending on the amount of glucose molecules present. Monomeric ETs are HHDP es ter groups bound to one molecule of glucose. The coupling between HHDP groups and glucose generally occurs at the 4,6carbon position and/or 2,3carbon position of the glucose molecule. 1,6-, 1,3-, 3,6-, and 2,4arrangements can also take place. These diverse combinations generate numerous monomeric ellagitanins. The most prominent ellagitannins are 4,6-HHDP (t ellimagrandin I and II), 2,3-HHDP (sanguin H4 anf H5 ) and 4,6-2,3-HHDP (pedunculag in and potentellin) (Lei 2002). Oligomeric ETs are created by the polyme rization of monomeric ETs. The most predominant oligomeric ETs are dimeric and tetr americ ETs. The polymerization of monomeric ETs occurs through oxidative C-O couplings be tween galloyl and HHDP moieties or the C-C interaction s between glucose and HHDP. Exam ples of oligomeric ETs with C-O pairing, coriariin and rugosin D, which ar e dimmers of tellimagradin I. Oligomeric ETs with C-C pairing, roburin A and D, which are dimmers of vescagalin/castalagin (Lei 2002). Figure 2-9. Ellagitannins: Tellimagrandin II (mon omeric ET) (left), Sang uiin H-6 (oligomeric ET) (right) (Meyers and others 2006, Clifford 2000, Lei 2002).

PAGE 35

35 2.4.3 Ellagic Acid and Its Deri vatives in Muscadine Grape EA was measured in muscadine grapes and it wa s found that it is one of the most abundant non-colored polyphenolics in the skins along with my ricetin (Pastrana-Bonilla and others 2003). Another investigation (Lee and Talcott 2004) showed an average of 700.5mg/Kg and 1080.9mg/Kg of total EA in the skins of eight di fferent cultivars of unripe and ripe muscadine grapes respectively. Of this total EA, 3.4% was free EA and 5.4% was EAG in unripe fruit. In ripe fruit, 9.2% constituted free EA and 8.7% was EAG of the total EA, proving that a large portion of the ellagic acid was in the form of ETs. Three EAGs were identified in muscadine gr ape (EA-rhamnoside, EA-xyloside, and EAglucoside), and in this study (L ee and others 2005), EAGs repr esented around 12.7% of the total ellagic acid in Noble muscadine grapes. Due to lack of chromatographic standards a nd the diversity of ETs, it is challenging to identify these compounds. Commonly, the concentr ation of ellagic acid de rivatives is measured by the amount of free EA released after acid hyd rolysis. ETs are measured by the difference between total EA and EAG and free EA. Invest igations have shown that ETs represent around 80%-90% of the ellagic acid present in muscad ine grapes and it depends on the stage and maturity of the fruit (Lee and Talcott 2004; Lee a nd others 2005). Four ETs were reported but not completely identified in muscadine grapes. Moreover, two other ETs were identified as Sanguiin H4 or H5 depending on the position of the galloyl group (Lee and others 2005). 2.5 Processing Effects on Polyphenolics Numerous changes in properties, both physical and chemical, can occur during fruit and vegetable processing. Some oxidative reactions may occur where electrons are removed from molecules to form oxidized compounds. These ox idative reactions lead to browning, changes in flavor and odor, changes in textur e, and most importantly loss of nutritional value. The overall

PAGE 36

36 concentration of nutrients could modify the antiox idant potential of fruit and vegetable products. Therefore, fruit and vegetable processing is direct ly related to the changes in concentration and functionality of phytochemicals (Wang 2006). As a result of simple processes such as peeling, grating, cutting, and slicing, commodities that usually had a shelf-life of weeks or months ar e perishable due to the disruption of plant cells, thus liberating intracellular products and enzy mes that will degrade polyphenolics and other compounds of nutritional interest in fruits a nd vegetables (Laurila and Ahvenainen 2002; Clifford 2000). Some commodities need peeling as part of their process. This step could be accomplished in several ways, but in an industria l scale usually mechanical peeling, chemical peeling, or high-pressure steam peeling is used. If this process is not gentle, the cell walls near the peel may be disrupted and some com pound may cause browning, and other degradation procedures (Laurila and Ahvena inen 2002). Studies have shown that simple procedures should be conducted with stainless steel materials th at wont oxidize the compounds in fruits and vegetables. Furthermore, these materials shou ld be disinfected constantly during operation (Laurila and Ahvenainen 2002). 2.5.1 Heat Procedures Although the main objective of heat processing is safety, it is commonly known that this procedure negatively affects the original properties of raw mate rials. Specifically, thermal processing is responsible for the decrease of co mpounds of nutritional valu e such as ascorbate, tocopherols and anthocyanins (Wang 2006). Howeve r, thermal process can be beneficial. Heat treatments have proven to enhance the avai lability of some compounds due to thermal destruction of cell walls and subcellular compar tments with the release of nutritional compounds and the denaturation of degrad ing enzymes such as polyphenol oxidases (PPO) and peroxidases (PO) (Takamura and others 2002; Wang 2006).

PAGE 37

37 Food canning is an important heat treatment wi dely used in the food industry to prevent the presence of dangerous microorganisms such as Clostridium botulinum which is an anaerobic organism that creates a leth al toxin. This microorganism grows at pH over 4.6 and is thermolabile. Even t hough the possibility of C. botulinum occurrence in high acid foods is uncommon, an F-value of 5D must be accomp lished to properly eliminate the risk of C. botulinum activity. To accomplish this task, canne d products have to be exposed to a high temperature retort that will have detrimental effects on its nutritional compounds (Pflug and Esselen 1979). Pasteurization is one of the most common h eat procedures used in food products. The most common pasteurization protocol, used to ensure proper heat tran smission while reducing the time of exposure, is high temperature sh ort time system (HTST) used as a continuous system for pasteurization in milk products and juices. A study (Klopotek and others 2005) showed that pasteurization wa s highly influential in the to tal phenolic and anthocyanin concentration of strawberry products. 2.5.2 Drying Procedures Drying is one of the most ancient processes us ed to preserve foods. The purpose of this procedure is to reduce the water activity of a fruit or vegetabl e to a level where growth of spoilage microorganisms and occurrence of degrad ing chemical reactions are halted or slowed down. Furthermore, drying was used to reduce the volume and weight of commodities for easier distribution and extended shelflife (Barbosa-Cnovas and others 2005). However, drying can diminish the nutritional content in foods. Due to thermal degrad ation, polyphenolics may be lost or their bioactivity may be reduced. A study (S chmidt and others 2005) showed that different processes did not decrease the phenolic content significantly but their bioactivity was reduced. Another investigation (Rababah and others 2005) showed that even though concentration of

PAGE 38

38 polyphenolics in strawberries, peaches and a pples was not significantly changed after dehydration, their antioxidant capacity decreased significantly. Spray drying is one of the most well-known and widely used drying procedures. This process take places at te mperatures between 150-200oC (Orsat and Raghavan 2006). The particles have a spherical shape. Due to the di minutive size of the particles, the drying procedure is shorter compared to other drying protocols and is a suitable met hod for sensitive compounds to heat deterioration. Powdered milk and whey concentrates are among the most common products produced by spray drying. Other product s include coffee, tea extracts, baby formula, enzymes, and yeast (Barbosa-Cnovas and others 2005). It is important to control the feed rate, drying air temperature, and pre ssure of the air in the nozzle in spray drying as these characteristics determine the final quality of the powder. Moreover, carriers such as maltodextrins can be used to improve agglomeration and provide stability (Orsat and Raghavan 2006). The best sp ray drying characteristic s for higher yields of roselle extract were analyzed (Andrade and Flores 2004) and results showed that the extract did not differ in pH from the original liquid feed, but the flavor was lost. The best results for color, pH, and yield recovery were detect ed at a temperature range of 178-190oC, and a pressure of 5-6 bar in the atomizer. Also known as liophilizati on, freeze drying is another major drying procedure. Liophilization was introduced to the food industr y in 1954. Later, in 1964 coffee was subjected to freeze drying. Freeze-dried products maintain most of their physico-chemical and sensory characteristics due to the lack of heat expos ure (Barbosa-Cnovas and others 2005). Freeze drying consists of two main steps: freezing the product and drying it under vacuum to cause sublimation. For water, sublimation is accomplis hed when the temperature is lower or equal to

PAGE 39

39 0oC and the pressure is below 672 Pa. This is called the triple point where ice could be evaporated without melting (Figure 210). It is important to reali ze that the amount and nature of solids in food play an important role in the sublimation temperature. Commercial freeze drying is carried out at -10oC and absolute pressure of 2mm Hg or less (Barbosa-Cnovas and others 2005). Figure 2-10. Phase diagram of water (adapt ed from Barbosa-Cnovas and others 2005). Characteristics such as rec onstitution, retention of vol atile compounds, rehydration and others are maintained by freeze drying. An i nvestigation (van Golde and others 2004) showed that the polyphenols of wine that were freeze dr ied had around a 70% recovery with the same qualities as the polyphenols from the original wi ne. Freeze-drying appears to be a good process for the conservation of large polyphenols like tannins whereas small polyphenols might not be protected (Abascal and others 2005). Another investigati on (Tambunan and others 2001) demonstrated that the quality of freeze-dried he rbal products was slight ly decreased but the overall quality was still higher than oven-dried sa mples. Freeze drying is considered the best drying procedure because the initial material is frozen and the atmosphere around the sample has

PAGE 40

40 a low concentration of oxygen. However, this procedure can be expensive and time consuming (Barbosa-Cnovas and others 2005). Drum drying is a technique where heat is tr ansferred to a material by conduction from a rotating drum. After the product is dried, it is separated from the drum with a scraper (Orsat and Raghavan 2006). This protocol is one of the chea pest drying methods. It is energy efficient, saves space and is more economical than spray dr ying for small volumes. The disadvantages of this procedure are: the product needs to be liquid; it has to adhere to a metal surface, resist an oxygen exposure and high temperatures (Barbosa-C novas and others 2005). Furthermore, a study (Hsu and others 2003) showed that drum drying had the highest losses in antioxidant capacity compared to freeze drying and hot air drying. Other drying procedures, not as common as th e ones previously discussed, can be found in the industry. Convective drying is a procedure where a layer of pr oduct is exposed to heated air. Vacuum drying is a procedure where steam heat s the products under low pressure. Due to the use of low pressure, vacuum drying improves the quality of a product by using lower temperature (Orsat and Raghavan 2006). 2.5.3 Extraction Procedures and Storage Pressing is a common procedure used for extracti ng juice from fruits. In this procedure a significant concentration of polyphenolics may rema in in the fruit solids. An investigation (Klopotek and others 2005) showed step by st ep how the phenolic profile was changing during strawberry processing. Total phe nolics were reduced by 44% dur ing mashing and pressing. On the other hand, the anthocyanin content was main tained during initial steps of strawberry processing. Storage can also have severe effects on f ood quality if its temperature is not properly controlled. Numerous studies have explored the detrimental effect of storage on polyphenolics.

PAGE 41

41 Storage had more impact than thermal process in guava juice production (Fender 2005). Turker and others (2004) showed a decrease in anthocya nin content and color density were decreased during a 90 days storage study at 40oC. Another study (Kirca and others 2006) showed similar results where the largest lost of compounds was at 37oC, followed by 20oC. 2.5.4 Enzymatic Procedures The use of enzymes to achieve desirable chan ges in food products has been utilized for centuries. Enzymes are catalysts that aid in the incr ease of yield, facilitate processes, and play an important role on sensory characte ristics. Pectinases are used to increase yield of pigments extracted from grapes in wine production, Naringinases are used to reduce the bitter flavor in citrus juices, pectin methyl esterase (PME) is us e to increase the yield and clarify citrus juices, and the list of enzymes and processes they ar e used in is vast (Whitaker 1994; Wang 2006). Enzymes could be classified as endoge nous and exogenous depending on whether the enzyme was found in the substrate or was in tentionally added to accomplish an activity. Enzymes could be further divided into six main groups depending on the reaction they catalyze (Whitaker 1994). However, not all enzymes are beneficial in the food industry. There are enzymes that are found naturally in fruits and vegeta bles that need to be inactivated in order to preserve the quality and prolong th e shelf-life of a product. The main enzymes studied that are closely related to fruit and ve getable deterioration are PPO a nd PO which are oxidoreductases (Whitaker 1994; Kader and others 1997; Kader and others 1998).

PAGE 42

42 CHAPTER 3 PHYTOCHEMICAL, ANTIOXIDANT AND PIGMENT STABILITY OF MUSCADINE GRAPE POMACE AS AFFECTED BY CONCENTRATION AND DEHYDRATION 3.1 Introduction Interest in phytochemicals has increased in recent years due to their association with human health benefits as well as their role in foods as functio nal ingredients (Talcott and Lee 2002; Wang 2006). The major mechanism by whic h these compounds enhance food quality and aid human health is radical scavenging (Robbi ns 2003), other mechanisms include enzymatic inhibition, enzymatic co-factoring, growth se lectivity and inhibition for deleterious gastrointestinal bacteria, and essential nutrien ts absorption enhancement (Reviewed by Dillard and German 2000). Radical s cavenging stops a degradation chain reaction caused by free radicals that are formed inside and outside th e body. Around 100 radicals have been associated with degenerative diseases such as cancer, atherosclerosis, arthri tis, and cataracts, and polyphenolics donate a hydrogen atom, obstructing the development of such diseases (Shahidi and Naczk 2003; Parr and Bowell 2000). Muscadine grapes ( Vitis rotundifolia ) are a significant fru it crop in the south and southeastern U.S. and are unique among Vitis species not only for their increased tolerance to Pierces disease ( Xylella fastidiosa ), but also in their chemical composition. These grapes are known to possess a diversity of polyphenolics, such as anthocyanin diglycosides, ellagic acid and its derivatives, numerous phenolic acids and fl avonoids (Pooling 1996; Mizell and others 2003; Lee and others 2005). The severity of Pierces di sease in the southeaste rn region of U.S. has limited the production of Vitis species other than Vitis rotundifolia which has demonstrated to be suitable for a commercial indus try (Ruel and Walker 2006). A growing concern for muscadine grape juice and wine producers is the handling of byproducts (skin and seeds) and th e desire to produce a food-grad e product with added value.

PAGE 43

43 There are numerous phytochemical compounds in grape pomace that may have significance for the food industry due to their association to hum an health, thus sustained efforts are currently underway to produce value-added food ingredient s from these otherwis e waste products (Sort 2003). The unique properties of muscadine grapes that have presented technological challenges include their thick pectin-laden skins, the selectiv e recovery of polyphenolics, and, for juice pomace, their high residual sugar content sin ce wine by-product contains little or no sugar concentration after fermentation. Recently, the literature on antioxi dant compounds from residual sources has been increa sing steadily with investigations on grape pomace, leaves, and skins (Monagas and others 2006; Bonilla and others 1999), olive mill waste (Visioli and others 1999), wine industry (Makris and ot hers 2007) and several other fru its and vegetables (Peschel and others 2006). Therefore, the purpose of this study was to de termine isolation techniques and subsequent stability of polyphenolics recove red from muscadine grape skins using various extractions and processing techniques to obtain a dry powder or a concentrate. 3.2 Materials and Methods 3.2.1 Materials and Processing Grape pomace was obtained from Paulk Vineyards (Wray, GA) from deseeded muscadine grapes grown in 2006. The pomace was obtaine d following a freeze-thaw cycle and a hydraulic pressing for juice recovery in th e absence of rice hulls as a pre ssing aid. The resulting pomace was frozen and transported overnight to the Food Science and Human Nu trition Department at the University of Florida, Gainesville, FL and held in frozen storage at -20oC until further processing. Upon thawing and removal of residual free-r un juice grape skins were thawed and mixed (1:1, 1:2, 1:5, and 1:10 w/w) with hot water (90-95oC). Extracts were manua lly stirred three or

PAGE 44

44 four times a day to improve contact between th e skins and water for a three-day assessment period. Extracts were pressed a nd filtered through cheesecloth daily and mixed with new grape skins to increase the phenolic con centration. For the fi rst extraction grape skins were mixed with hot water while the second and third extracti on were not submitted to heat to minimize degradation in compounds already extracted. After the third day of concentr ation the extract was pressed and filtered to get rid of the skins. Extr acts were analyzed for total anthocyanin content as a marker for completion of the polyphenolic extraction (appendix A). The disadvantage of a long concentration pr ocess was that the ju ice already started undesired fermentation process and the color extr action in the second and third day was not as important as the first one. Due to the long time spent in compound concentration, the mixture process was reduced to only one day with hot wa ter and likewise assessed for total anthocyanin content as a marker for polyphenolic extraction (appendix B). At this point, the ratios of pomace to water 1:1, 1:2, 1:5 were assessed. The hi gh volume of water in dilution 1:10 would have represented extensive drying in a future proc essing and was eliminated. After a one-day extraction, dilution 1:5 was also eliminated due to a low concentration of compounds and high volume of water. On the other hand, the most concentrated sample (1:1) did not have enough water to facilitate handling of the product and minimal extraction of compounds, thus excluded from further experimentation. Consequently, th e dilution (1:2) was used for further processing and investigation. After the 24-hour process was c onducted and the grape skin to water ratio was selected, the mixture was assessed with samples every 30 minutes to determine the maximum compound extraction time by total anthocyanin evaluation (appendix C). The extract was manually stirred every 15 minutes to improve contact between the skins and water, and allowed to extract. The

PAGE 45

45 maximum color extraction was accomplished at 3.5 hours and samples collection was conducted for 90 more minutes to assure this maximum ex traction. Therefore, the process was reduced from 24 hours of water skin exposure to about 3.5 hours at which, a maximum concentration of 1,200 mg/kg total anthocyanins was accomplished. Af ter this point the cont ent of anthocyanins was relatively stable. Based on previous results, an experiment was run at a semi-industrial scale. Grape skins were thawed and mixed (1:2 w/w) with 45.4 Kg of hot water (90-95oC) for 3.5 hours. The free run extract was collected and skins pressed in a hydraulic press at (500 bar) to obtain an aqueous extract. This extract was filtered through cheesec loth and a 1-cm bed of diatomaceous earth to remove insoluble agents and to clarify the extract. This clarified extract was used as the starting material for subsequent procedures to eliminate or reduce the presence of soluble sugars. Three isolation methods were utilized including tw o solid phase extractions and a fermentation procedure followed by three concentration prot ocols that included spray drying, freeze drying and vacuum concentration. All handling and proc essing methods were compared to a control of the starting clarified extract for calculation of phytochemical recove ry of changes due to process techniques. Upon completion of each isolation or processing prot ocol, samples were held at 20oC until analysis. 3.2.2 Solid Phase Isolation A batch of extract was separated as a control and for affinity column isolation treatments (Amberlite XAD-4, reverse phase C18). This batch was clarifie d through diatomaceous earth and no further processing was done to a control sample of the extract that was stored frozen at -20oC until analysis. Reversed phase C18 is a model system used commonly at a laboratory scale due to its high cost. Moreover, particle size is also an impediment for its usage on a larger scale (Kraemer-Schafhalter and others 1998). SEP-PAK C18 columns proved to efficiently separate

PAGE 46

46 major phenolic compounds to improve analysis by HPLC and be very practical and easy to clean (Jaworski and Lee 1987; Oszmianski and others 1988; Kraemer-Schafhalter and others 1998). Amberlite copolymers, on the other hand, are used at laboratory scale but could also be used as industrial alternatives for affinity column isolatio n due to their commercial availability and price (Pietrzyk and Chu 1977b). Various types of Am berlite XAD copolymers have been industrially utilized in the removal of impurities from waste and potable water as well as isolation of carotenoids, steroids, and othe r biologically important compounds (Pietrzyk and Chu 1977a; Fritz and Willis 1973). Amberlite copolymers, wh ich vary in surface area, porosity, and activity, where shown to vary in their mode of se paration, quality of co mpound retention based upon nature of the target compounds, pH of the envi ronment, amount of Amberlite resin used as adsorbent and the type of copolymer present in the resin (Kraemer-Sch afhalter and others 1998; Pietrzyk and Chu 1977a, Pietrzyk and Chu 1977b) Amberlite resin XAD-2 was partially satisfactory in retaining polar solutes from a queous extract and it was shown that Amberlite XAD-7 could adsorb substances with both li pophillic and polar inte raction even though Amberlite XAD-2 showed greater affinity with aromatic compounds (McRae and others 1982). Another work illustrated the efficiency and feas ibility of Amberlite XAD-8 in the extraction of polyphenolics because such resin was used for 2 years showing reproducible results. However, when the Amberlite resin was overloaded with compounds, the solvent started to remove material from the resin (Lalaguna 1993). Kraemer-S chafhalter and others (1998) explained that a type of Amberlite XAD-7 showed poor separation and was a difficult column to clean, while other Amberlite resins showed better separation but still s howed cleaning complications. Furthermore, Amberlite XAD-2 showed insufficient pigment retention. For present experiments,

PAGE 47

47 reversed phase C18 column was used as a comparison to Amberlite XAD-4 as an industrial solidphase separation and isolation technique. Amberlite XAD-4 resin (10g) previously washed with methanol and thoroughly cleaned with deionized water was loaded into a small column whereby 2mL of extract was loaded and allowed to adsorb for 1 hour. Following ad sorption, unbound compounds were washed with water (200mL) and desorbed with 100% meth anol. Following evaporation to dryness, compounds were re-dissolved in a known volume of 0.1M citrate buffer at pH 3.0. Those compounds not retained on the resin were subseque ntly adsorbed onto 1-gram of Sephedex LH20 (normal phase) placed in a mini-column, washed with water, eluted with 100% methanol, evaporated, and likewise re-dissolved in citrate buffer. Likewise, 5mL of extract was loaded onto a 5g reversed phase C18 mini-column and allowed to adsorb by gravity feed. The colu mn was then washed with water (200mL) and phytochemicals desorbed with 100% methanol. Following evaporation, compounds were redissolved to a known volume of the citrate buffe r. Non-retained co mpounds were likewise adsorbed onto a Sephedex LH-20 (normal phase) mi ni-column and re-dissolved in citrate buffer. A third procedure to remove re sidual sugars involved fermen tation of simple sugars by inoculation with wine yeast ( Saccharomyces cereviseae strain Premium Cuve) at a rate of 2.5g/L and allowing aerobic fermentation to occur at 20-25oC until soluble solids content was decreased to a constant amount by monitoring oBrix values every 12 hours. After fermentation, the extract was clarified by passi ng through a 1-cm bed of diatomaceous earth with the aid of vacuum. Each of the three phytochemical isolation tec hniques that also served to remove residual sugars was compared to a non-isol ated control. To determine the effects of common processing

PAGE 48

48 or concentration steps, three pro cessing techniques were evaluated for the fermented isolate. The isolate was sub-divided into equal portions for vacuum concentration, spray drying, and freeze drying. A control sample was retained and frozen at -20oC until analysis. For spray drying, 2L of the fermented extract was spray dried (Anhydr o, Copenhagen, Denmark) at a temperature of 220-230oC and an exhaust temperature of 100-110oC over a 4 hour period. The resulting powder obtained was re-dissolved in a known volume of 0.1M citrate bu ffer at pH 3.0 for subsequent analysis. A freeze dried sample of the same fermented extract (300mL) was accomplished in a Freeze Drier 5 unit (Labconco, Kansas City, MO) at -100oC and 1 Torr inside the drying chamber over an 8 hour period. The resulting powd er was likewise re-disso lved in citrate buffer for analysis. Lastly, 15mL of ferm ented extract was evaporated at 60oC using a rotary evaporator over a 40 minute period and re-disso lved in a known volume of citrate buffer for analysis. 3.2.3 Chemical Analysis 3.2.3.1 Spectrophotometric determination of total anthocyanins Total anthocyanin content was determined spectrophotometrically by the pH differential method (Wrolstad 1976). Isolation and processing treatments were appropriately diluted with buffer solutions at pH 1.0 and pH 4.5. Absorbance was read on a UV-Vis microplate reader (Molecular Devices Spectra Max 190, Sunnyvale, CA ) at a fixed wavelength of 520 nm and total anthocyanin concentration calculated and reported in mg/kg equivalents of cyanidin-3-glucoside with an extinction coefficient of 29,600 (Jurd and Asen 1966). 3.2.3.2 Determination of polymeric anthocyanins The percentage of polymeric anthocyanins was determined based on color retention in presence of sodium sulfite (Rodr iguez-Saona 1999). Treatments were diluted in pH 3.0 buffer and each sample subdivided into two fractions. A solution containing 5% sodium sulfite was

PAGE 49

49 added to one fraction while an equivalent volum e of pH 3.0 buffer was added to the remaining fraction. Absorbance at 520 nm was recorded for each on a UV-Vis microplate reader (Molecular Devices Spectra Max 190, Sunnyvale CA). Concentration of polymeric anthocyanins was calculated and reported as the percentage of absorbance remaining after the addition of sodium sulfite. 3.2.3.3 Determination of total soluble phenolics Total soluble phenolics were de termined by the Folin-Ciocalteu assay (Singleton and Rossi 1965). Samples were diluted in water and 100 L of each were loaded into test tube for reaction with 0.25N Folin-Ciocalteu reagent (Sigma Chemical Co. St. Louis, MO). After a 3 min reaction of the reagent and the sample, 1N sodium carbona te was added to form a blue chromophore that was read after 30 minutes at 726 nm on a UV-Vis microplate reader (Molec ular Devices Spectra Max 190, Sunnyvale, CA). Total soluble phenolics were quantified in equi valents of a gallic acid standard with data expressed in mg/kg of gallic acid equivalents. 3.2.3.4 Quantification of antioxidant capacity Antioxidant capacity was determined by the oxygen radical absorbance capacity (ORAC) method (Cao and others 1996), adapted to be performed with a 96-well Molecular Devices fmax fluorescent microplate reader (485 nm excitation and 538 nm emission). The assay measures the ability of an antioxidant to inhibi t the decay of fluorescein induced by the peroxyl radical generator 2,2-azobis (2-amidinopropane dihydrochloride) as compared to Trolox, a synthetic, water-soluble vitamin E analog. Fo r analysis, samples were diluted in pH 7.0 phosphate buffer and 50 L of each sample was then transferred to a microplate along with a Trolox standard curve (0, 6.25, 12.5, 25, 50 M Trolox) and phosphate buffer blanks. 100 L of flourescein and 50 L of peroxyl radical generator were adde d to all samples, standard curve, and blanks. Readings were taken every 2 min over a 70 min period at 37C. Antioxidant capacity

PAGE 50

50 was quantified by linear regression based on th e Trolox standard curve and results were expressed in mol of Trolox equivalents per gram ( mol TE/g). 3.2.3.5 Half life determination Samples were diluted with pH 3.0 citrate buffer and placed in a 96-cuvette microplate subdivided into two fractions. A solution cont aining 3% hydrogen peroxide was added to one fraction while an equivalent volume of pH 3.0 buffer was added to the remaining fraction. Absorbance at 520nm was recoded using a UVVis microplate reader (Molecular Devices Spectra Max 190, Sunnyvale, CA). The assay was carried out at 45oC for 60 minutes with readings every 2 minutes to quant ify color loss over time. Results were expressed as minutes of half-life. 3.2.3.6 Analysis of polyphenolics by HPLC Polyphenolic compounds were analyzed by reverse phase HPLC using modified chromatographic conditions (Lee and others 2005) with a Dionex system equipped with an ASI100 Autosampler injector, a P-680 HPLC pump, and a PDA-100 Photodiode Array Detector. Separations were performed on a 250 x 4.6 mm Acclaim 120-C 18 column (Dionex, Sunnyvale, CA) with a C18 guard column. Mobile pha se A consisted of water acidified with o -phosphoric acid (pH 2.4) and Mobile phase B consisted of 60:40 methanol and water acidified with o phosphoric acid (pH 2.4). Samples were hydroly zed in 2N HCl (adjusted to contain 50% methanol) for 90 min at 95oC before injection. The gradient solvent program held Phase A for 3 min; then phase B from 0 to 30% in 3 min; 30 to 50% in 2 min, 50 to 70% in 5 min, 70 to 70.63% in 3 min, 70.63 to 70.7% in 1 min, 70.7 to 70.81% in 0.5 min, 70.81 to 71.2% in 2.1 min, 71.2 to 71.3% in 2 min, 71.3 to 85% in 1.4 min and 85 to 100% in 10 min for a total run time of 30 minutes for both set of samples at a flow ra te of 1mL/min. Polyphe nolics were identified by UV/VIS spectral interpretation, re tention time and comparison to authentic standards (Sigma

PAGE 51

51 Chemical Co., St. Louis, MO). All treatments were filtered through a 0.45 M filter and directly injected into the HPLC. Data was reported as mg/L of each compound. Anthocyanins were compared to a cyanidin aglycone standard, flavon ols were compared to a quercetin standard, and ellagic acid was compared to an ellagic acid standard. 3.2.4 Statistical analysis The study was designed as a completely ra ndomized design (CRD) that included seven treatments (Amberlite, C18, spray drying, freeze drying, vacuum drying, and controls for both the fermented and non-fermented extracts). Data for each treatment is the mean of three replicates. Analysis of variance and means separations by LSD test (P < 0.05) were conducted using JMP software (SAS Institute, Cary, NC). 3.3 Results and Discussion The effects of processing were evaluated and re sults for all analyses were reported in units from each assay mentioned earlier in the method section (Table 3-1). Table 3-1. Quality analyses of muscadine pomace extract (polyphenols) as affected by various processing protocols. Process Total Phenolics1 Total Anthocyanins2 Polymeric Anthocyanins3 Half life4 ORAC5 E8 1640 a6 1470 a 7.65 e 17.2 d 34.3 a F 1580 a 1520 a 9.75 de 17.9 cd 28,7 b BC18 1130 bc 996 c 25.8 a 24.7 a 16.7 d UBC18 4.55 e 0.25 e NA7 NA >0.1 f BA 1030 c 873 d 18.6 b 21.4 b 22.6 c UBA 186 d 1.65 e NA NA 1.91 e SD 1240 b 1060 c 14.3 bcd 20.5 b 22.0 c FD 1270 b 1230 b 11.3 cde 19.8 bc 18.1 d VD 1620 a 1450 b 14.8 bc 20.5 b 28.8 b 1Expresed in gallic acid equivalents (mg/kg). 2Expressed in cyaniding-3-glucoside equivalents (mg/kg). 3Expressed in percentage of polymeric anthocyanins (%). 4Expressed in time (min). 5Expressed in Trolox equivalents ( mol TE/g). 6Similar letters within columns for each analysis are not significantly different (LSD test P < 0.05). 7NA (not applicable) samples did not contain the analyte. 8Treatment abbravietions: (E) Ex tract, (F) fermented extract, (BC18) bound to reversed phase C18 column, (UBC18) unbound to reversed phase C18 column, (BA) bound to Amberlite XAD-4, (UBA) unbound to Am berlite XAD-4, (SD) spray dried, (FD) freeze dried, and (VD) vacuum dried.

PAGE 52

52 3.3.1 Anthocyanin Color Stability Anthocyanin stability was assessed spectropho tometrically and is shown in Figure 3-1. Anthocyanin content was maintained during fermen tation and vacuum drying (Table 3-1). Initial anthocyanin concentration in extract (1470 102 mg/kg) was somewhat affected by freeze drying (1230 114 mg/kg), but experienced a 28% decrease after spray drying (1060 26.3 mg/kg). Samples after affinity co lumn isolation (reversed phase C18 column and Amberlite resin) also showed significant decrease in their anthocyanin content (996 24.4 mg/kg and 873 78.9 mg/kg respectively). The extensive loss of color in both reversed C18 column and Amberlite XAD-4 procedures (32 and 41% respectively) presumably occurred mainly while anthocyanins were exposed to high pH enviro nment since less than 0.03% of the initial materials anthocyanins we re found in the unbound fraction from reversed phase C18 and less than 0.2% was found in the unbound fraction from th e Amberlite resin. Another possible reason for loss of anthocyanin by solid pha se isolation might be column efficiency. Perhaps, a single elution with methanol was not sufficient to di ssociate polyphenolics from either column and, after desorption, a fraction of th ese compounds remained in associ ation with the column and was washed away during cleaning of the resin and not collected for analysis. Thus, recovery from reversed phase C18 bound anthocyanins were more effici ent than those from Amberlite resin. Another possible explanation is that Amber lite XAD-4 might have higher affinity with polyphenolics than reversed phase C18 thus, making it difficult to recover compounds on a simple desorption step. Furthermore, in both affi nity column isolation te chniques, anthocyanins were subjected to copious amounts of solvent to separate polyphenolics from sugars and other compounds that were washed away. After adso rption, anthocyanins were desorbed with 100% methanol followed by evaporation. Although evaporat ion uses mild temperatures, in the last phase when most of the solvent has been eva porated, compounds could have been subjected to

PAGE 53

53 heat. Stability of anthocyanins is known to be jeopardized by lowering concentration of anthocyanins in a medium (Giusti and Wrolst ad 2003), decreasing acidity (Clifford 2000), and applying heat (Klopotek and ot hers 2005; Clifford 2000), explai ning why both affinity column isolation protocols significantly decreased an thocyanin concentration in muscadine pomace extract. a a bc c a b b 0 500 1000 1500 2000 E F BC18BASDFDVD TreatmentsCy-3-glu equivalents (mg/kg ) Figure 3-1. Total anthocyanin content of mu scadine pomace extract as affected by various processing methods. Error bars represent the standard error of each mean, n=3. Reasons for color loss were oxygen exposure, lo w acidity levels in the environment and heat. Vacuum drying maintained the concentrat ion since some temperature was substituted by low pressure and oxygen was removed from the medium to generate vacuum, thus making this process mild and effective. On the othe r hand, freeze drying showed more than 16% anthocyanin content loss despite the fact that no heat was applie d to the extract. The exposure time for the sample to freeze dry, and possibly erro rs at the recovery phase, might have had a negative effect on anthocyanin conc entration. Affinity column is olation techniques showed the highest color losses due not onl y to heat and oxygen exposur e, but also to higher pH

PAGE 54

54 environment and low anthocyanin concentration due to massive amount of solvent used during adsorption and desorption, thus illustrating the im portant role of pH in anthocyanin stability. Polymerization of anthocyanins present in muscadine pomace extract was significantly influenced by processing. Th e bound fraction of the reversed C18 column showed the highest polymerization index (25.8 3.25%) followed by bound fraction of Amberlite resin, vacuum dried, and spray dried that showed no significan t difference between each other. At the same time, the freeze dried, spray dried and the fermented extract samples showed no difference between each other (Table 3-1). The extract showed the lowest polymerization index (7.65 0.52%) due to the minimum processing (concentra tion and clarification) it was subjected to (Figure 3-2). When comparing the concentration of anthocya nins to polymeric anthocyanin content, it was concluded that both affinity column isol ation techniques had th e lowest anthocyanin concentration and yet had the highe st polymerization index (Table 31). Therefore, anthocyanins might be forming new high molecular wei ght compounds by associ ation with other polyphenolics such as condensed tannins and ot her anthocyanins. Processing was directly related to the polymerization process since pr ocessed samples had higher polymerization index while the starting material (extra ct) showed the lowest polymer formation, and as processing was milder, polymer formation was decreased. A co rrelation between anthocyanin concentration and polymeric index (r = -0.73) confirmed that as processes were conducted, polymers were forming in the media. As compared to other studies (Weinert and others 1990), processing induced polymerization which explained some of the losses in total anthocyanins attributed to this process.

PAGE 55

55 bc cde bcd b a de e 0% 5% 10% 15% 20% 25% 30% 35% E F BC18BASDFDVD TreatmentsPolimerization index (%) Figure 3-2. Polymeric anthocyanins (%) in mu scadine pomace extract as affected by various processing methods. Error bars represent the standard error of each mean, n=3. Half life determinations were made unde r accelerated conditions of storage (45oC) in the presence of hydrogen peroxide, a strong oxidizing agent. Conc entrations of peroxide and holding temperatures were determined to create a slow decay curve, suitable to testing a widerange of anthocyanin concentra tions with varying degrees of stability. Analogous to the formation of the polymeric pigments, the effects of anthocyanin isolation and processing slightly increased protection against hydrogen peroxi de-induced oxidation. The bound fraction of C18 showed the highest resistance (24.7 1.01 min) followed by all th ree drying protocols and the bound fraction of Amberlite resin (Table 3-1). The in itial extract showed shor test half life of all treatments (17.2 0.55 min) and was due to the predominance of monomeric anthocyanins in this fraction. Correlations between anthocyanin concentration and half li fe (r = -0.67) and half life and polymeric index (r = 0.86) confirmed that as polymerization index increases, anthocyanins become more stable, thus, increasi ng their durability (Figur e 3-3). Stability of anthocyanins has been enhanced due to polim erization (Weinert and others 1990; Rein and Heinonen 2004), and copigmentation of anthocyani ns showed reduction in pigment degradation

PAGE 56

56 in grape anthocyanins (Brenes and others 2005), which explains the increas e in anthocyanin half life in processed muscadine extract samples. bb bc b a cd d 0 5 10 15 20 25 30 E F BC18BASDFDVD TreatmentsTime (min) Figure 3-3. Half life (min) of muscadine pom ace extract as affected by various processing methods. Error bars represent the standard error of each mean, n=3. Although processing reduced the concentrati on of anthocyanins, the anthocyanins remaining in the matrix were more resistant. Po lymeric anthocyanin proved to be more stable to processing but that may affect the final qual ity of a food product si nce the color of the anthocyanin might change due to this process. An investigation showed that some copigments enhanced the a* value by retaining more red but also increased the b* value showing a yellowing in the sample during storage (Rein and He inonen 2004). Moreover, polymerization could decrease the antioxidant capacity of phenolic compounds by variations in the hydroxyl groups arrangement and availability which are related to their radical scavenging ability (Miller and Ruz-Larrea 2002), thus polymeric anthocyanins may not have as important benefit as compared to monomeric anthocyanins. In addition, abso rption of phenolic compounds might be conducted by hydrolysis to obtain aglycone forms or simp le phenols are likely to be absorbed more efficiently in the human body (Reviewe d by Miller and Ruz-Larrea 2002).

PAGE 57

57 3.3.2 Polyphenolic Concentration and Stability Total soluble phenolics were assessed spectroph otometrically as affected by isolation and food processes (Figure 3-4). Polyphenolic co ncentration in muscadine pomace extract and processed samples experienced somewhat simila r behavior as compared to the anthocyanin content. Polyphenolic conten t was maintained during fermen tation and vacuum dehydration while affected by freeze drying and spray drying (Table 3-1 & Figure 3-4). Affinity column isolation protocols (reversed phase C18 column and Amberlite resin) significantly decreased the concentration of polyphenolics in the extract (1130 43.8 and 1030 17.8 mg/kg respectively). The unbound fraction of C18 did not show significant polyphe nolic concentration (4.55 2.72 mg/kg) while the unbound fractions from Amber lite resin processing showed an important concentration of polyphenolics (186 122 mg/kg). Amberlite was less effective in binding polyphenolics than reversed phase C18 since the unbound fraction showed a much higher concentration (15.3%) than the unbound fraction from C18 (<0.5%). The unbound fraction of Amberlite resin contained mos tly polyphenolics that did not absorb light at 520nm since previous analysis showed a small concentrati on of anthocyanins in this fraction. Such compounds might tentatively be ellagitannins since previous investigations have shown occurrence of ellagitann ins in unbound fractions of solid pha se separation techniques (Lee 2004). Polyphenolic losses in solid phase extraction techniques were possibly due to low acidity medium, oxygen exposure, and heat exposure. Only vacuum drying maintained the concentration due to temperature substitution an d removal of oxygen by vacuum. On the other hand, freeze drying showed almost 23% loss in polyphenolic content even though the product was frozen during the procedure. Furthermore, there were no signifi cant differences between freeze drying, spray drying and reversed phase C18 column process.

PAGE 58

58 Reversed phase C18 column effectively bound most pol yphenolics since less than 0.5% was detected in the unbound fraction. On the othe r hand, the Amberlite resin showed compound losses due to the presence of polyphenolics that did not bind with the resin and were washed away together with sugars and some other organic compounds. The unbound fraction had more than 15% of the phenolics that were exposed to the Amberlite resin and more than 11% of the polyphenolics from the starti ng material (extract). a b b a a bc e c d 0 500 1000 1500 2000 E F BC18UBC18BAUBASDFDVD TreatmentsGallic Acid equivalents (mg/kg) Figure 3-4. Total phenolic content in muscad ine pomace extract as affected by various processing methods. Error bars represent the standard error of each mean, n=3. 3.3.3 Polyphenolics by HPLC Polyphenolics present in muscadine pomace ex tract and various processing methods were analyzed and monitored by HPLC at 360 and 520 nm. Analysis was focused on total ellagic acid, anthocyanins, and flavonols. Compounds were identified and quantified in hydrolyzed samples. 3.3.3.1 Anthocyanins by HPLC HPLC analysis of muscadine pomace extracts co nfirmed the occurrence of six anthocyanin aglycone forms (Figure 3-5). Previous investig ations reported similar results (Talcott and Lee

PAGE 59

59 2002; Talcott and others 2003), although some reports on the specific identity of anthocyanins in muscadine grapes differed (Lee and Talcott 2004 ; Yi and others 2005; Yi and others 2006), detecting only five anthocyanins aglycones, ex cluding pelargonidin. Since samples from this study were hydrolyzed and six peaks eluted, it was concluded that the six main forms of anthocyanin aglycones were presen t and thus, anthocyanidins coul d be identified by their elution order based on their st ructure and polarity. Figure 3-5. HPLC chromatogram of anthocyanidins present in muscadine pomace: delphinidin (A), cyanidin (B), petunidi n (C), pelargonidin (D), pe onidin (E), malvidin (F). Identification (520 nm) was done based on sp ectral characteristics and comparison to cyanidin aglycone. Addition of the corresponding peak areas for all six anthocyanidins yielded the total anthocyanin content for each treatment whic h was used for genera l comparison between treatments (Table 3-2). Delphinidin, cyanidi n, petunidin, and peonidin constituted most of the muscadine skin extract anthocyanin profile while pelargonidin accounted for only 1.1% and malvidin for 8.2%. Low content of malvidin in the extract may be explained by its lower polarity compared to the other five anthocyani din bases. Possibly malvidin was not as well extracted with hot water from the skin as other anthocyanidins. In mus cadine grape juice, the concentration of malvidin is somewhat similar peonidin (Del Pozo-Insfran 2006). The unbound fraction from the Amberlite resin had small amounts of delphinidin and cyanidin on its compound profile. The presence of these two anthocyanidins in the unbound

PAGE 60

60 fraction from Amberlite can possibl y be explained by their higher sol ubility in water as they are the two most polar anthocyanindins. Due to su ch polarity, both anthocya nidins might not have bound to the Amberlite resin as well as the others. Data indicated that peonidin and malvidin were the most resistant anthocyanidins to degradation under the processing conditions si nce only the spray dried sample indicated significant differences with the starting material (extract). On the other hand, pelargonidin showed the highest instability since only fr eeze drying and vacuum drying maintained the concentration of this compound wh ile other processing techniques si gnificantly reduced it. The Amberlite resin technique indicated the lowest concentrations of three of the four major anthocyanidins (delphinidin, cyanid ing, and petunidin), while spray drying affected pelargonidin, peonidin, and malvidin the most. Fermentation pr eserved all anthocyanidins except pelargonidin which had lower concentration compared to the extr act. Previous investig ations have illustrated the unstable nature of pelargonidin during pr ocessing (Garzn and Wrosltad 2001; Garzn and Wrosltad 2002; Kammerer and others 2007) while malv idin has been proven to resist thermal processing (Del Pozo-Insfran 2006) and showed great stability in general due to the presence of only one hydroxyl group in the B ring (Hradzin a and others 1970, Talcott and others 2003; Lee and Talcott 2004). HPLC analysis results showed a slight disc repancy compared to the total anthocyanins assay. Such discrepancy might be explained by the limitation of colorimetric assays to account for copigmentation compared to single compounds examination (Talcott and Lee 2002; Talcott and others 2003). Moreover, after HPLC results, data suggested that heat was not a major issue in degradation since only Amberlite and spray dried samples indicated significantly lower concentration of total anthocyanins compared to the starting materi al (Table 3-2). These results

PAGE 61

61 illustrated the divergence of previous results co mpared to HPLC analysis data, opposing to early conclusions mentioning vacuum drying being th e only processing protocol that maintained anthocyanin concentration. High va riation on HPLC results and sli ght higher concentrations of some anthocyanindins in processed samples were due to the difficulties of analyzing anthocyanindins instead of ant hocyanin glycosides, on ce the sugar molecule is separated from the anthocyanidin, the aglycone fo rm is very susceptible to degr adation (Dao and others 1998). Furthermore, consistency at the hydrolysis st ep prior to HPLC analysis and poor compound separation might also explain these results. Table 3-2. Anthocyanidin concentr ations in the muscadine pomace extract as affected by various processing protocols. Anthocyanin concentration1 Process Dp2 Cy Pt Pg Pn Mv Total3 E4 316 ab5 333 bc 258 bc 15.7 a 367 ab 115 abc 1400 ab F 401 a 411 a 329 ab 12.2 b 449 a 143 a 1750 a BC18 243 bc 264 cd 219 cd 11.5 b 301 bc 95.2 bcd 1130 bc UBC18 ND6 ND ND ND ND ND ND BA 165 c 186 e 169 d 9.27 b 280 bc 87.6 cd 897 c UBA 12.7 d 7.74 f ND ND ND ND 20.4 d SD 207 c 216 de 185 d 5.40 c 250 c 75.2 d 939 c FD 313 ab 335 bc 283 abc 16.0 a 396 a 127 ab 1470 ab VD 401 a 374 ab 336 a 16.2 a 439 a 135 a 1700 a 1Expresed in cyanidin aglycone equivalents (mg/kg). 2Anthocyanidin abbreviations: (Dp) Delphinidin, (Cy) Cyanidin, (Pt) Petunidin, (Pg) Pelargonidin, (Pn) Peonidin, (Mv) Malvidin. 3Sum of all anthocyanidin bases. 4Treatment abbravietions: (E) Extr act, (F) fermented extract, (BC18) bound to reversed phase C18 column, (UBC18) unbound to reversed phase C18 column, (BA) bound to Amberlite XAD4, (UBA) unbound to Amberlite XAD-4, (SD) spray dried, (FD) freeze dr ied, and (VD) vacuum dried. 5Values with similar letters within columns are not significantly different (LSD P < 0.05). 6Compounds were not detected. 3.3.3.2 Ellagic acid and flavonols by HPLC HPLC analysis of non-anthocyanin polypheno lics in muscadine pomace extract confirmed the presence of free ellagic acid, as previously characterized in other studies (Lee and Talcott 2002; Talcott and Lee 2002; Lee and Talcott 2004 ; Lee and others 2005; Pastrana-Bonilla and others 2003; Yi and others 2006). However, only my ricetin and quercetin c ould be detected after hydrolysis, while previous invest igations (Talcott and Lee 2002; Lee and Talcott 2004; Pastrana-

PAGE 62

62 Bonilla and others 2003; Yi and others 2005; Yi and others 2006) have shown three flavonols in muscadine grapes (myricetin, quercetin, and kaem pferol). Identificati on was possible since the retention time and spectroscopic attributes concurred with those of the authentic standards of ellagic acid and quercetin aglycones (Figure 3-6). Figure 3-6. HPLC chromatogram of polyphenolics present in muscadine pomace: ellagic acid (A), myricetin (B), and quercetin (C). Identification (360 nm) was done based on spectral characteristics and comparison to au thentic standards of ellagic acid and quercetin. Total ellagic concentration was fairly st able and only affected by spray drying and Amberlite treatments (Table 3-3). The content of ellagic acid was expected to be maintained in most of the treatments since it has been proven to be quite stable to processing and increment its concentration due to hydrolysis of ellagitannins which are unstabl e to heat and acidic conditions (Amakura and others 2000; Zafr illa and others 2001; TomsBarbern and Clifford 2000). However, another investigation showed that el lagic acid was decrease d after cooking probably due to oxidation reactions (Hkkinen and others 2000). The unbound fraction from the Amberlite resin showed an important concentra tion of total ellagic acid (8.6%) that was not bound to the resin and thus thought to be ellagitannins (Lee 2004). Similar to the total soluble phenolics assay, reversed phase C18 was an efficient separation technique since negligible concentrations of free ellagic acid was dete cted in the unbound fraction from this technique. In the case of flavonols, myricetin and querc etin were observed in the muscadine [omace extract (10.2 mg/kg and 5.44 mg/kg respectively). No significant di fferences were detected in

PAGE 63

63 flavonols due to processing (Table 3-3). Re maining isolation protocols showed higher concentrations of flavonols, thus, the only conclusion that could be de rived from this data is that processes had no effect on the flavonol concentr ation of the muscadine pomace product. Such results differed with some investigations that illustrated the loss of flavonols due to heat processing (Amakura and others 2000; Hkkinen and others 2000). However, flavonols have indicated better stability than anthoc yanindins in storage at 20 and 37oC (Talcott and Lee 2002). Such results could be explained by the high variation detected in HP LC analysis due to the use of aglycone forms of flavonoids that are know to be very unstable making this assay difficult. Neither myricetin or quercetin were detected in the unbound fractions (C18 and Amberlite), thus flavonols might bind more effectively to bot h columns compared to other polyphenolics. Table 3-3. Ellagic acid and fla vonol concentrations in the muscadine pomace extract as affected by various processing protocols. Polyphenolic Concentration Process Ellagic acid1 Myricetin2 Quercetin2 E3 168 ab4 10.2 c 5.44 bc F 176 ab 22.2 ab 7.26 a BC18 123 cd 18.6 b 6.54 ab UBC18 ND5 ND ND BA 113 d 11.3 c 5.16 bc UBA 9.77 e ND ND SD 122 cd 13.8 c 4.94 c FD 155 bc 20.7 ab 7.13 a VD 202 a 22.8 a 7.67 a 1Expresed in ellagic acid equivalents (mg/kg). 2Expressed in quercetin equivalents (mg/kg). 3 Treatment abbravietions: (E) Extract, (F) fermented extract, (BC18) bound to reversed phase C18 column, (UBC18) unbound to reversed phase C18 column, (BA) bound to Amberlite XAD-4, (UBA) unbound to Amberlite XAD-4, (SD) spray dried, (FD) freeze dried, and (VD) vacuum dried. 4Values with similar letters within columns are not significantly different (LSD P < 0.05). 5Compounds were not detected. 3.3.4 Antioxidant Capacity Antioxidant capacity of muscadine pomace extract (34.3 0.57 mol Trolox equivalents (TE)/g) was found to be relatively high and comparab le to other fruits such as cherries (33.4 3.4 mol TE/g) and strawberries (35.4 4.2 mol TE/g), and vegetables such as red cabbage

PAGE 64

64 (31.5 6 mol TE/g) (Wu and others 2004). Processing in general had a negative effect on the antioxidant capacity of the ex tract (Figure 3-7). Fermentation, which maintained the concentration of polyphenolics from the starti ng extract, had signifi cant differences in antioxidant capacity (28.7 2.53 mol TE/g). Following the fermentation procedure only vacuum drying maintained an tioxidant capacity (28.8 1.23 mol TE/g) while spray and freeze drying significantly reduced it (22.0 0.75 mol TE/g and 18.1 3.13 mol TE/g respectively). Solid phase isolation process al so had a detrimental effect on the antioxidant capacity of the starting material since both affinity column tec hniques decrease the antioxidant capacity by more than 34% (Amberlite) and 51% (reversed phase C18). The unbound fraction of Amberlite showed a comparatively low antioxidant capacity probably due to the pres ence of ellagitannins in the fraction (1.91 3.64 mol TE/g) since more than 15% of polyphenolics that were subjected to the Amberlite were found in the unbound fract ion and were thought to contain comparable content of antioxidant capacity that was wa shed away. Data of the unbound fraction only indicated 8% of antioxidant capacity which conf irmed the previous assumption of ellagitannins occurrence in the fraction since its antioxidant cap acity is considered to be lower than ellagic acid aglycone as explained by Lee (2004) where antioxidant capacity was higher in aglycone forms and gradually reduced by polymerization of ellagic acid. Data from the unbound fraction of reversed phase C18 did not indicate presence of antioxi dant compounds. Furthermore, HPLC, total anthocyanins, and total phe nolics analyses confirmed a negligible concentration of anthocyanins and polyphenolics in this fraction (Table 3-1). All treatments affected the initial antioxidant capacity from the extract which agrees with Schmidt and others (2005), who suggested that even if the polyphenolic concentration of any sample is maintained after processing, its bi oactive characteristics are modified. Vacuum

PAGE 65

65 concentration resulted in a grea ter retention of antioxidant cap acity compared to spray drying and freeze drying which resulted in losses of 23% and 37% respectively. Also, vacuum concentration was the only isol ation procedure that preserved the antioxidant capacity following fermentation. Consequently, the vacuum drying process did not have a negative effect on the quality of the final extract. a b b c c d d e f -5 0 5 10 15 20 25 30 35 40 E F BC18UBC18BAUBASDFDVD TreatmentsTrolox equivalents ( mol/g ) Figure 3-7. Antioxidant capacity of muscadine po mace extract as affected by various processing protocols. Error bars represent th e standard error of the mean, n=3. 3.4 Conclusions Target compounds extracted from muscadine grape pomace showed fairly high antioxidant activity (34.3 0.57 mol TE/g) that is comparable to some fruits and vegetables. Processing positively influenced polymerization and stability of anthocyanins. In the first section of this work, high temperature together with low con centration of polyphenolics, oxygen exposure, and high pH environment were thought to be the most harmful factors that affected polyphenolic content and bioactive characteristics. However, later information from HPLC analysis illustrated the impact of polymerization was not accounted by colorimetric analysis and heat was not a major problem, thus showing biased results. Although processing did not show significant

PAGE 66

66 compound losses in all treatments except Amber lite and spray drying (by HPLC analysis), antioxidant capacity was significantly affected by processing. Vacuum drying proved to be the best treatment of all since it best maintain ed both anthocyanins and other polyphenolics concentration and also preserved the antioxidant capacity following fermentation. Fermentation showed better results than solid phase isolati on to get rid of sugars without jeopardizing the quality of the final product. Moreover, solid phase isolation, specifica lly the Amberlite resin technique, could not bind all po lyphenolics efficiently. Drying pr ocedures following an aerobic fermentation were more practical than solid pha se isolation followed by methanol evaporation, thus drying processes were not on ly statistically different but al so showed practicality to be considered for implementation by the industry. Fu rthermore, the use of methanol, a non edible alcohol, in both affinity column isolation techniques could repr esent an environmental downside and a potential operative cost compared to use of water in the fermentation procedure.

PAGE 67

67 CHAPTER 4 ECONOMIC ANALYSIS OF AN ISOLATED PRODUCT OBTAINED FROM MUSCADINE GRAPE POMACE Functional foods and beverages consumption is growing due to major consumer trends toward health consciousness (Milo 2005). As a result of consumer awareness, natural products such as food colorants and antioxidants have gain ed substantial attention in the market. In 1996, 26% of the food antioxidant market was occupied by natural antioxidants with a growth rate of 6-7% annually. The sources of natural antioxidants such as vitamin C, tocopherols, polyphenolics, and organic acids include fruits, ve getables, spices, and herbs (Meyer and others 2002). In berries and fruits, the most abundant antioxidants are vitamin C and polyphenolics. Companies such as Optiture (USA), Chr. Hansen (Denmark), Overseal Natural Ingredients (GB), Quim Dis (France), Inheda (France), and Fole xco (USA) share the mark et for extracts and concentrates from fruits and be rries (Meyer and others 2002). A growing demand for natural products has crea ted an opportunity to substitute synthetic antioxidants by natural compounds. Among the tr aditional antioxidants used are butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) which have been associated with potential toxicity. Furthermore, the manufacturing costs of these compounds are more expensive than natural antioxidants from fruits and vegetables (Moure and others 2001). Polyphenolics can be widely found in pomace or wastes of numerous fruits and vegetables after processing (Koplotek and others 2005; Pastra na-Bonilla and others 2 003; Visioli and others 1999; Bonilla and others 1999) and these sources repr esent an inexpensive material to potentially create food ingredients. As a result, the intere st in natural antioxidants and the occurrence of these compounds in by-products have driven fruit a nd vegetable processors to attempt to extract polyphenolics from their wastes and increase the profitabi lity of their operati ons. In the case of muscadine grapes, pomace constitutes around 40% of th e total fruit and it is an important source

PAGE 68

68 of polyphenolics (Pastrana-Bonilla and others 20 03; Morris and Brady 2004) that has not been utilized, but could be an importa nt source to generate food ingredients. Research has focused on converting muscadine and blueberry pomace into nutra ceutical products that could be sold for as much as $100/lb. In Georgia alone, around 1.5 million pounds of dry muscadine skins are produced yearly and could be transformed into a very profitable product (Phillips 2006). Therefore, the aim of this part of the study was to analyze the prof itability of proposed technologies to extract and concentrate polyph enolics from muscadine grape pomace as an incremental operation to a typical grape juice production facility. 4.2 Materials and Methods 4.2.1 Data Collection Primary data, regarding the nature of the muscadine industry and its operations, was collected by interviewing muscadine processors of South Ge orgia. Other primary information, regarding equipment specification, capacity and pricing, was collected by interviewing some companies responsible for selling used equipment for food industries. Assumptions used in this study were drawn from interview co mments and educated estimations. 4.2.2 Economic Analysis An economic analysis was conducted based on profitability, sensitivity, and economic return of three altern atives of polyphenolic isolation following fermentation. Potential profitability was evaluated by comp aring total revenue and total co sts by a breakeven analysis of both prices and volumes of production. Resu lts were reported as minimum price per pound ($/lb) and minimum volume (lb) needed to cover total annual costs respectively. A ratio between production volume of extract and the volume of dried residue from the skins following fermentation was used for relevant calculations. A ratio between prices of the extract and the dried skins was also applied assuming that prices would fluctuate proporti onally. Variations in

PAGE 69

69 the output of the model were assessed by a se nsitivity analysis conducted by modifying the production and price of both the extract and the dried skins (%). Th e gross revenue and the profit were displayed in the analysis for better i llustration of the outcome. Results were reported in US dollars ($) per season. Finally, the econo mic return to the business was assessed over a period of ten years by a cash flow analysis. Ne t Present Value (NPV) was calculated based on a rate of 12% which was compared to a calculated Internal Rate of Return (IRR). Inflation was estimated as 3.43%. Depreciation of equipment was conducted by the single line method with a 10% salvage value. A loan is calculated based on a fixed interest rate with higher payments towards interest at the beginni ng, paying the loan in 10 years. Results were expressed as US currency per season. 4.2.2.1 Description of the operation The intended operation is an extension of a juic e process facility illustrated in Figure 4-1. After grapes have been pressed for juice manufac ture, the waste is collected and mixed with hot water (1:2) to allow soluble compounds to migr ate to water creating a liquid extract. A pressing procedure following the initial extraction is necessary to remove skins to facilitate fermentation. Furthermore, skins can be dried and sold as animal feed. Next, sugars which have migrated into the liquid extract together with other compounds must be removed to facilitate further processing. Sugars are removed by an aerobic fermentation followed by a series of filtrations that allow separation of insoluble compounds. Lastly, a drying protocol is necessary to obtain an isolated product ri ch in polyphenolics. Three con centration protocols have been considered for the process: spray drying, freeze drying, and vacuum evaporation. 4.2.2.2 Economic assumptions The analyses in this study are based on the following assumptions: Land is previously owned and is not included in the capital investment.

PAGE 70

70 Figure 4-1. Operation flow for a typical grape juice processor planning to process its byproduct. The company already has some equipment needed for the operation and it is not included in the capital investment. Muscadine pomace has no cost since it is a by-product of their own operation and no costs were considered for elimination of this material. 90 tons of fruit will go through juice pro cessing and the pomace will be the by-product operation which will be used as the starting material for polyphenolic extraction. This operation lasts two months due to the harvesting season of mu scadine grapes (61 days). The final consumer is a processor that will use the product as a food ingredient. Equipment specifications (Table 4-1) are approximates drawn from interviews with equipment vendors. Residue Skins Juice production Water (90-95oC) Drying Pomace Press (filtered) Isolated extract (final product) Concentration Filtration Liquid Extract Fermentation Drying Dried Skins (animal feed)

PAGE 71

71 The process needs 4 employees working part tim e (4h/day) on the proc ess with a wage of $8/h. Employees already work in the company. Proposed price for yeast required for fermentatio n is $5.45/lb and intended to be used at a ratio of 0.25lb yeast/ 100lb of extract. Price for diatomaceous earth (DE) for clarific ation is quoted at $0.80/lb and planned to be used at a ratio of 1lb of DE/100lb of extract. Most of the working time will be spent in the extract manufacturing (70%) while 30% is spent on skin drying. Currently no costs in hauling or marketing of the final product are taken into account. The final product is intended to be sold to formulator of dietary supplements and/or functional foods. The estimated price used for the ex tract in the analysis is $70/lb. The dried skins after this process ar e going to be sold as animal feed. The estimated price of the dried skins in the analysis is $1.5/lb. Depreciation is calculated at 5 years for equipment, 20 years for land, and 10 years for other materials. The rate used for taxes is 20% for a self employed operation according to IRS. There is a loan for the buildi ng construction of $70,000 to be paid in ten years at a 7.5% interest rate (Table 4-2). For other activities such us cleaning and maintenance needed in the operation, 1% of the total revenue was assign ed in the cash flow. Installation cost for drye rs are 40% of the total cost of the machinery. Installation cost for filters are 70% of the total cost of the machinery. Pumps have an electrical consumption of 17KW each. Operation time was calculated based on th e volume produced per day, for electrical calculations, extra time (1 hour) was added for basic maintenance and warming up of equipment.

PAGE 72

72 Table 4-1. Generalities of drying equipments. Equipment Estimated cost ($US) Estimated capacity (lb/h) Estimated electrical demand (KW) Evaporator $ 150,000 600 39.0 Spray dryer $ 84,000 200 25.0 Freeze Dryer $ 77,000 150 18.4 Residue dryer $ 100,000 500 16.0 Table 4-2. Loan payment plan for ten years at a fixed rate of 7.5%. Intial debt (ID) Rate1 Payment2 Interest Amortization (A) Debt3 (ID A) $ 70,000 7.50% $ 7,525 $ 998 $ 6,528 $ 63,473 $ 63,473 7.50% $ 7,525 $ 893 $ 6,633 $ 56,840 $ 56,840 7.50% $ 7,525 $ 788 $ 6,738 $ 50,103 $ 50,103 7.50% $ 7,525 $ 683 $ 6,843 $ 43,260 $ 43,260 7.50% $ 7,525 $ 578 $ 6,948 $ 36,313 $ 36,313 7.50% $ 7,525 $ 473 $ 7,053 $ 29,260 $ 29,260 7.50% $ 7,525 $ 368 $ 7,158 $ 22,103 $ 22,103 7.50% $ 7,525 $ 263 $ 7,263 $ 14,840 $ 14,840 7.50% $ 7,525 $ 158 $ 7,368 $ 7,473 $ 7,473 7.50% $ 7,525 $ 53 $ 7,473 $ 1Rate estimated from a normal bank for a fixed rate loan. 2Payment is constituted by amortization and interest. 3Debt is the result of subtracting annual amortization from previous period debt. 4.3 Results and Discussion Proposed technologies were economically eval uated and results were reported in US currency ($US). 4.3.1 Economic Analysis For all three operations (spray, freeze and v acuum drying), the process was identical until the final step where isolation of polyphenolics took place via three different methods. According to results from Chapter 3, the extract has a concentration of polyphenol ics (1640 18.1 mg/kg) that is intended to be isolated as a final product (extract). From this number we consider that the final product will be at least 0.16% concentrated and that all cal culations are based on this proportion of the liquid extr acted from the process. With 90 tons of grapes expected to be used in this process, 40% represent skins (Pastrana-B onilla and others 2002) that are directly intended

PAGE 73

73 for the secondary process to obtain the desired ex tract. This results in a season skin production of 72,000 lb of fresh skin that is intended to be mixed with hot water for a production of 144,000 lb of liquid extract and 72,000 lb of residual skins. If 0.16% of the liquid extract represents polyphenolics and we are trying to obtain a product with 5% moisture, the final concentration of extract in a season would be 249 lb. From th e remaining skins after fermentation, 30% is considered solids (Phillips, 2006) and is proposed to be sold as animal feed as part of the byproduct operation, which will also contain 5% moistu re as a final product. From the remaining skins obtained, a total of 22,619 lb of dry skin can be produced in a season. Currently, efforts to process muscadine pomace are still emerging and the amount of time and capital intended for such an operation is limited. Therefore, proposed processes are basic and economically feasible for a typical muscadine producer. 4.3.1.1 Spray drying operation A used spray dryer with a capacity to remove 200 lb of water per hour was considered for the process. An estimated price ($60,000) wa s quoted for the equipment which needed to operate 13 hours daily to remove the amount of wa ter required to obtain a 5% moisture isolated product. The initial investment was $120,640 which included the spray dryer, its installation fee ($24,000), and other required materials (Table 4-3). Additional equipment necessary for this specific operation would tipically already be owne d by a juice processor. As mentioned earlier in the assumptions, the new building constructio n is intended to be built with a bank loan. From the seasonal production of extract and dried skins (249lb and 22,619lb respectively) and the medium price estimated and expected by an average muscadine grape producer for such extract and dried skins production ($70/lb and $1 .5/lb respectively ), total income for the incremental operation was calculated. Furtherm ore, with knowledge of the equipment and material required by this operation, total costs were also calculated and used for further

PAGE 74

74 economic analysis. Once income and cost sources were determined, a production volume breakeven point established the minimum amoun t required to sustai n the intended by-product operation. From the break even equation [1], where I represents income from the extract (IE) and the residue skins (IR), C represents cost both fixed (FC) a nd variable from the two products (VCE and VCR) and the ratio between extract (QE) and dried skins (QR) productions (lb per season) [2], 161 lb of extract and 14,616 lb of dried skins mu st be produced to cover both annual fixed costs and variable costs of an operation using a spray dryer with similar technical characteristics as the method for polyphenolic isolation. Fi gure 4-2 illustrates that at pr ices $70/lb for the extract (pE) and $1.50/lb for the dried skins (pR), the total cost to cover for a season is $33,168 which is covered by 161 lb of extracts and proportionally by 14,616 lb of dried skins. The production of dried skins was not shown in the x-axis of this figure to avoid confusion. Table 4-3. Capital expenditure to initiate a marginal process obtaining extract and dried skins from muscadine grape skins using spra y drying as the isolation technique. Description Unit Unit cost Quantity Total estimated cost 1. EQUIPMENT COSTS1 Filter Each $ 25,000 1 $ 25,000 Spray Dryer Each $ 84,000 1 $ 84,000 Pumps Each $ 1,200 2 $ 2,400 Containers (bins) Each $ 220 42 $ 9,240 Sub-total $ 120,640 2. CONSTRUCTION COSTS2 New Building3 Each $ 70,000 1 $ 70,000 Sub-total $ 70,000 GRAND TOTAL$ 190,640 1Equipment listed in the table is applied directly to the expenditure of the operation since the rest of equipment needed for the operation is already owned. 2The total construction cost for the new building includes wiring, electrical and tubing installations. 3New building will be constructed with a bank loan. R E R EVC VC FC I I [1] R EQ Q 011 0 [2] R R E E R R E EQ VC Q VC FC Q p Q p R E R EQ Q Q Q 11 0 82 39 162 25 5 1 70

PAGE 75

75 R R R RQ Q Q Q 11 0 011 0 82 39 162 25 5 1 011 0 70 R R R RQ Q Q Q 11 0 44 0 162 25 5 1 77 0 162 25 55 0 27 2 R RQ Q 162 25 72 1 RQ 72 1 162 25 RQ lb QR56 615 14 lb QE63 160 $0 $10,000 $20,000 $30,000 $40,000 $50,000 $60,000 $70,000 050100150200250300 Volume of extract production (lb)US dollars ($) FC VC TC I Figure 4-2. Volume break-even point for a facility using a spray dryer as a final step for product isolation. Abbreviations: FC Fixed costs, VC variable costs, TC total costs, I income. Another useful analysis is to keep produc tion volume steady and to vary prices to determine the minimum price needed to cover total costs. For a price break even point calculation, the break even equati on [1] and the ratio between extr act and dried skins productions [2] were also employed. Furthermore, a ratio between prices ($/lb) [3] was used under the assumption that prices of extract and dried skin s would maintain a differe nce between each other and that such difference would re main proportional. With such assumptions in mind, the prices where the actual production of extract (249 lb) an d dried skins (22,619 lb) cover the annual total cost are $51.21/lb and $1.10/lb, respectively. Figure 4-3 illustrates five diverse scenarios with

PAGE 76

76 different incomes (I) illustrating the significan t impact price alterations can have on the operation. Prices ranged from $20/lb (I-1) to $100/lb (I-5) with increments of $20 for the extract and proportionally from $0.43/lb to $2.14/lb with in crements of 43 cents for the dried skins. When prices were below $40/lb (extract) and $0. 86/lb (dried skins), the minimum production to cover annual costs exceeded 300 lb of extract and more than 27,000 lb of dried skins. On the other hand, when prices reached $100/lb (extract) and $2.14/lb (dried skins), net earnings can be reached after only 100 lb of extr act and 9,000 lb of dried skins pr oduced. With such prices and the intended season production vo lume, gross income of almost $90,000 would be generated and profit would increase up to $48,379 in a single season. R E R EVC VC FC I I [1] R R E E R R E EQ VC Q VC FC Q p Q p R R R E R R R EQ VC Q VC FC Q p Q p 011 0 011 0 R E R R R E RVC VC Q FC Q p p Q 011 0 011 0 R E R R EVC VC Q FC p p 011 0 011 0 R Ep p 67 46 [3] 11 0 82 39 011 0 619 22 162 25 67 46 011 0 R Rp p lb pR/ 10 1 $ lb pE/ 21 51 $ A sensitivity analysis of the circumstances when a spray dryer (with characteristics previously described) is used as the isolation method was conducte d (Table 4-4). Data suggested that a loss of more than $11,000 could be anti cipated if production and prices dropped 40%. Losses were still registered when one characteri stic, either price or volume, was reduced 40%.

PAGE 77

77 Under initial circumstances (medium price and volume), a profit of almost $14,000 in a season was registered. The remaining situations also showed positive numbers Therefore, this operation is viable when the combination of pr oduction volume and pri ces surpasses low-low and low-medium combinations. $0 $10,000 $20,000 $30,000 $40,000 $50,000 $60,000 $70,000 $80,000 $90,000 $100,000 050100150200250300 Volume (lb)US Dollars ($) TC I-1 I-2 I-3 I-4 I-5 Figure 4-3. Break-even point as affected by price fo r a facility using a spra y dryer as a final step for product isolation. Abbreviati ons: I income, TC total costs. Table 4-4. Sensitivity analysis for a marginal process obtaining extract and dried skins from muscadine grape skins using spray drying as the isolation technique. Prices ($US) Low1 Medium High2 Ext / DS3 pExt / pDS4 $50 / $1.07 $70 / $1.50 $100 / $2.10 Sales5 $ 6,188 $ 36,664 $ 51,685 Low1 178 / 16,156 Profit6 $ (11,363)7 $ (887) $ 14,133 Sales $ 36,664 $ 51,329 $ 72,358 Medium 249 / 22,619 Profit $ (887) $ 13,778 $ 34,807 Sales $ 51,329 $ 71,861 $ 101,302 Production (lb) High2 348 / 31,666 Profit $ 13,778 $ 34,310 $ 63,750 1Low represent 40% less of both medium prices and volumes. 2High represent 40% more of both medium prices and volumes. 3Extract and dried skins productions are displayed vertically in the table. 4Prices for extract and dried skins are displayed horizontally in the table. 5Sales are shown for all 9 different supposed scenarios. 6Profits are shown for all 9 different supposed scenarios. 7Number in parenthesis indicate profit loss. The economic return was asse ssed after 10 years of opera tion by a cash flow analysis (Table 4-5). With an investment of $120, 640 and a bank loan of $70,000, the operation could

PAGE 78

78 return the investment and genera te almost $60,000 of net earnings in ten years. With average annual income of more th an $30,000, the internal rate of return (IRR) was 10% higher than the rate used for the net present value (NPV) calculation, thus showing thriving results for this additional operation for the muscadine industry. It is important to denote that more compounds could be extracted from the skins if processes ar e to be optimized resulting in a higher yield, extract production and thus, gene rating profit for selling more product at $70/lb than $1.5/lb. 4.3.1.2 Freeze drying operation A used freeze dryer with a capacity to remove 150 lb of water per hour was evaluated for this isolation technique and according to its spec ifications, it needed 15.7 hours to remove water from the daily liquid extract produced. An estimated price ($55,000) was quoted for the equipment. The initial investment ($113,640) included the freeze dryer, its installation fee ($4,400), and other required materials (Table 46). Additional equipment necessary for this specific operation was assumed to be already owned by a typical juice proce ssor. The facilities required to conduct a by-product isolation process we re intended to be built with a bank loan as described in the assumption section of this chapter. Table 4-6. Capital expenditure to initiate a ma rginal process obtaining extract and dried skins from muscadine grape skins using freeze drying as the isolation technique. Description Unit Unit cost Quantity Total estimated cost 1. EQUIPMENT COSTS1 Filter Each $ 25,000 1 $ 25,000 Freeze Dryer Each $ 77,000 1 $ 77,000 Pumps Each $ 1,200 2 $ 2,400 Containers (bins) Each $ 220 42 $ 9,240 Sub-total $ 113,640 2. CONSTRUCTION COSTS2 New Building3 Each $ 70,000 1 $ 70,000 Sub-total $ 70,000 GRAND TOTAL$ 183,640 1Equipment listed in the table is applied directly to the expenditure of the operation since the rest of equipment needed for the operation is already owned. 2The total construction cost for the new building includes wiring, electrical and tubing installations. 3New building will be constructed with a bank loan.

PAGE 79

79Table 4-5. Cash flow analysis for a ma rginal process obtaining extract and dried skins from muscad ine grape skins using spray drying as the isolation technique. Periods (years) 0 1 2 3 4 5 6 7 8 9 10 Investment $120,640 Income Extract $ 17,401 $ 17,998 $ 18,615 $ 19,254 $ 19,914 $ 20,597 $ 21,304 $ 22,035 $ 22,790 $ 23,572 Residue $ 33,928 $ 35,092 $ 36,296 $ 37,540 $ 38,828 $ 40,160 $ 41,537 $ 42,962 $ 44,436 $ 45,960 Expenses Labor $ 7,168 $ 7,412 $ 7,664 $ 7,924 $ 8,194 $ 8,472 $ 8,760 $ 9,058 $ 9,366 $ 9,685 Materials $ 3,341 $ 3,455 $ 3,572 $ 3,694 $ 3,819 $ 3,949 $ 4,083 $ 4,222 $ 4,366 $ 4 ,514 General costs $ 1,753 $ 1,813 $ 1,874 $ 1,938 $ 2,004 $ 2,072 $ 2,142 $ 2,215 $ 2,291 $ 2,369 Depreciation $ 18,303 $ 18,303 $ 18,303 $ 18,303 $ 18,303 $ 4,424 $ 4,424 $ 4,424 $ 4,424 $ 4,4 24 Interest (7.5%) $ 998 $ 998 $ 893 $ 788 $ 683 $ 578 $ 473 $ 368 $ 263 $ 158 Gross Income $ 19,767 $ 21,215 $ 22,710 $ 24,253 $ 25,845 $ 41,367 $ 43,064 $ 44,815 $ 46,622 $ 48,488 Tax (20%) $ 3,953 $ 4,243 $ 4,542 $ 4,851 $ 5,169 $ 8,273 $ 8,613 $ 8,963 $ 9,324 $ 9 ,698 Utilities after Taxes $ 15,813 $ 16,972 $ 18,168 $ 19,402 $ 20,676 $ 33,094 $ 34,451 $ 35,852 $ 37,298 $ 3 8,791 (+) Depreciation $ 18,303 $ 18,303 $ 18,303 $ 18,303 $ 18,303 $ 4,424 $ 4,424 $ 4,424 $ 4,424 $ 4,424 Amortization $ 6,528 $ 6,633 $ 6,738 $ 6,843 $ 6,948 $ 7,053 $ 7,158 $ 7,263 $ 7,368 $ 7,473 Salvage Value $ 7,711 Net Income $ 27,589 $ 28,643 $ 29,734 $ 30,863 $ 39,742 $ 30,465 $ 31,717 $ 33,013 $ 34,354 $ 35,742 Net Present Value (NPV) $177,808 rate 12.00% Intern Rate of Return (IRR) 22.2%

PAGE 80

80 When income and cost sources were determined, a production volume breakeven point was conducted to determine the minimum amount of extract and dried skins required for the byproduct operation to be feasible. Analogous to the previous tec hnology (spray drying), the break even equation (US dollars) [1] and the proportion between extract and drie d skins productions (lb per season) [2] were used to conduc t a break even analysis Data suggested that 152 lb of extract and 13,847 lb of dried skins were needed to c over total annual costs ($31,424) of the operation when prices for the extract and the dried skin s were $70/lb and $1.50/lb respectively (Figure 44). R E R EVC VC FC I I [1] R EQ Q 011 0 [2] R R E E R R E EQ VC Q VC FC Q p Q p R E R EQ Q Q Q 11 0 68 39 862 23 5 1 70 R R R RQ Q Q Q 11 0 011 0 68 39 862 23 5 1 011 0 70 R R R RQ Q Q Q 11 0 44 0 862 23 5 1 77 0 862 23 55 0 27 2 R RQ Q 862 23 72 1 RQ 72 1 862 23 RQ lb QR41 847 13 lb QE18 152

PAGE 81

81 $0 $10,000 $20,000 $30,000 $40,000 $50,000 $60,000 $70,000 050100150200250300 Volume of extract production (lb)US dollars ($) FC VC TC I Figure 4-4. Volume break-even point for a facil ity using a freeze dryer as a final step for product isolation. Abbreviations: FC Fixed costs, VC variable costs, TC total costs, I income. For the break even price analysis, the break even equation US currency [1] and the ratio between extract and dried skins pr oductions in pounds per season [2 ] were used for estimations. A ratio between prices ($/lb) [3 ] was calculated and also used under the assumption that prices would maintain a proportional difference between each other. To cover total annual costs when medium production volumes of extract (249 lb) and dried skin s (22,619 lb) are produced, the prices should be at least $39/lb a nd $1.06/lb, respectively. Five scenarios in which prices varied from $20/lb (I-1) to $/100/lb (I-5) with increm ents of $20 for the extr act and $0.43/lb (I-1) and $2.14/lb (I-5) with increments of 43 cents for the dried skins were analyzed (Figure 4-5). Similar to an operation with spray drying as the isolati on method, when prices ar e below $40/lb (extract) and $0.86/lb (dried skins), the minimum producti on to cover annual costs would have to be greater than 300 lb of extract and more than 27,000 lb of dried skins. In contrast, when prices just surpass $40/lb (extract) a nd $0.86/lb (dried skins) profit is reached under such production volumes. This slight difference in the cal culation between spray drying and freeze drying operations is attributable to th e reduction in the investment. The freeze dryer suggested in this chapter is 10% cheaper.

PAGE 82

82 R E R EVC VC FC I I [1] R R E E R R E EQ VC Q VC FC Q p Q p R R R E R R R EQ VC Q VC FC Q p Q p 011 0 011 0 R E R R R E RVC VC Q FC Q p p Q 011 0 011 0 R E R R EVC VC Q FC p p 011 0 011 0 R Ep p 67 46 11 0 68 39 011 0 619 22 862 23 67 46 011 0 R Rp p lb pR/ 06 1 $ lb pE/ 39 49 $ $0 $10,000 $20,000 $30,000 $40,000 $50,000 $60,000 $70,000 $80,000 $90,000 $100,000 050100150200250300 Volume (lb)US Dollars ($) TC I-1 I-2 I-3 I-4 I-5 Figure 4-5. Break-even point as a ffected by price for a facility us ing a freeze dryer as a final step for product isolation. Abbreviati ons: I income, TC total costs. A sensitivity analysis of the circumstances wh ere freeze drying is proposed as the isolation method (Table 4-7) indicated loss of $10,025 wh en production and prices dropped 40%. The low-medium combinations showed a small prof it ($450) in a single year. The rest of the sensitivity analysis indicated positive numbers (p rofit) for the eight remaining situations.

PAGE 83

83 Therefore, the only circumstance under which this operation could not be viable was when both production and prices were 40% lower th an the expected values (medium). Table 4-7. Sensitivity analysis for a marginal process obtaining extract and dried skins from muscadine grape skins using freeze dr ying as the isolation technique. Prices ($US) Low1 Medium High2 Ext / DS3 pExt / pDS4 $50 / $1.07 $70 / $1.50 $100 / $2.10 Sales5 $ 26,188 $ 36,664 $ 51,685 Low1 178 / 16,156 Profit6 $ (10,025) $ 450 $ 15,471 Sales $ 36,664 $ 51,329 $ 72,358 Medium 249 / 22,619 Profit $ 450 $ 15,116 $ 36,145 Sales $ 51,329 $ 71,861 $ 101,302 Production (lb) High2 348 / 31,666 Profit $ 15,116 $ 35,648 $ 65,088 1Low represent 40% less of both medium prices and volumes. 2High represent 40% more of both medium prices and volumes. 3Extract and dried skins productions are displayed vertically in the table. 4Prices for extract and dried skins are displayed horizontally in the table. 5Sales are shown for all 9 different supposed scenarios. 6Profits are shown for all 9 different supposed scenarios. 7Number in parenthesis indicate profit loss. Economic return of this operation assessed th rough a ten-year peri od suggested that the operation was profitable and could return the investment in less than 6 years with and NPV of more than 177,000 with a IRR of almost 24% (Table 4-8). This increase in IRR compared to the spray drying operation is consequenc e of a reduction in the investment. 4.3.1.3 Vacuum drying operation The evaporator cited in this situation had a capacity of 600 lb of water removed per hour and water produced from the daily operation coul d be evaporated in less than 4 hours. An estimated price ($107,143) was quoted for the eq uipment, and initial investment of $186,640 included the evaporator, its insta llation fee ($42,857), and other requ ired materials (Table 4-9). Additional equipment necessary fo r the isolation operation was t hought to be previously owned by a typical juice processor and thus not included in the current analysis.

PAGE 84

84Table 4-8. Cash flow analysis for a ma rginal process obtaining extract and dried sk ins from muscadine grape skins using freeze drying as the isolation technique. Periods (years) 0 1 2 3 4 5 6 7 8 9 10 Investment $113,640 Income Extract $ 17,401 $ 17,998 $ 18,615 $ 19,254 $ 19,914 $ 20,597 $ 21,304 $ 22,035 $ 22,790 $ 23,572 Residue $ 33,928 $ 35,092 $ 36,296 $ 37,540 $ 38,828 $ 40,160 $ 41,537 $ 42,962 $ 44,436 $ 45,960 Expenses Labor $ 7,168 $ 7,412 $ 7,664 $ 7,924 $ 8,194 $ 8,472 $ 8,760 $ 9,058 $ 9,366 $ 9,685 Materials $ 3,341 $ 3,455 $ 3,572 $ 3,694 $ 3,819 $ 3,949 $ 4,083 $ 4,222 $ 4,366 $ 4 ,514 General costs $ 1,716 $ 1,774 $ 1,835 $ 1,897 $ 1,961 $ 2,028 $ 2,097 $ 2,168 $ 2,242 $ 2,318 Depreciation $ 17,403 $ 17,403 $ 17,403 $ 17,403 $ 17,403 $ 4,424 $ 4,424 $ 4,424 $ 4,424 $ 4,4 24 Interest (7.5%) $ 998 $ 998 $ 893 $ 788 $ 683 $ 578 $ 473 $ 368 $ 263 $ 158 Gross Income $ 20,704 $ 22,154 $ 23,650 $ 25,194 $ 26,788 $ 41,411 $ 43,109 $ 44,862 $ 46,671 $ 48,538 Tax (20%) $ 4,141 $ 4,431 $ 4,730 $ 5,039 $ 5,358 $ 8,282 $ 8,622 $ 8,972 $ 9,334 $ 9 ,708 Utilities after Taxes $ 16,563 $ 17,723 $ 18,920 $ 20,155 $ 21,430 $ 33,129 $ 34,487 $ 35,889 $ 37,337 $ 3 8,831 (+) Depreciation $ 17,403 $ 17,403 $ 17,403 $ 17,403 $ 17,403 $ 4,424 $ 4,424 $ 4,424 $ 4,424 $ 4,424 Amortization $ 6,528 $ 6,633 $ 6,738 $ 6,843 $ 6,948 $ 7,053 $ 7,158 $ 7,263 $ 7,368 $ 7,473 Salvage Value $ 7,211 Net Income $ 27,439 $ 28,494 $ 29,586 $ 30,716 $ 39,096 $ 30,501 $ 31,754 $ 33,051 $ 34,393 $ 35,782 Net Present Value (NPV) $177,065 rate 12.00% Intern Rate of Return (IRR) 23.8%

PAGE 85

85 Table 4-9. Capital expenditure to initiate a ma rginal process obtaining extract and dried skins from muscadine grape skins using vac uum drying as the isolation technique. Description Unit Unit cost Quantity Total estimated cost 1. EQUIPMENT COSTS1 Filter Each $ 25,000 1 $ 25,000 Vaccum Dryer Each $ 150,000 1 $ 150,000 Pumps Each $ 1,200 2 $ 2,400 Containers (bins) Each $ 220 42 $ 9,240 Sub-total $ 186,640 2. CONSTRUCTION COSTS2 New Building3 Each $ 70,000 1 $ 70,000 Sub-total $ 70,000 GRAND TOTAL$ 256,640 1Equipment listed in the table is applied directly to the expenditure of the operation since the rest of equipment needed for the operation is already owned. 2The total construction cost for the new building includes wiring, electrical and tubing installations. 3New building will be constructed with a bank loan. Once revenue and cost sources have been determined, a producti on volume breakeven analysis was used to determine the minimum amo unt required to sustain the intended by-product operation. The break even equation [1] and the proportion between extract and dried skins productions [2] were used as part of the analysis. Calculations i ndicated that in order to cover both annual fixed and variable co sts of the operation, 236 lb of extract and 21,516 lb of dried skins should be produced at $70/lb and $1.50/lb respectively. Figure 4-6 illustrates these calculations by showing the point at which the gross income gene rated covers season costs of $48,828. When using an evaporator of such specifications, the demand of volume of both products is high due to the elevat ed cost of the equipment (investment). According to these calculations and the medium produc tion, only 13 lb of extract and 103 lb of dried skins represent earnings in the season. R E R EVC VC FC I I [1] R EQ Q 011 0 [2] R R E E R R E EQ VC Q VC FC Q p Q p R E R EQ Q Q Q 11 0 75 37 419 37 5 1 70

PAGE 86

86 R R R RQ Q Q Q 11 0 011 0 23 38 419 37 5 1 011 0 70 R R R RQ Q Q Q 11 0 42 0 419 37 5 1 77 0 419 37 53 0 27 2 R RQ Q 419 37 74 1 RQ 74 1 419 37 RQ lb QR44 516 21 lb QE47 236 $0 $10,000 $20,000 $30,000 $40,000 $50,000 $60,000 $70,000 050100150200250300 Volume of extract production (lb)US dollars ($) FC VC TC I Figure 4-6. Volume break-even point for a facil ity using a vacuum dryer as a final step for product isolation. Abbreviations: FC Fixed cost s, VC variable costs, TC total costs, I income. A price break even analysis for an operati on with an evaporator was also conducted. Similar to previous break even analysis, the break even equation [1], the ratio between extract and dried skins productions [2], and a ratio between prices [3] were used for calculations. Total annual costs could be covered if prices of extr act (249 lb) and dried skins (22,619 lb) surpassed $67/lb and $1.44/lb respectively. Figure 4-7 illu strates five scenarios in which prices varied from $20/lb (I-1) to $/100/lb (I-5) with increm ents of $20 for the extr act and $0.43/lb (I-1) and $2.14/lb (I-5) with increments of 43 cents for the dried skins. Unlike the two previous analysis

PAGE 87

87 (spray drying and freeze drying), profit with a production under 300 lb of extract and over 27,000 lb of dried skins requires a pr ice of at least $60/lb (extract) and $1.29/lb (dried skins). At such prices a profit of only $1,203 could be accomp lished. In contrast, net season earnings of $36,600 can be reached when 300 lb of extract a nd 27,000 lb of dried skins are produced at the highest prices analyzed ($100/lb and $2.14/lb respectively). A lthough price of the evaporator was high, profit can be reached in the first year and cover season co sts. Moreover, if the process is successful and projected to grow, the evaporator analyzed in this scenario has enough capacity to support such growth while the other two equi pments (spray and freez e dryers) operate more than 10 hours daily while the evaporator works only 4. Thus, while this isolation technique is capital demanding, if implemented it has room fo r growth without severe changes in the process line. R E R EVC VC FC I I R R E E R R E EQ VC Q VC FC Q p Q p R R R E R R R EQ VC Q VC FC Q p Q p 011 0 011 0 R E R R R E RVC VC Q FC Q p p Q 011 0 011 0 R E R R EVC VC Q FC p p 011 0 011 0 R Ep p 67 46 11 0 75 37 011 0 619 22 419 37 67 46 011 0 R Rp p lb pR/ 44 1 $ lb pE/ 22 67 $

PAGE 88

88 $0 $10,000 $20,000 $30,000 $40,000 $50,000 $60,000 $70,000 $80,000 $90,000 $100,000 050100150200250300 Volume of extract production (lb)US Dollars ($) TC I-1 I-2 I-3 I-4 I-5 Figure 4-7. Break-even point as affected by price for a facility using a vacuum dryer as a final step for product isolation. Abbreviations: I income, TC total costs. Table 4-10. Sensitivity analysis for a marginal process obtaining extract and dried skins from muscadine grape skins using vaccum drying as the isolation technique. Prices ($US) Low1 Medium High2 Ext / DS3 pExt / pDS4 $50 / $1.07 $70 / $1.50 $100 / $2.10 Sales5 $ 6,188 $ 36,664 $ 51,685 Low1 178 / 16,156 Profit6 $ (23,105)7 $ (12,630) $ 2,391 Sales $ 36,664 $ 51,329 $ 72,358 Medium 249 / 22,619 Profit $ (12,630) $ 2,036 $ 23,065 Sales $ 51,329 $ 71,861 $ 101,302 Production (lb) High2 348 / 31,666 Profit $ 2,036 $ 22,568 $ 52,008 1Low represent 40% less of both medium prices and volumes. 2High represent 40% more of both medium prices and volumes. 3Extract and dried skins productions are displayed vertically in the table. 4Prices for extract and dried skins are displayed horizontally in the table. 5Sales are shown for all 9 different supposed scenarios. 6Profits are shown for all 9 different supposed scenarios. 7Number in parenthesis indicate profit loss. When an evaporator is proposed as the isolati on method (Table 4-10), there were losses in the low-low and medium-low combinations of pr oduction and prices. Th e remaining situations showed positive numbers but only the medium-h igh and high-high combinations showed fivedigit figures of profit. At th e original circumstances (medium price and volume), resulted in only $2,000 are registered in profit per season. As mentioned before, extract production is likely

PAGE 89

89 to increase if process is optimized and therefor e this isolation method is also profitable and convenient. Vacuum drying, due to the characteristics of the equipment quoted, resulted in a low IRR (12.3%) and NPV of 188,627 (Table 4-11). A lthough the NPV calculated for this operation was higher than the last two, the investment de manded most of the money earned in a 10 yearoperation period. However as mentioned earlier, th is process did generate profit and had room for expansion if the process was successfully implemented by a muscadine processor. The machine at the early stages of the operation is going to be sub-utilized while spray dryer and freeze dryer quoted for previous ope rations are going to be working at almost full capacity. With the actual liquid extract volume, the vacuum ev aporator worked less than 4 hours while spray dryer and freeze dryer were working 12 hours and 16 hours respectively.

PAGE 90

90Table 4-11. Cash flow analysis for a ma rginal process obtaining extract and dried skins from muscadine grape skins using vacuu m drying as the isolation technique. Periods (years) 0 1 2 3 4 5 6 7 8 9 10 Investment $186,640 Income Extract $ 17,401 $ 17,998 $ 18,615 $ 19,254 $ 19,914 $ 20,597 $ 21,304 $ 22,035 $ 22,790 $ 23,572 Residue $ 33,928 $ 35,092 $ 36,296 $ 37,540 $ 38,828 $ 40,160 $ 41,537 $ 42,962 $ 44,436 $ 45,960 Expenses Labor $ 7,168 $ 7,412 $ 7,664 $ 7,924 $ 8,194 $ 8,472 $ 8,760 $ 9,058 $ 9,366 $ 9,685 Materials $ 3,341 $ 3,455 $ 3,572 $ 3,694 $ 3,819 $ 3,949 $ 4,083 $ 4,222 $ 4,366 $ 4 ,514 General costs $ 1,357 $ 1,403 $ 1,451 $ 1,500 $ 1,551 $ 1,604 $ 1,659 $ 1,715 $ 1,773 $ 1,834 Depreciation $ 26,789 $ 26,789 $ 26,789 $ 26,789 $ 26,789 $ 4,424 $ 4,424 $ 4,424 $ 4,424 $ 4,4 24 Interest (7.5%) $ 998 $ 998 $ 893 $ 788 $ 683 $ 578 $ 473 $ 368 $ 263 $ 158 Gross Income $ 11,677 $ 13,139 $ 14,648 $ 16,205 $ 17,812 $ 41,836 $ 43,548 $ 45,315 $ 47,140 $ 49,023 Tax (20%) $ 2,335 $ 2,628 $ 2,930 $ 3,241 $ 3,562 $ 8,367 $ 8,710 $ 9,063 $ 9,428 $ 9 ,805 Utilities after Taxes $ 9,342 $ 10,511 $ 11,718 $ 12,964 $ 14,250 $ 33,468 $ 34,838 $ 36,252 $ 37,712 $ 39,219 (+) Depreciation $ 26,789 $ 26,789 $ 26,789 $ 26,789 $ 26,789 $ 4,424 $ 4,424 $ 4,424 $ 4,424 $ 4,424 Amortization $ 6,528 $ 6,633 $ 6,738 $ 6,843 $ 6,948 $ 7,053 $ 7,158 $ 7,263 $ 7,368 $ 7,473 Salvage Value $ 12,425 Net Income $ 29,603 $ 30,668 $ 31,770 $ 32,910 $ 46,516 $ 30,840 $ 32,105 $ 33,414 $ 34,768 $ 36,170 Net Present Value (NPV) $188,627 rate 12.00% Intern Rate of Return (IRR) 12.3%

PAGE 91

91 4.4 Conclusions Aerobic fermentation following a simple concentration step was an inexpensive way to obtain polyphenolics from muscad ine grape skins since most of the investment was focused in the latter procedure (iso lation). Results from three isola tion techniques of polyphenolic from muscadine grape skins suggested that this addi tional operation can be suitable and profitable for a typical muscadine producer. Further inves tigation is needed to optimize the polyphenolic concentration procedure prior to fermentation to increment the extract produced and, consequently increase profit. Using initial production conditions (medium price and volume), the profit generated for each of the techniques (i n order from most to least) was freeze drying > spray drying > vacuum drying. In contrast, the vacuum evaporator used as an example for this chapter had three times th e capacity of the spray dryer and fo ur times the capacity of the spray dryer, thus only the evaporat or could support a growth in ex tract production if the volume processed is increased significan tly. Another advantage of vac uum evaporation for a muscadine grape processor is the ve rsatility to obtain both powder and/or concentrate product depending on the purchaser requirements whereas spray dryi ng and freeze drying will produce only a powder limiting the market for the isolated extract. Moreover, to implement a freeze drying line, refrigeration is needed to freeze the liquid extract before it is submitted to drying. This chapter illustrated various assumptions that could be changed to recalculate and to adjust the investment and costs of any processor. Due to limited info rmation regarding equipments, this chapter only contained general information for engineeri ng and equipment specifications, thus, many specifications can be added to predict economic information more accurately. Furthermore, this process could be adjusted and implemented for by -products from various fruits and vegetables sources to estimate the increase in profit of their industries.

PAGE 92

92 CHAPTER 5 SUMMARY AND CONCLUSIONS Interest in by-product utilization has encour aged fruit and vegetable processors to investigate ways to efficiently and economically add value to otherwise low-benefit processing residues. For muscadine grapes, the potential ex ists to extract valuable compounds that remain in the by-product and bring these novel products to a revenue-gene rating market. In parallel, interest in natural antioxidants has driven the production of nutraceuticals from fruit and vegetable sources. Prior studies have focused on extracting polyphenolics from diverse fruit and vegetable sources, but the presen t studies are the first to expl ore the potential economic and processing enviroment impact on polyphenolic conten t and stability. The st udies illustrate the ability to extract polyphenolics from muscad ine grape pomace and measuring the effect of various processes on chemical composition, chemical reactivity and oxidative and thermal stability. Furthermore, these studies tried to illustrate the feasibility of obtaining by-product isolates rich in polyphenolics from muscadine gr ape pomace. Such feasibility was determined by simple economic indicators, using logical assu mptions and the best economic predictors for success of a by-product extraction venue. Polyphenolics extracted from muscadine gr ape pomace showed antioxidant activity comparable to fruits and vegetables such as cherries, strawberries, and red cabbage. Fermentation was better than solid phase isolation in getting rid of residual sugars with minimal damage to the chemical profile and antioxidant ac tivity of the final product, as well as showing more practicality on a larger scale. Vacuum drying proved to be the best treatment following fermentation since it best maintained polypheno lic content and preser ved the antioxidant capacity. Initial concentration followed by aerob ic fermentation was an inexpensive way to obtain polyphenolics from muscad ine grape skins. Isolation of polyphenolics from muscadine

PAGE 93

93 grape pomace can be suitable and profitable for a typical muscadine juice producer if their circumstances are somewhat similar with the ones indicted in these studies. Assumptions can be adjusted to any fruit or vegetable processor s ituation for more accurate calculations. Further research is needed to support the work shown in these studies. Investigations need to be focused on the optimization of polyphenolic concentration prior to fermentation, the impact of processing on phytochemical characteristics at an industrial scale, and more detailed economic evaluation.

PAGE 94

94 APPENDIX A PRELIMINARY STUDY I 0 500 1000 1500 2000 2500 0123 DaysCyanidin-3-glucoside equivalents (mg/kg) 1/10x 1/5x 1/2x 1/1x Figure A-1. Total anthocyanins dur ing a 3-day extraction procedure. 0 2 4 6 8 10 12 0123 DaysoBrix 1/10x 1/5x 1/2x 1/1x Figure A-2. oBrix values during a 3-da y concentration procedure.

PAGE 95

95 APPENDIX B PRELIMINARY STUDY II 1,472.44 1,183.38 667.10 0 200 400 600 800 1,000 1,200 1,400 1,600 1/5x1/2x1/1x Concentration ratio (skin/water)Cyanidin-3-glucoside equivalents (mg/kg ) Figure B-1. Total anthocyanin conten t in a 1-day extraction procedure. 6.2 9.5 3.4 0 1 2 3 4 5 6 7 8 9 10 1/5x1/2x1/1x Concentration ratio (skin/water)oBrix Figure B-2. oBrix values in a 1-da y extraction procedure.

PAGE 96

96 APPENDIX C PRELIMINARY STUDY III 0 200 400 600 800 1000 1200 1400 0306090120150180210240270300330 MinutesCyanidin-3-glucoside equivalents (mg/kg) Figure C-1. Total anthocyanin content after a 5-hour concentration procedure.

PAGE 97

97 LIST OF REFERENCES Aaby K, Skrede G, Wrolstad R. 2005. Phenolic composition and antioxidant activities in flesh achenes of strawberries ( Fragaria ananassa ). J Agric Food Chem 53:4032-40. Abascal K, Ganora L, Yarnell E. 2005. The effect of freeze-dryi ng and its implications for botanical medicine: a review Phytother Res 19:655-60. Amakura Y, Umino, Y, Tsuji S, Tonogai Y. 2000. Influence of jam processing on the radical scavenging activity and phenolic content in berries. J Aric Food Chem 48:6292-97. Andrade I, Flores H. 2004. Optimizati on of spray drying of roselle extract ( Hibiscus sabdarrifa L.). Proceedings of the 14th International Drying Sympos ium A: 597-604. So Paulo, Brazil. Atkinson CJ, Nestby R, Ford YY, Dodds PAA. 2005. Enhancing th e beneficial antioxidants in fruits: a plant physiological pe rspective. Biofactors 23:229-34. Barbosa-Cnovas GV, Ortega-Rivas E, Juliano P, Yan H. 2005. Drying. In: Food Powders, Physical Properties, Processing, and Functionality. New York, NY. Kluwer Academic/Plenum Publishers. p 271-304. Bonilla F, Mayen M, Merida J, Medina M. 1999. Extraction of phenolic compounds from red grape marc for use as food lipid antioxidants. Food Chem 66:209-15. Brenes CH, Del Pozo-Insfran, D, Talcott, ST. 2005. Stability of copigmented anthocyanins and ascorbic in a grape juice model sy stem. J Agric Food Chem 53:49-56. Bridle P, Timberlake C. 1997. Anthocyanins as food colors, selected aspects. Food Chem 58(12):103-9. Brouillard R, Mazza G, Saad Z, Albrecht-Ga ry AM, Cheminat A. 1989. The copigmentation reaction of anthocyanins: a microprobe for th e structural study of aqueous solutions. J Am Chem Soc 111:2604-10. Bruneton J. 1995. Pharmacognosy, Phytochemistry, Medical Plants. Tec & Doc-Lavoisier, Paris. Buettner GR. 1993. The pecking order of free ra dicals and antioxidants: lipid peroxidation, alpha-tocopherol, and ascorbate. Archives of biochemistry and biophysics 300(2):535-43. Cabrita L, Andersen M. 1999. Anthocyanins in blue berries of Vaccinium padifolium Phytochem 52:1693-96. Cao G, Wang G, Prior R. 1996. Total antioxidant capacity of fruits. J Agric Food Chem 44:7015. Clifford MN. 2000. Anthocyanins: nature, occu rrence and dietary burden. J Sci Food Agric 80:1063-72.

PAGE 98

98 Clifford MN, Scalbert A. 2000. Ellagitannins-nature, occurrence and dietary burde n. J. Sci Food Agric 80:1118-25. Cline B, Fisk C. 2006. Overview of muscad ine acreage, cultivars and production areas in southeastern US. Plant Pathology and Hort icultural Science. North Carolina State University. Crocker TE, Mortensen JA. 2001 The Muscadine Grape. Extension. Institute of Food and Agricultural Sciences. University of Florida. Croft KD. 1999. Antioxidant effects of plant phenolic compounds. In: Basu TK, Temple NJ, Garg ML editors. Antioxidant in Human Hea lth and Disease. New York NY. CABi Pub. p 109-22. Dao LT, Takeoka GR, Edwards RH, Berrios JDJ. 1998. Improved method for the stabilization of anthocyanidins. J Agric Food Chem 46:3564-9. De Bruyne T, Pieters L, Deelstra H, Vlie tinck A. 1999. Condensed vegetable tannins: biodiversity in structure a nd biological activities. Bi ochem Syst Ecol 27:445-59. Degner RL, Rodan LW, Mathis K. 1981. Farmer to Consumer, direct marketing of grapes in Florida: producer and consumer benefits. Fl orida Agricultural Market Research Center. Institute of Food and Agricultural Sciences. University of Florida. Del Pozo-Insfran D. 2006. Emerging technologi es and strategies to enhance anthocyanin stability. Dissertation. University of Florida Del Pozo-Insfran D, Brenes C, Talcott S. 2004. Phytochemical composition and pigment stability of aai ( Euterpe oleracea Mart.). J Agric Food Chem 52:1539-45. Dillard CJ, German JB. 2000. Re view: Phytochemicals: nutraceutic als and human health. J Sci Food Agric 80 (12):1744-56. Ector BJ. 2001. Compositional and nutritional char acteristics. In: Basiouny FM Himelrick DG, editors. Muscadine Grapes. Alexandria VA.: ASHS ASHS Press. p 341-67. Erlund I. 2004. Review of the fl avonoids quercetin, hesperetin, and naringenin. Dietary sources, bioactivities, bioavailability, and epidem iology. Nutrition Research 24:851-74. Fang Z, Zhang M, Sun Y, Sun J. 2006. How to improve bayberry ( Myrica rubra Sieb. et Zucc.) juice color quality: effect of juice pr ocessing on bay berry anthocyanins and polyphenolics. J Agric Food Chem 54:99-106. Feldman KS, Sambandam A.1995. Ellagitannin Chemis try. The first total chemical synthesis of an O (2), O (3)-galloyl-coupled ellagitannin, Sa nguiin H-5. J Prg Chem 60:8171-8. Fender L. 2005. Phytochemical, antioxidant, and st orage stability of ther mally processed guava ( Psidium guajava ) and guava juice blends. Thes is. University of Florida.

PAGE 99

99 Francis FJ. 1989. Food colorants: anthoc yanins. Crit Rev Food Sci Nutr 28:273-314. Fritz JS, Willis RB. 1973. Chromatography sepa ration of phenols using an acrylic resin. J Chromatography 79:107-19. Furtado P, Figueiredo P, Chaves das Neves H, Pina F. 1993. Photochemical and thermal degradation of anthocyanidins. J Photochem Photobiol A 75:113-8. Garzn GA, Wrolstad RE. 2001. The stability of pelargonidin-based ant hocyanins at varying water activity. Food Chem 75 185-96. Garzn GA, Wrolstad RE. 2002. Comparison of the stability of pelargonidi n-based anthocyanins in strawberry juice and concentrate. J ournal of Food Science 67:(4) 1289-99. Gil MI, Toms-Barbern FA, Hess-Pierce B, Holcroft DM, Kader AA. 2000. Antioxidant activity of pomegranate jui ce and its relationship w ith phenolic composition and processing. J Agric Food Chem 48:4581-9. Giusti MM, Wrolstad RE. 2003. Acylated anthocyanins from edible sources and their applications in food system s. Biochem Eng J 14:217-25. Gregory III JF. 1996. Vitamins. Chapter 8 in Food Chemistry 3rd Edition, Marcel Dekker, Inc. New York NY. Hkkinen SH, Krenlampi SO, Mykknen HM, Heinonen IM, Trrnen AR. 2000. Ellagic acid content in berries: Influence of domestic processing and storage. Eur Food Res Technol 212:75-80. Halbrooks MC. 1998. Alternativ e opportunities for small farms: muscadine grape production review. Cooperative Extension Service. Inst itute of Food and Agricultural Sciences. University of Florida. Helm RF, Zhentian L, Ranatunga T, Jervis J, Elder T. 1999. Toward understanding monomeric ellagitannin byosynthesis. In Plant Polyphenol ics 2: chemistry, biology, pharmacology, ecology. Academic/Plenum Publishers, New York, NY. Himelrick DG. 2003. Handling, storage, and pos tharvest physiology of muscadine grapes: a review. Small Fruits Review 2(4):45. Hradzina G, Borzell AJ, Robi nson WB. 1970. Studies on the stab ility of the anthocyanin-3,5diglucosides. Am J Enol Vitic 21:201 Hsu CL, Chen W, Weng YM, Tseng CY. 2003. Ch emical composition, physical properties, and antioxidant activities of yam flours as affected by differe nt drying methods. Food Chem 83:85-92. Huang HT. 1955. Decolorization of anthocyani ns by fungal enzymes. J Agric Food Chem 3:141-6.

PAGE 100

100 Jaworski AW, Lee CY. 1987. Fractionation and HPLC determination of grape phenolics. J Agric Food Chem 35:257-9. Jurd L, Asen S. 1966. The formation of meta l and co-pigment complexes of cyanidin 3glucoside. Phytochem 5:1263-71. Kader F, Haluk JP, Nicolas JP, Metche M. 1998. Degradation of cyan idin-3-glucoside by blueberry polyphenol oxidase: kinetic stud ies and mechanisms. J Agric food Chem 46:3060-5. Kader F, Rovel B, Girardin M, Metche M. 1997. Mechanism of br owning in fresh highbush blueberry fruit (Vaccinium corymbosum L). Pa rtial purification and characterization of blueberry polyphenol oxidase. J Sci Food Agric 73:513-6. Kammerer DR, Schillmller S, Maier O, Schieber A, Carle R. 2007. Colour stability of canned strawberries using black carrot and elderberry juice concentr ates as natural colourants. Eur Food Res Technol 224:667-9. Khanbabaee K, Ree TV. 2001. Tannins: classifi cation and definition. Nat Prod Rep 18:641-9. Kirca A, Ozkan M, Cemeroglu B. 2006. Stability of black carrot anthocya nins in various fruit juices and nectars. Food Chem 97:598-605. Klopotek Y, Otto K, Bohm V. 2005. Processing stra wberries to different pr oducts alters contents of vitamin C, total phenolics, total anthocya nins and antioxidant capacity. J Agric Food Chem 53:5640-6. Kong J, Chia L, Goh N, Chia T, Brouillard R. 2003. Analysis and biological activities of anthocyanins. Phytochem 64:923-33. Kraemer-Schafhalter A, Fuchs H, Pfannhauser W. 1998. Solid-phase extraction (SPE) a comparison of 16 materials for the pur ification of anthocyanins from Aronia melanocarpa var Nero. J Sci Food Agric 78:435-40. Kraus TEC, Dahlgren RA, Zasoski RJ. 2003. Tannins in nutrient dynamics of forest ecosystemsa review. Plant and Soil 256:41-66. Lalaguna F. 1993 Purification of fresh cassava ro ot polyphenolics by solid-phase extraction with Amberlite XAD-8 resin. J Chromatography 657:445-9. Laleh GH, Frydoonfar H, Heidary R, Jameei R, Zare S. 2006. The effect of light, temperature, pH and species on stability of anthocyanin pigments in four Berberis species. Pakistan Journal of Nutrition. 5(1):90-2. Laurila E, Ahvenainen R. 2002. Minimal proce ssing in practice. In: Ohlsson T, Bengtsson N, editors. Minimal processing technologies in the food industry. Boca Raton, FL. CRC Press. p 219-44.

PAGE 101

101 Lee JH, Talcott ST. 2004. Fruit maturity and jui ce extraction influences ellagic acid derivatives and other antioxidant polyphenolics in mus cadine grapes. J Agric Food Chem 52:361-6. Lee JH. 2004. Hydrolytic and antioxidant propertie s of ellagic acid and its precursors present in muscadine grape. Dissertation. University of Florida. Lee JH, Johnson JV, Talcott ST. 2005. Identification of ellagi c acid conjugates and other polyphenolics in muscadine grapes by HPLC -ESI-MS. J Agric Food Chem 53:6003-10. Lei Z. 2002. Monomeric ellagitannins in oaks an d sweetgum. Dissertation. Virginia Polytechnic Institute and State University. Le Marchand L. 2002. Cancer preventive effects of flavonoidsa review. Biomed Pharmacother 56:296-301. Lindsay RC. 1996. Food Additives. In: Fenemma, OR editor. Food Chemistry 3rd Edition. New York NY. Marcel Dekker, Inc. p 767-824. Makris DP, Boskou G, Andrikopoul os NK. 2007. Polyphenolic cont ent and in vitro antioxidant characteristics of wine industry and other ag ri-food solid waste extrac ts. Journal of Food Composition and Analysis 20:125-32. Marais JPJ, Deavours B, Dixon RA, Ferreira D. 2006. The stereo chemistry of flavonoids. In: Grotewold E, editor. The Science of Fl avonoids. New York, NY. Springer. p 1-46. Markakis P. 1982. Stability of anthocyanins in foods. In: Anthocyanins as Food Colors. New York, NY. Academic Press, Inc. p 163-80. Marti N, Perez-Vicente A, Garcia-Viguera C. 2001. Influence of storage temperature and ascorbic acid addition on pomegran ate juice. J Sci Food Agric 82:217-21. McRae TG, Gregson RP, Quinn RJ. 1982. Amberl ite XAD-7 as a chromatographic absorbent. Journal Chromatographic Science 20:475-9. Meyer AS, Suhr KI, Nielsen P, Lyngby, Holm F. 2002. Natural food preservatives. In: Ohlsson T, Bengtsson N, editors. Minimal processing technologies in the food industry. Boca Raton, FL. CRC Press. p 124-76. Meyers KJ, Swiecki TJ, Mitchell AE. 2006. Understanding the native californian diet: identification of condensed hydrolazable tannins in Tanoak Acorns ( Lithocarpus densiflorus ). J Agric Food Chem 54:7686-91. Miller NJ, Ruiz-Larrea MB. 2002. Flavonoids a nd other plant phenols in the diet: their significance as antioxidants. Journal of Nutr itional & Enviromental Medicine 12:39-51. Milo L. 2005. Nutraceuticals & Functi onal Foods. Food Technology 59(5):65-7.

PAGE 102

102 Mizell R, Andersen P, Tipping C, Brodbeck B. 2003. Xylella fastidiosa diseases and their leafhopper vectors. Institute of Food and Agri cultural Sciences (IFAS). University of Florida. Monagas M, Hernndez-Ledesma B, Gmez-Cordov s C, Bartolom B. 2006. Comercial dietary ingredients from Vitis vinifera L. leaves and grape skins: Antioxidant and Chemical Characterization. J Agric Food Chem 54:319-27. Morata A, Gmez-Cordovs MC, Caldern F, Sur ez JA. 2006. Effects of temperature, pH and SO2 on the formation of pyranoanthocyanins during red wine fermentation with two species of Saccharomyces Int J Food Micro 106:123-9. Morris JR, Brady PL. 2004. The muscadine expe rience: Adding value to enhance profits. Arkansas Agricultural Experiment Station. In stitute of Food Science and Engineering. University of Arkansas. Moure A, Cruz JM, Franco D, Domnguez JM, Si neiro J, Domnguez H, Nez MJ, Paraj JC. 2001. Natural antioxidants from resi dual sources. Food Chem 72:145-71. Mullen W, Stewart AJ, Lean MEJ, Gardner P, Du thie GG, Crozier A. 2002. Effect of freezing and storage on the phenolics, ellagitannins, flavonoids, and antioxidant capacity of red raspberries. J Agric Food Chem 50:5197-201. Mullen W, Yokota T, Lean ME, Cr ozier A. 2003. Analysis of e llagitannins and conjugates of ellagic acid and quercetin in raspberry fruits by LC-MSn. Phytochemistry 64:617-24. Orsat V, Raghavan GSV. 2006. Dehydration technologi es to retain bioactive components. In: Shi J, editor. Functional Food Ingredients and Nutraceuticals: Processing and Technologies. Boca Raton, FL. CRC Taylor & Francis Group. p 173-192. Oszmianski J, Ramos T, Bourzeix M. 1988. Fr actionation of phenolic compounds in red wine. Am J Enol Vitic 39(3):260-2. Pacheco LA. 2006. Phytochemical, antioxida nt and color stab ility of Aai ( Euterpe oleracea Mart.) as affected by processing and storage in juice model systems. Thesis. University of Florida. Parr A, Bowell G. 2000. Review: Phenols in the plant and in man. The potential for possible nutritional enhancement of th e diet by modifying the phenol content or profile. J Sci Food Agric 80:985-1012. Pastrana-Bonilla E, Akoh C, Sellapan S, Krewer G. 2003. Phenolic content and antioxidant capacity of muscadine grapes. J Agric Food Chem 51:5497-503. Peschel W, Snchez-Rabaneda F, Diekmann W, Plescher A, Gartzia I, Jimenez D, LamuelaRaventos R, Buxaderas S, Codina C. 2006. An industrial approach in the search of natural antioxidants from vegetable a nd fruit wastes. Food Chem 97(1):137-50.

PAGE 103

103 Pflug IJ, Esselen WB. 1979. Heat sterilization of canned food. In: Jackson JM, Shinn BM, editors. Fundamentals of Food Canning T echnology. Westport, CT. AVI Publishing Company, Inc. p 10-94. Phillips RD. 2006. Pilot-scale, pre-commerci al production of nutraceuticals from Georgia commodities. In Fiscal Year 2005-2006 Repor t to industry. Georgias Traditional Industries Program for Food Processing. F ood Processing Advisory Council. p 10. Pietrzyk DJ, Chu CH. 1977a. Amberlite XAD copolym ers in reversed phase gravity flow and high pressure liquid chromatogra phy. Anal Chem 49(6):757-64. Pietrzyk DJ, Chu CH. 1977b. Separation of or ganic acids on amberlite XAD copolymers by reversed phase high pressure liquid ch romatography. Anal Chem 49(6):860-7. Pietta PG. 2000. Flavonoids as an tioxidants. J Nat Prod 63:1035-42. Poling EB. 1996. Muscadine grapes in the home ga rden. Department of Horticultural Science. North Carolina Cooperative Extension Serv ice. North Carolina State University. Rababah TM, Ereifej KI, Howard L. 2005. Eff ect of ascorbic acid and dehydration on concentration of total phenolic s, antioxidant capacity, anthocya nins, and color in fruits. J Agric Food Chem 53:4444-7. Rein MJ, Heinonen M. 2004. Stability and enhan cement of berry juice co lor. J Agric Food Chem 52:3106-14. Rein MJ. 2005. Copigmentation reactions and color stability of berry ant hocyanins. Dissertation. University of Helsinki. Robbins R. 2003. Phenolic acids in foods: An ov erview of analytical methodology. J Agric Food Chem 51:2666-87. Rodriguez-Saona LE, Guisti MM, Wrolstad RE. 1999. Color and pigment stability of red radish and red fleshed potato anthoc yanins in juice model systems. J Food Sci 64:451-6. Rommel A, Wrolstad RE. 1993. Ella gic acid content of raspberry juice as in fluenced by cultivar processing, and enviromental fact ors. J Agric Food Chem 41:1951-60. Ruel J, Walker A. 2006. Resist ance to Pierces disease in Muscadinia rotundifolia and other native grape species. Am J Enol Vitic 57:158-66. Saito N, Tatsuzawa F, Yoda K, Yokoi M, Kasaha ra K, Iida S, Shigihara A, Honda T. 1995. Acylated cyanidin glycosides in the violet-blue flowers of Ipomoea purpurea Phytochem 40(4):1283-9. Schmidt BM, Erdman JW, Lila MA. 2005. Effects of food processing on blueberry antiproliferation and antioxidant capacity. Journa l of Food Science 70:389-94.

PAGE 104

104 Shahidi F, Naczk M. 2003. Phenolics in Food and Nutraceuticals. Boca Raton, FL. CRC Press p 558. Shi B, He Q, Yao K, Huang W, Li Q. 2005. Pr oduction of ellagic acid from degradation of valonea tannins by Aspergillus niger and Candida utilis J Chem Technol Biotechnol 80:1154-9. Sims CA. Morris JR. 1985. pH eff ects on the color of wine from two grape species. Ark Farm Res 34(2):9. Singleton VL, Rossi J. 1965. Colorimetry of total phenolics with phosphomolybdicphosphotungstic acid reagents. Am J Enol Vit 16: 144-53. Skrede G, Wrolstad RE. 2000. Flavonoids from berries and grapes. In: Mazza G, editor. Functional Foods: Biochemical and Processing Aspects. Lancaster, PA. Technomic Pub. p 71-134. Sort X. 2003. Enviromental problem of agro industrial spills. Enviromental Management. Electron. J Environ Agric Food Chem 2:205-7. Stintzing F, Stintzing A, Carle R, Frei B, Wrol stad R. 2002. Color and antioxidant properties of cyanidin-based anthocyanin pigmen ts. J Agric Food Chem, 50: 6172-81. Takamura H, Yamaguchi T, Terao J, Matoba T. 2002. Change in radical-scavenging activity of spices and vegetables during cooking. In: Lee TC, Ho CT, editors. Bioactive Compounds in Foods: Effects of Processing and Storag e. Washington, DC. American Chemical Society. p 34-43. Takeda F, Saunders MS, Saunders JA. 1983. Physic al and chemical changes in muscadine grape during postharvest storage Vitis rotundifolia Am J Enol Vit 34(3):180-5. Talcott ST, Lee JH. 2002. Ellagic acid and fla vonoid antioxidant content of muscadine wine and juice. J Agric Food Chem 50:3186-92. Talcott ST, Brenes CH, Pires DM, Del Pozo-Insfra n D. 2003. Phytochemical stability and color retention of copigmented and processed mu scadine grape juice. J Agric Food Chem 51:957-63. Tambunan AH, Yudistira, Kisdiyani, Hernani. 2001. Freeze drying charac teristics of medical herbs. Drying Tech 19(2):325-31. Toms-Barbern FA, Clifford MN. 2000. Diet ary hydroxybenzoic acid derivatives-nature, occurrence and dietary burden J Sci Food Agric 80:1024-32. Torreggiani D, Bertolo G. 2001. Osmotic pre-treat ments in fruit processing: chemical, physical and structural effects. J Food Eng 49:247-53.

PAGE 105

105 Tsai PJ, Delva L, Yu TY, Huang YT, Dufoss L. 2005. Effect of sucrose on the anthocyanin capacity of mulberry extrac t during high temperature heat ing. Food Res Int 38:1059-65. Turker N, Aksay S, Ekiz I. 2004. Effect of stor age temperature on the stab ility of anthocyanins of a fermented black carrot (Daucus carota.) beverage: Shalgam. J Agric Food Chem 52: 3807-13. Van Golde PH, Van der Westelaken M, Bouma BN Van de Wiel A. 2004. Characteristics of piraltin, a polyphenolc concentrate, produced by freeze-drying of red wine. Life Sci 74:1159-66. Visioli F, Romani A, Mulinacci N, Zarini S, Conte D, Vincieri FF, Galli C. 1999. Antioxidant and other biological activities of olive mill waste waters. J Agric Food Chem 47:3397401. Wang SY. 2006. Fruits with high antioxidant activity as functi onal foods. In: Shi J, editor. Functional Food Ingredients and Nutraceutic als: Processing and Technologies. Boca Raton, FL. CRC Taylor & Francis Group. p 371-414. Weinert IAG, Solms J, Escher F. 1990. Polyme rization of anthocyanins during processing and storage of canned plums. Lebensm-Wiss u Technol 23:445-50. Winkel BSJ. 2006. The biosynthesis of flavonoids In: Grotewold E, editor. The Science of Flavonoids. New York, NY. Springer. p 71-96. Whitaker JR. 1994. Principles of en zymology for the food sciences 2nd edition. Ney York, NY. Marcel Dekker. p 625. Wrolstad RE. 1976. Color and pigment analys is in fruit products. Oregon Agricultural Experimental Station Corvallis. Bulletin 624. Wu H, Haig T, Prately J, Lemerle D, An M. 2000. Allelochemicals in wheat ( Triticum aesvestum L.): Variation of phenolic acids in ro ot tissues. J Agric Food Chem 48:5321-5. Wu X, Beecher GR, Holden JM, Haytowitz DB Gebhardt SE, Prior RL. 2004. Lipophillic and Hydrophillic Antioxidant Cap acities of Common Food in the United States. J Agric Food Chem 52:4026-37. Yi W, Fischer J, Akoh CC. 2005. Study of anti cancer activities of mu scadine grape phenolics in vitro J Agric Food Chem 53:8804-12. Yi W, Akoh, CC, Fischer J, Kr ewer G. 2006. Effect of phenolic compounds in blueberries and muscadine grapes on HepG2 cell viability and apoptosis. Food Res Int 39:628-38. Zafrilla P, Ferreres F, Toms-Barbern FA. 2001. Effects of processing and storage on the antioxidant ellagic acid derivatives and flavonoids of red raspberry ( Rubus idaeus ) jams. J Agric Food Chem 49:3651-5.

PAGE 106

106 BIOGRAPHICAL SKETCH Jorge Cardona was born on October 13, 1983, in Cochabamba, Bolivia. Before graduating from high school in 2000, he traveled to Devon, PA as an exchange student for 11 months with AFS exchange program. He went back to Bolivia to graduate from high school in 2001. After high school, he entered Zamorano University (E scuale Agrcola Panamericana) in Honduras, Central America to obtain his bach elors degree in agroindustry. After his graduation in 2005, he was offered an assistantship to pursue his grad uate education at the Food Science and Human Nutrition Department at University of Florida, under the supervision of Dr. Stephen Talcott and Dr. Charles Sims. In August 2007, he earned a Master of Scie nce in Food Science and Human Nutrition and will continue his studies towards a doctoral degree.


xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E20101114_AAAADQ INGEST_TIME 2010-11-14T12:29:39Z PACKAGE UFE0021495_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 2355 DFID F20101114_AABIHF ORIGIN DEPOSITOR PATH cardonaponce_j_Page_088.txt GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
662ff6fae553307edef0c413d9657c28
SHA-1
874f5836aff2010ee9770f00c78424dec1a59554
2174 F20101114_AABIGQ cardonaponce_j_Page_001thm.jpg
7ea41a5282133a94380e77baab648845
613ed7f2ff05497e1b9579f2e70ac805f5b135ab
2090 F20101114_AABIHG cardonaponce_j_Page_057.txt
0155b88d039804c0b033747b0a891d7a
bd005c3f950a94b10d17d1f01279a22d65b204d7
29784 F20101114_AABIGR cardonaponce_j_Page_041.QC.jpg
556405f3e24b434ac9f361e1c0702e40
5a56bb2b0e8fe8bcba8c7eeae5fee0621b84eb83
4072 F20101114_AABIGS cardonaponce_j_Page_079thm.jpg
be7976f0b29c61699353d37e1ea4e417
1f3f83833e511df9637539b63c32ae0aa46f0d59
1051913 F20101114_AABIHH cardonaponce_j_Page_009.jp2
9e0c56523fbe1d2bd6bd333d7f3902d2
2d192d5162ffd4fa911c0191859f75b9555cafdc
1053954 F20101114_AABIGT cardonaponce_j_Page_080.tif
ff807c2405486d87e73bf47274da942c
6866e4a630cb4b2097f5ce2bdb0fe28600f64271
72816 F20101114_AABIHI cardonaponce_j_Page_004.jp2
6f7a26c59d40eaf1f9cca0c6bfdf9e68
a52bbd5bfd8d4c10e5ff2275dd4fbc843fed2aed
90945 F20101114_AABIGU cardonaponce_j_Page_041.jpg
fc40a0eace4be0d4b6edb195fe53ceff
37c5e4edb2718ff25ffcf1270d03122a6a42cb52
58695 F20101114_AABIHJ cardonaponce_j_Page_051.pro
c5504f5da625411cb4c5b324a5e9a56c
b1722cf16ba29413726b8d7ccb467a4308de1792
2452 F20101114_AABIGV cardonaponce_j_Page_099.txt
fc98c497f68558c7b4a7beefd91ffb54
b677abefc7300a8c765d73f20c8b4a52a3e90e43
118137 F20101114_AABIHK cardonaponce_j_Page_045.jp2
22e125ef8dbb75b9ca87682721e2ef99
9626053eac096bcdcc506c0db09267d23737a966
F20101114_AABIGW cardonaponce_j_Page_004.tif
63ec5edd79296de72345c81cd3317182
6085c5b2a3c14f868f221f6bd5c5bdf63f5c2b4c
26020 F20101114_AABIIA cardonaponce_j_Page_082.pro
465a7be3066950340042654ca0f16a58
54ecb5a7c55354d0fd86b9f304e00bee6a134b3e
8166 F20101114_AABIHL cardonaponce_j_Page_072thm.jpg
1ff1476c4d1d0a06508dac48b2c422e8
a871a0a4d8e48ea36f282b39f902ad08cf7668be
32031 F20101114_AABIGX cardonaponce_j_Page_086.pro
d57b4cd1f32c8cf67c69e76c54db8cee
3a8dbbf9d9e0d2abe897f4b12cd736010c1d6d80
8917 F20101114_AABIIB cardonaponce_j_Page_064thm.jpg
51c7c02a24dfaa71c58bb2c669fcc53e
e27eeed417b9ccef416edf51100b9e978ce481d7
111837 F20101114_AABIHM cardonaponce_j_Page_018.jp2
6a50c17c719e539085a2342bb10377c6
2aa0b2563a4379e4039953290f3163d4c4a3eb12
F20101114_AABIGY cardonaponce_j_Page_073.tif
d047417261ced7474eb73ae55fadca5c
d0da2f55ccef129c776b95bf4cf495ef5b176bce
2766 F20101114_AABIIC cardonaponce_j_Page_007.txt
2167d0098af6ac66cf1c8f230b25ec94
ef4e4bf12b7c5e9dd7f51704d6882de78b7e6af2
55678 F20101114_AABIHN cardonaponce_j_Page_067.pro
c5de1ae22e01c2416038847ce0f6a456
bfbcb50d4a346a2829ae1ad1371d8e4a681dade8
1695 F20101114_AABIGZ cardonaponce_j_Page_076.txt
32a95bb5b9014dfa84cc17aa9c4ea0aa
679abdd90d40a9c1df75b1096ea338404d714990
6365 F20101114_AABIID cardonaponce_j_Page_087thm.jpg
0504e56b011886cb18d88f3eaf361cad
5e167ccf8975772a4c887567c7ff1921be7980a7
8610 F20101114_AABIHO cardonaponce_j_Page_057thm.jpg
e3a5aa5ee7d001cf718e025a142b251f
be5fa05188d316194cc907c3f07c60cba899233f
33324 F20101114_AABIIE cardonaponce_j_Page_027.pro
1f9989722bdd538d7e0307d41cc82dfd
d2b7776c70a42e410c75cf293e429a59e295d3c7
29212 F20101114_AABIHP cardonaponce_j_Page_019.QC.jpg
2fcbc80cd83eb95ea6333702867b5d52
a89b1edf31ae1bc6a74117d8b8d6bdb957c7f37d
53660 F20101114_AABIIF cardonaponce_j_Page_046.pro
7396db0a53ea24516f9ea79292d302fc
48401a8d3ca882e78bb5e0982fa0b16e748b3d69
4261 F20101114_AABIHQ cardonaponce_j_Page_089thm.jpg
8ecddf278308b909e18d3ea772a74e09
95cd963c62cb3712e72c6ef0e4add47513dd9873
447 F20101114_AABIIG cardonaponce_j_Page_095.txt
6ccf539691a50b4317cb523fd14e2bde
8588778003598e1607245bbbf0b8c6942b6ab8ff
46718 F20101114_AABIHR cardonaponce_j_Page_071.pro
13a1e85e4223188b3c73c4c3e78d143e
2d83d9e5b8e37125f1f4c23e935b944123a7140b
35335 F20101114_AABIIH cardonaponce_j_Page_044.QC.jpg
31cadc8154bbe321ccd26cc4431ceee2
0e3048ce64428b341164adc33f38f2620661d1cc
8423998 F20101114_AABIHS cardonaponce_j_Page_082.tif
9a12691357d407901734a248f29997e0
135608022dc991d0d84c93f1d23bb9caf9c40c53
F20101114_AABIHT cardonaponce_j_Page_046.tif
929b4b905cdac8c40a03baeee834d1ab
7bba22079e39badfb5554fcb4d7ebd10db9024d2
12616 F20101114_AABIII cardonaponce_j_Page_014.QC.jpg
e859aedc121479208cba6dc06becf3fd
a031c2f95f79bcc71cb29b271412e84ec53b2006
F20101114_AABIHU cardonaponce_j_Page_106.tif
17cc12756acc9cf053ec9f1b54b0a9f9
a64422e3ab4be00d4607aca831c3afe05112a7d6
F20101114_AABIIJ cardonaponce_j_Page_037.tif
99fcfe477fbeba85fa9796bd1d2a3f22
2077a2b130e9d00904305cbb633638b47abf5cf8
115314 F20101114_AABIHV cardonaponce_j_Page_037.jp2
df1ce6e17b3f5776b0d0d15e152478dd
a2743a4ac114b1f4a24dae51ff6b4a2e666204f9
1051981 F20101114_AABIIK cardonaponce_j_Page_008.jp2
23ab8c26b6388e689faf4713d257a0f6
2fa190f19445181f6862cfb967d00162815d117b
2050 F20101114_AABIHW cardonaponce_j_Page_048.txt
487d3fec0ec33ece96fc00bcd5cef1f8
59282bd0eeae1a81b6884e8d1ed80047846eb5eb
F20101114_AABIIL cardonaponce_j_Page_067.tif
2132cd0501d38ab2c15706191a6061a5
c9474a178629f944bed27c0da9e5244c6b832645
20807 F20101114_AABIHX cardonaponce_j_Page_066.QC.jpg
5d30e774fc5705b0aa337796392e9b5f
6dd30bf23fb612033dd7904bf4b89512c0c4fff8
83194 F20101114_AABIJA cardonaponce_j_Page_039.jpg
7c395951d93c7f5b3856f20d1a3462dc
db491b5bfc03cb527ceaec68443765ff0b204e44
F20101114_AABIIM cardonaponce_j_Page_105.tif
3d8e09b5ecae6328f39f304164958010
265289b3825996372aaafad2ed8348bb3d182431
1734 F20101114_AABIHY cardonaponce_j_Page_041.txt
c08e8cb6badbab82b44d5aaa7bf4e41c
b8b4dd0ce66551480af73d6be07a66eb60a42b90
8850 F20101114_AABIJB cardonaponce_j_Page_036thm.jpg
0c0d8b3c62c7e23dc9d4b47cd6820128
abf4def4c97a64ff03d260cc9f7ad90b309aa8dd
2018 F20101114_AABIIN cardonaponce_j_Page_020.txt
7f3a39fafb8a68ead9bb0005900c5fe5
576d7f76e8d38e9d4a36d21b6906346b2e3095bb
52865 F20101114_AABIHZ cardonaponce_j_Page_015.pro
c1fae9690c7f473a446c3da68b0f8130
517f5e70088f8cb1abca9f1b4864de6fb82f5ab7
103554 F20101114_AABIJC cardonaponce_j_Page_010.jpg
c23f346e0fa2ae03560b4a383da3de8c
69f1f3b8efa17943022c6954e0072f2ecb30314c
92464 F20101114_AABIIO cardonaponce_j_Page_055.jpg
06a4b439e8beb9b205aa5c059abdf089
102664a49e5e7d0fb5badacaf23f6c572ed7caf7
54392 F20101114_AABIJD cardonaponce_j_Page_044.pro
53913681d9504c7b9d901db7be404eb7
ec5e5e3ecb3498498875aa425ab57d2331f80e44
1054428 F20101114_AABIIP cardonaponce_j_Page_084.tif
2c3ca4f5bab4fe509d56c80d4d444756
5406c542f81fc2a11d5c881e61d64f6938e2f996
1371 F20101114_AABIJE cardonaponce_j_Page_002.QC.jpg
bc8a982941a828d77aab8212c34397e3
32ee598e8ffbe0efc2918d1a25c1b5b2e4093f8b
25271604 F20101114_AABIIQ cardonaponce_j_Page_034.tif
7207d27620e7e423505b7281a5039b68
95dd1d6b5ac061775373790ffb0f59361a9aab54
F20101114_AABIJF cardonaponce_j_Page_095.tif
61682bbbf4f4f33d5ac2b2005bc8b19d
8ec6ac397d9135d1fd3de0ec79d90236ae92949f
F20101114_AABIIR cardonaponce_j_Page_031.tif
4e99ebc3dee2c8545266c8024dc189af
4e12bb83f1d884841851a632172fb2cb14020ff4
53160 F20101114_AABIJG cardonaponce_j_Page_042.pro
3ddb25737a222670fdf29087d3b12c72
ff42817d9655530ac76bfc181688e6937f36639c
18797 F20101114_AABIIS cardonaponce_j_Page_080.QC.jpg
b5adec30167abf809e3143a3acef7b17
cb1a05603cf5da8c6fabcb6e93a2655d0d893f0d
1684 F20101114_AABIJH cardonaponce_j_Page_065.txt
8fa59d034ac6c1bcc99add7dcbb1b80d
3138cf26a087724352138500e90788f36eee9590
54101 F20101114_AABIJI cardonaponce_j_Page_084.jpg
0ab93f8588ce6bb27b787985995f5bf8
dcbce39d4609c2dbba0ca7c8ea3d718e5455728e
52303 F20101114_AABIIT cardonaponce_j_Page_074.pro
730d2f91fd9d1d5ff9f6430368e8e18b
14e00e4f419b9c951dc9f40ad8513e52e336909b
25517 F20101114_AABIIU cardonaponce_j_Page_080.pro
e3b6b1162ed4ce6d8ec51a1416adb29e
e3ef6c89962887e4882a61945e7afa02e143e429
41974 F20101114_AABIJJ cardonaponce_j_Page_014.jp2
22f368e668aa788e6b3dc80573383c17
8aa44bb7a4e263509d5e166a38a26160e0be05a8
78993 F20101114_AABIIV cardonaponce_j_Page_011.jp2
ce7a29cb825c3e2ffdd9ff60244cf24a
01d61f56d5bd5437e8a1728cf93062490e00cb3d
F20101114_AABIJK cardonaponce_j_Page_089.tif
a3ccbac981811f6f65632dec8fe602d4
9251c3c669491d897734b3bfc4b6f0839f6f3d2c
38936 F20101114_AABIIW cardonaponce_j_Page_098.QC.jpg
630cc936e0c112e02b0af11c2ccd93e9
a6a229107e89f469b5e0cbe01b1d3365ddc6d1ca
33998 F20101114_AABIKA cardonaponce_j_Page_047.QC.jpg
e7fb37c97bc8a15a956a5613eb26925c
1435032bffad6c24e8dc7f6a8375386cc366cf1d
F20101114_AABIJL cardonaponce_j_Page_054.tif
f633e9d73f8ca72a2f5711d0f39182ab
12a78dcff8c619a969322a609bd85a55d0e34a50
1664 F20101114_AABIIX cardonaponce_j_Page_039.txt
c36346b8e9fd79815944f9b5ca929ee1
d23a00d3a1e5f2355814666a08c1aa3880a8dbee
26525 F20101114_AABIKB cardonaponce_j_Page_001.jpg
8570976fe963d54078ae12b792b076e0
5fd66e20ea1b6a5a6fdd270f35c5e045b736cede
F20101114_AABIJM cardonaponce_j_Page_008.tif
5adc1ef4bcc250cf86cb7d366c72573d
039a91b4c49c576403de7bbdcd8d8043bd920342
998869 F20101114_AABIIY cardonaponce_j_Page_021.jp2
72508d3751e528079de8b6845049722d
98b16e178aeb5a8143cb5de05e98fc4a1b4e9454
100587 F20101114_AABIKC cardonaponce_j_Page_081.jpg
5b7382cc7ead3c76ae5eadd388498df4
d6c60c33c31b6fb6472c9b6a4457e3d6951bcc02
F20101114_AABIJN cardonaponce_j_Page_057.tif
a91a59d7500778069e3ce7cb5ac1a541
23414f0102e95d021098335f637e540621cefa6f
124814 F20101114_AABIIZ cardonaponce_j_Page_061.jpg
855d2a7fcaf1133e4c9e4875571b59b8
2d0aff960bcd4cf5a0fe1e33bebfd5dbf084aa46
1965 F20101114_AABIKD cardonaponce_j_Page_028.txt
54ca9370ea89b20bfdf1b24ca9055ef2
88a26de1d8e49e2eec70775362e31fe7f9a28050
4986 F20101114_AABIJO cardonaponce_j_Page_080thm.jpg
b6c10d8b5139b93ce962e6de10bfbfde
4dd0ae708008a34f63d3c5a316066ddece26592e
77722 F20101114_AABIKE cardonaponce_j_Page_086.jpg
8d63bb90a4e535b405429003971235c5
5a6cdc37409ad177992a0abe716945df5e863128
1545 F20101114_AABIJP cardonaponce_j_Page_075.txt
20cf7f07890225d163670b73683c8cb7
73d1a28b5c5fef050d1f828520bcf81501f4e503
2117 F20101114_AABIKF cardonaponce_j_Page_026.txt
905d71917ab5152f42081e5f95c13bfd
4d3b9dce88006bd45bee5f7fd9455ac6e34dd020
967829 F20101114_AABIJQ cardonaponce_j_Page_025.jp2
e7c8e1043d194915e8a382eabf4bbe7f
4ff6a3c89015613830c882a38f28bc8a8a4b2881
2380 F20101114_AABIKG cardonaponce_j_Page_077.txt
ab72c2beadf1fdcd0e357d91c31dee6f
7e0d13c11e81531e1449058294bdcd22a0eb7f09
900 F20101114_AABIJR cardonaponce_j_Page_106.txt
bf11b02c65254fad6c14dbac667871f4
d07b05e6da1a7318b37d4052e5a3343881120510
118706 F20101114_AABIKH cardonaponce_j_Page_042.jp2
c15fca7a300344eb7fbb2a7b3ff6b473
9cea3c2132604bddf885cd33310e26214af62762
110786 F20101114_AABIJS cardonaponce_j_Page_083.jpg
61a576e98487a5be9a3cc34aff246e91
f0d4def886a3a6b8a42561c6cb98270318f0943a
5674 F20101114_AABIKI cardonaponce_j_Page_096.pro
a2cafde38df5ade517382814fafd737f
846c1ad7bbe70b036b20bb372379a6b5fcc27475
2115 F20101114_AABIJT cardonaponce_j_Page_010.txt
7231d4a2031461011f8bbe2ab0ebe109
ff2b8436dcf598850274ae85452c2614b8df450c
109142 F20101114_AABIKJ cardonaponce_j_Page_036.jpg
5323aba79467e0fc766fddacc07bf707
ffe192dd951be5a658af8fcc1b9c64a4b980268a
2360 F20101114_AABIJU cardonaponce_j_Page_009.txt
5911cd8bf136f39976a5a583b07f7aad
548d10325e7fcaaa19268c1af321bad6698f8631
35566 F20101114_AABIJV cardonaponce_j_Page_030.QC.jpg
0635b30b43b904122cad3378f62e80b4
48b3c22a25432b371b58c04b45ef21c4b035a973
111336 F20101114_AABIKK cardonaponce_j_Page_026.jpg
c9d0d5120175a0258153c86d37ec291f
ece05f627bc37621441039661aa46c761a9311f1
8572 F20101114_AABIJW cardonaponce_j_Page_022thm.jpg
fc9a2a95eba2336dc04a7c6a4fc3b388
3fd270f7cc73c8f15c14f12b9510f77dc486d1e1
2641 F20101114_AABIKL cardonaponce_j_Page_061.txt
dc36db9f813e6b917137aa964cd47385
0c6ac8139abb0a40a345ea368acb9169635153f4
1403 F20101114_AABIJX cardonaponce_j_Page_011.txt
f8844e755ba2249752cde7052e88e799
8015e1a94fe4f87b53f437f2dd37bfe57d3ab4f5
143421 F20101114_AABILA cardonaponce_j_Page_102.jp2
35d7abc368f6c4476b7bc0f529dfa319
a4a96c72c4a4d86354dcc42a48df34482405d5e7
94651 F20101114_AABIKM cardonaponce_j_Page_021.jpg
cda13b4342b2c0bb7ecd197b7db1d77e
ccf7892c8d8e4b064ac7f9c2dd5ad2cd8d51b419
48705 F20101114_AABIJY cardonaponce_j_Page_054.pro
d20d7ce1cbf0c050637078908904ed55
5df50d5a7b3ae17104961a3015e15d3bf645eb22
7790 F20101114_AABILB cardonaponce_j_Page_065thm.jpg
c6a63c2542bcb0725bdb9b7a5906414e
744c3027e4d51b00ba8eedaa116406863eeeb9de
832229 F20101114_AABIKN cardonaponce_j_Page_027.jp2
ffeb19223e107d53451abec84f1d08f0
a9dc24fc6d7e2a95e03ed857b4ef679560241a01
F20101114_AABIJZ cardonaponce_j_Page_085.QC.jpg
df76007bb4342e034f0b43f5edf6fa80
b81c65f0928fef55fbae1b24451aa0809dd8b944
121224 F20101114_AABILC cardonaponce_j_Page_073.jp2
c6b8e8ed8c7e7eaeca19070e71194e4a
752c6c00587301239edccae56cfa3da2c83b34a3
35238 F20101114_AABIKO cardonaponce_j_Page_023.QC.jpg
87bf8b7f0b0e7f672851f749266dcc37
9b898452b4c8c2d76838d355f23db1f7e606f48e
34769 F20101114_AABILD cardonaponce_j_Page_040.QC.jpg
6256f8eab7431ba251ecdc01febf63de
edf61e085f500a7ea4afda4ce128a3310c80b8fd
90260 F20101114_AABIKP cardonaponce_j_Page_019.jpg
f74049a3f97ca34420bb1429063d7f91
1aefd626f29013d2e564b745343bcdcfb53abc6a
2068 F20101114_AABILE cardonaponce_j_Page_013.txt
ccd425af9663cbd50dfd969adf468c50
dd7927d57b7ed13e0994696ec73fb948e892fa4a
109769 F20101114_AABIKQ cardonaponce_j_Page_048.jpg
724ab3ac1ee2a39077300d64293ccb05
ea72cbbe9514e75c9ddb32f75d61c628ae1fc329
8215 F20101114_AABILF cardonaponce_j_Page_059thm.jpg
c22a44dabc7e42c47d83ea126aad1a5f
f0440d27109252be6c7995c01c717f57ba985896
6155 F20101114_AABIKR cardonaponce_j_Page_002.jp2
978b89431666dd3a3952ca969ffc4945
a32b648397d11f00b124b3296fdef60fe1d46032
976469 F20101114_AABILG cardonaponce_j_Page_055.jp2
c766d7f23bbeb48adb867204776c630e
a9c344c3fedd1a160640473db2da01dbe3185844
138454 F20101114_AABIKS cardonaponce_j_Page_008.jpg
947499b8ac94477483e4e9db1b2bad1e
f7a4913154e82ebc6f4ef0e66d2b1f5b45197b91
99 F20101114_AABILH cardonaponce_j_Page_002.txt
57b5405178551dcf332f7a9b1b561a53
cb7441e1bf7ea998fe27ebdc6b885aea8f922913
32712 F20101114_AABIKT cardonaponce_j_Page_072.QC.jpg
fd510a81976dee2a2164b84690b3de50
c767d5d50e16036df62232e05834a464c2fc8311
611 F20101114_AABILI cardonaponce_j_Page_002thm.jpg
06f435c3ba697ef14346523f43bf028c
ed0dc0da61e9630be266d8c54c66ed8994057411
F20101114_AABIKU cardonaponce_j_Page_066.tif
c1fce216f0eb08103e54a963fade44bb
326066f26e5c3c0b30e851b897e99b0839bb031d
111472 F20101114_AABILJ cardonaponce_j_Page_047.jp2
6ae1b198942b6cc4249cf8558ca1f1d6
671bf93a14ee7789b432a47a68c0f52363d77c3b
F20101114_AABIKV cardonaponce_j_Page_083.tif
770f924ff5fa72fc5165a9a7514fb33b
1e345b8e968d0caf1a0371accfd3416e9fae5f38
36946 F20101114_AABILK cardonaponce_j_Page_035.QC.jpg
dde85c637c9d4ceb0b88e0e6dd435652
77a1d58e0e840933781b858c07da2e387029932b
8601 F20101114_AABIKW cardonaponce_j_Page_088thm.jpg
4fa5f6c7826f61c4f8a05d3348cb67a1
1a349185770708c60415e5c33537301c148f183b
36085 F20101114_AABIKX cardonaponce_j_Page_050.QC.jpg
a3b5a9f4e7b66dc26ddd96b0d5700cb1
a26cf4babe4452c4b9b86df78df817a903a9b9ec
63068 F20101114_AABIMA cardonaponce_j_Page_105.pro
0f5ae7ad78bef558e9ec01adc8026789
b1f4fd04696830098acbd317ed8957dd748e2cf1
115023 F20101114_AABILL cardonaponce_j_Page_038.jp2
1af06332ce992386b884a00747d898ed
3a577e8778bb35992847e8e3cff43e1e790c6633
139165 F20101114_AABIKY cardonaponce_j_Page_098.jp2
157b929ce732dfc0429aa4bff11b092e
440ba7a53348b793377916cfeaf7af8e6ec56c37
F20101114_AABIMB cardonaponce_j_Page_030.tif
ebc9edbde893378b1f9eb0e257455ccd
c453033d3207c3a88d1dfa85984f966e0dede16b
2221 F20101114_AABILM cardonaponce_j_Page_073.txt
2faf342a089488b2407fa1abafa8b26b
5c0171e4e6b00dd3d596d6c9efaa4d7382257bea
F20101114_AABIKZ cardonaponce_j_Page_069.tif
1c9234e78a6e8f8fd51e76de74fc3841
b8c6fd3b32cbd2214f32498e45b018757a2dc3b5
97350 F20101114_AABIMC cardonaponce_j_Page_041.jp2
50d4b400de40a1a9d8fac4f3573a83c3
169dfa30203893a26919e726fdf61cc09e2863c8
33676 F20101114_AABILN cardonaponce_j_Page_093.jpg
9300419f6448c8f47962fbb2215dba85
c07a41323d0bbf9f965af9c4018e52f0cecbac07
40726 F20101114_AABIMD cardonaponce_j_Page_014.jpg
259e6c8427a3a0a1d7bee9fea137e660
427d81f173c209bf09f6ce077be080570d9d2b46
108417 F20101114_AABILO cardonaponce_j_Page_009.jpg
2ab07bf6ef8730aeb5f67d5849a71496
8d598fc2fae361388075266ac90cf1dd7faa4e0a
8800 F20101114_AABIME cardonaponce_j_Page_030thm.jpg
15ea5896ca05829904724898a578e6a4
7e2b30d00282f5587c5d4e5f0a141ba7b5cbd135
116334 F20101114_AABILP cardonaponce_j_Page_023.jp2
67f32095898f5c0b671364001c8aebc6
2f54fae779e1baaa7039d9701fe2c9e599a0b69f
36433 F20101114_AABIMF cardonaponce_j_Page_048.QC.jpg
781b781eb661d229c6402b67920612f5
b645223cb02a4418c2800a333566058db75b00ae
38176 F20101114_AABILQ cardonaponce_j_Page_103.QC.jpg
a68a7ebaf98d7f9d78a683720ec3be35
c0ba348d32576c9d1c434dc2de82daaf6afb1235
126536 F20101114_AABIMG UFE0021495_00001.mets FULL
8ffb1dc0ee49588928a809942d6fa838
5bf965576815a449741b4ec2fb2b53974125bf4f
9593 F20101114_AABILR cardonaponce_j_Page_097thm.jpg
0b337c71f5f5145e624664aee4b1a9f9
268b0f60ffc414c010fa3c6cd59f37920c0056f2
F20101114_AABILS cardonaponce_j_Page_076.tif
f53134830bd6a526597e6a5426a7443c
a2efffb07174ab4a26b0dac433ce07d9b734aca2
24255 F20101114_AABILT cardonaponce_j_Page_089.pro
62389a2c7fc30ba0855899f429b89b0b
168dd34351c6177d46d8faaccfc5177f3367ba17
4805 F20101114_AABIMJ cardonaponce_j_Page_002.jpg
76f6d3380e429e966510ccc48e8f78b1
faeb592626df85357e54fecf0c87e9f56e9fee5e
123527 F20101114_AABILU cardonaponce_j_Page_067.jp2
feeb1e343ba0abdde28534fa2158f30f
f290b3506605175b4b5becfc548e70b455117c8e
5945 F20101114_AABIMK cardonaponce_j_Page_003.jpg
46d07faf0b94e3263d277b98039e9ccd
d38c4eb046cd9841297fa1fc7c714221fec6dbef
8512 F20101114_AABILV cardonaponce_j_Page_047thm.jpg
ea0c568807d56fea3a8f3101c9a40070
304f575cbe9d84e758e6b36fb028e86821e43956
125529 F20101114_AABIML cardonaponce_j_Page_005.jpg
a291dcb82ab19b15e3182395721d0b7f
959c8f22ab5f614d605da6a978db5d1007d4d28d
55489 F20101114_AABILW cardonaponce_j_Page_064.pro
6a2be46a3acf3f586e4170444b8e5166
3308739ea1ddd7fe831e4d56f693ba0a6c0e50db
108130 F20101114_AABINA cardonaponce_j_Page_037.jpg
5dacf0d08d6d8ec7dba15e5248b2985e
bd940dfc54ea06ac2d4cb75489a5f89c3943bcbd
8700 F20101114_AABILX cardonaponce_j_Page_083thm.jpg
59ce991cb13db2b675f1954315cc4acd
6f3b54a2b7d81f7592895b2403b35aba95caf088
106173 F20101114_AABINB cardonaponce_j_Page_038.jpg
0a6c61b2b53503d2b0c382c20cc7e815
ef4a5d5cb0310265befcb00019145be3cd266c4d
132485 F20101114_AABIMM cardonaponce_j_Page_006.jpg
e21ce2dc115019e6f1acef86f9d52928
9172d0b95e75b31ad8ac4d3f731ebbafd3c43998
118731 F20101114_AABILY cardonaponce_j_Page_063.jp2
9bfa1918a300e4289a2509aeb4a19f8c
875db6c4ba6861af6155c42740140a5d87d0c2dd
104677 F20101114_AABINC cardonaponce_j_Page_040.jpg
18d11b37f5e9eb89450490717f5b222d
bb53f71495ee8b74050d2a4b9b9975b843281ccd
73867 F20101114_AABIMN cardonaponce_j_Page_011.jpg
ba1a8aa7f41f69f15cab4c30e7ff0d64
fced1965c198451f6ae2af5eb91286f76044635a
9616 F20101114_AABILZ cardonaponce_j_Page_094.pro
136bfeb433c3dbb3ba7bf2d79323f667
86e1a7325356eb6f0d13cb679fd62c8e412d4f05
112394 F20101114_AABIND cardonaponce_j_Page_042.jpg
67225cf3e2228265c0b5bcb8917d2ac8
82bff3ed118187ed90647dd1deea4fdb3d9598f4
101846 F20101114_AABIMO cardonaponce_j_Page_012.jpg
78a4a243c405dfda51925c6ba6b7abe6
cc29bb0a8792a4ac0e48d52d745caec9834a67dc
104596 F20101114_AABINE cardonaponce_j_Page_043.jpg
f91adbf24905ec6a2b505855db358474
e32485e3fb904aa53d3858c6a36b6fe957daf90d
108494 F20101114_AABIMP cardonaponce_j_Page_013.jpg
39c170f51948eca7cf620a084feab9f1
0e474055ff5621ff99601a3ab96891a85d55f5ab
109531 F20101114_AABINF cardonaponce_j_Page_044.jpg
7f0886378d4fd19687fb4a419a330239
0bf0a6e5972a35c91c342f2d351e2c7282facc4c
88958 F20101114_AABIMQ cardonaponce_j_Page_016.jpg
0aca2af070bc8a956f6ad3e730e6c270
9fa55858c7ed99a5812960d14ffd020cd059a82f
109999 F20101114_AABING cardonaponce_j_Page_045.jpg
fa0cf49dd4ca4601ad56cbda164606ea
d20fad552fab6b5336f7cd81876588c01cf14ce6
105381 F20101114_AABIMR cardonaponce_j_Page_018.jpg
e1940a3fc309620f1d66a3138779a135
7ffa9bedc7df7a0dd0c7059c65413176aab21fe6
112602 F20101114_AABINH cardonaponce_j_Page_046.jpg
7661001812a12eabdcdff6b7778ec9f7
3f3fa218e3208ae4b95f39796c44c9b407bc467c
108449 F20101114_AABIMS cardonaponce_j_Page_023.jpg
a614902f4193f1a7e8aec9d5ef93405b
2f99c2fed33649451502c67d809a1fbdf8dcb7ee
104179 F20101114_AABINI cardonaponce_j_Page_047.jpg
9cfa6a204059381a8a201e5799abf670
5aa74e26ef600845fad78764ca2015d4237fd035
107491 F20101114_AABIMT cardonaponce_j_Page_024.jpg
6153d03aa8051ac1988f6ad2fa23caac
6d2a966cab657288714785f4e40e69fbd342d770
111983 F20101114_AABINJ cardonaponce_j_Page_049.jpg
2732a6cfedbaa1f562afcfae113e3882
b1bc8a013cfc08041bd296b271fdbea4d3c4b31f
90658 F20101114_AABIMU cardonaponce_j_Page_025.jpg
752baab7f810ef6136dd59c34b72bcc8
70b4ad7480e3022b44f2ac93298838a234f1db31
108136 F20101114_AABINK cardonaponce_j_Page_051.jpg
6b2076275f39375bc85645ecb8ee4624
77a265bd914eee0cf674306f8828e809b27e5253
89067 F20101114_AABIMV cardonaponce_j_Page_028.jpg
bd44f4124af5513e7e7de5a5be756d99
b216b5f6fddfb4d93018aa7ef2407c0ad25ac8e6
117719 F20101114_AABINL cardonaponce_j_Page_052.jpg
7c73cf065d0981171eff5c44992d090a
bee1d80cc5f2085ab3a784455b1fc4aef1b84330
110056 F20101114_AABIMW cardonaponce_j_Page_030.jpg
284a0281bc465599c901c6d02cd37024
d2ce2b3530c30e9a209e487c37d29ea69ede6038
85589 F20101114_AABINM cardonaponce_j_Page_053.jpg
f27f2d52a394a0500f3bc4383466f4b8
2bfa7edf62f2af7f12d06249afbc93da8f5ac3fd
73153 F20101114_AABIMX cardonaponce_j_Page_031.jpg
38adef89772ad41268ce5a3031f4dfe9
f40b553c02f6566071623d55362e12fe0b9ed069
108936 F20101114_AABIOA cardonaponce_j_Page_077.jpg
4ca90e5b3ec6ff0c0a7d15f4ded4c216
17e49ae6edc4458ecdaf80af7b59bc1855cb9b4d
96382 F20101114_AABIMY cardonaponce_j_Page_034.jpg
19ffb3765f299084d86b1f411af2f247
e745f80313ef56f79bfc9b44b9c3e0247ada2a0e
113856 F20101114_AABIOB cardonaponce_j_Page_078.jpg
ee1393ccc6eeed88ea9100a50ac120fe
94af945baeffaf2cb7aeeb7a45cf43be66bfef87
101578 F20101114_AABINN cardonaponce_j_Page_054.jpg
ee147cf48368052b7ab3cb0f1fb783aa
82a47919569ef999c8defb37bb390a98b5555f3c
111142 F20101114_AABIMZ cardonaponce_j_Page_035.jpg
c5b35709ab3eefb09e6c4b473c623f3f
f6e6180940d3b3f56b385d247e8bf0c8135ba80d
71874 F20101114_AABIOC cardonaponce_j_Page_082.jpg
581966395e42c4b5676d919a533f2772
d3c8fd75f12fe2174c6c6bb83da1040f9094cd1f
110714 F20101114_AABINO cardonaponce_j_Page_057.jpg
f762b48afbc972a5fa2ec28f0b2b0293
85d98566d5107dc41512a1fcbd2b9c8ea78963cc
108750 F20101114_AABIOD cardonaponce_j_Page_092.jpg
aae0c34bf0c24aa24e0e98c4c0692760
5cdb1fd62f91a80e50289a42105e399614817139
81891 F20101114_AABINP cardonaponce_j_Page_058.jpg
35315dbdc89cd4b2e12f38b98f498389
a980b064e6af745e68d7bb4f4ded6ecd0e319c00
25307 F20101114_AABIOE cardonaponce_j_Page_096.jpg
167e406219c8e1c3757d0cddcdc5d2bf
33394e7fc339b718f71148854434ed0cf80e363f
122919 F20101114_AABIOF cardonaponce_j_Page_097.jpg
2f562ab31a348da3b8b80ae27a3a0156
9f6d177eaffb04ee7ab5615922ccf90c7fcee3c0
101984 F20101114_AABINQ cardonaponce_j_Page_059.jpg
b0674ccf77a22fc53cc568be53e5ad7e
64c2cd6d494fe46809d511050bf1fe083119aa45
131472 F20101114_AABIOG cardonaponce_j_Page_098.jpg
162185d3b12b696ec2b097e9b9fa02e7
a18cc81266e4b2bbdd6ffc24e0becc7c4234740a
108569 F20101114_AABINR cardonaponce_j_Page_062.jpg
dc4da77ef6618d486d94bad8c3eff9a1
3c205816b31446048612b1e2808da2aaf7404289
125300 F20101114_AABIOH cardonaponce_j_Page_099.jpg
a199fd1538f44e48bc2cd0f5cde0cdf7
5a01a95251dcb0a14ce78f98dc9a1fd933fa07e1
110328 F20101114_AABINS cardonaponce_j_Page_063.jpg
766bfc3d322178bcd431f9bcaa0fe003
3a04edffd39645e3021b95a33915348f43180151
125676 F20101114_AABIOI cardonaponce_j_Page_100.jpg
27ac0a804622c35a242b2387dfe0435c
cbca4706781522eca1a223d412b4e6ffb28b507f
113714 F20101114_AABINT cardonaponce_j_Page_064.jpg
75a1507644c3183b38e894d2f356c4eb
7bcc7931276f3f23fbca9f1f8ca0eeed154d24d9
128560 F20101114_AABIOJ cardonaponce_j_Page_101.jpg
f7c5b064b3d13b1d2978de4a132d7c4c
fd8d18c35277fc4ac8af05caad3cd4ab855f0a7b
86931 F20101114_AABINU cardonaponce_j_Page_065.jpg
35ed8c1818a3ffe2fa829e049d715496
4c0e66e2b521122b1d009d762838473b074cf358
135054 F20101114_AABIOK cardonaponce_j_Page_102.jpg
2a11c9689283c53a0582eea3559bbda9
7ecaa6068c4ff1ca074b50dd65807068c6cadfbe
116662 F20101114_AABINV cardonaponce_j_Page_067.jpg
f32fc7c63444ea7f4671091fa39d9af5
4adde1ddb60380bd00c8840204c34a76a6a56acc
132164 F20101114_AABIOL cardonaponce_j_Page_103.jpg
51477110dc492c38cc7491355bcc4775
299b433825af0b272d1b5fd1c910c90add78c525
108551 F20101114_AABINW cardonaponce_j_Page_069.jpg
2b96f9b76a5176b164f0430b7cf06e3b
59c99b127835974f986e90f1ed0338eb105dbcc0
789085 F20101114_AABIPA cardonaponce_j_Page_031.jp2
aa7e8fa8b7b579d43068d90da0ee856a
495390e4a13629a2e592e3f0006505b3b0f7be7e
122596 F20101114_AABIOM cardonaponce_j_Page_104.jpg
91cb0c4a2a7792ad92bf5b1b8b2faf19
7a1cb253e69aad39d0fcc7115296a885f1074fd0
66692 F20101114_AABINX cardonaponce_j_Page_070.jpg
8c1631695616e390ead61532ffbfc600
2b0f672637c145bcf09472a922ba350ec0bdc9ec
825789 F20101114_AABIPB cardonaponce_j_Page_033.jp2
b637f34da521134086346cc039a0ec93
ccc3f9b8d2dc50dcc91c969b825c55386baf9c05
135339 F20101114_AABION cardonaponce_j_Page_105.jpg
14ba0faaf26de57abc023df9d559eea7
0a363c35af2317a8ce3270ed8cc0193b77eca5e5
107365 F20101114_AABINY cardonaponce_j_Page_074.jpg
36e439c65f4c69c6f73c5b5f5022cec5
e9c04b140e7083ed3a1ee4670d50d07b8b670c88
117901 F20101114_AABIPC cardonaponce_j_Page_035.jp2
d82a4eeb3b904d50ed5129a4770af208
551c56ee9cfafb7963c240e67710735a9a0768e2
86275 F20101114_AABINZ cardonaponce_j_Page_076.jpg
3b49b6be03ae563374dee41c50858049
e884608bd36759acb1c14ef3625323b12eb2b3c7
868853 F20101114_AABIPD cardonaponce_j_Page_039.jp2
21663764e22d0185fe87dfb5d7db8325
d54a63a5b68a2d0284ca30871d48f043ad06959b
49382 F20101114_AABIOO cardonaponce_j_Page_106.jpg
3b977560f551ffb576aa0f5cd1d42f7f
5b3c146550a5ffedd3fd9d453060188ff596ddf3
112889 F20101114_AABIPE cardonaponce_j_Page_040.jp2
d52fcb173aadca0bff89324862458513
445393ba8074e9c735558c3ee364272ccd2a02e2
1051963 F20101114_AABIOP cardonaponce_j_Page_006.jp2
1c8d723bdb554484b943d8ec58488e45
3e159495da971d59f5741a7bc804c7b41c0ba8ba
116762 F20101114_AABIPF cardonaponce_j_Page_044.jp2
76dcffdfecda12c62505397e42cfefbc
8c2d36fc48113aa844ed64eb4d802ecb2b0e40b4
108288 F20101114_AABIOQ cardonaponce_j_Page_010.jp2
ff0d580148dcf9eb37eefc4fde49cccb
2544c90829f920f50135c27a0f9c31ad84ef3522
117155 F20101114_AABIPG cardonaponce_j_Page_046.jp2
c39c1aeb8f5ec165fa01ea22e7f91a3b
5e298575fbfb9e2c84ea1a7c08b8b5729bd2840e
110046 F20101114_AABIOR cardonaponce_j_Page_012.jp2
0007dce60028138f904cc50755d116f6
fcc635b3b599fcceb88e01d3c32c30f6572d96b3
118618 F20101114_AABIPH cardonaponce_j_Page_049.jp2
20b703be6ac7328ced898bfea91971f4
1231f6cdc99f53d4aa01cfedfa4c37904d277477
114865 F20101114_AABIOS cardonaponce_j_Page_015.jp2
ed5f2c0dc72cac8547645cddef865263
6256fabdceab379a023ba02baa63c47afe31db91
909222 F20101114_AABIPI cardonaponce_j_Page_056.jp2
c8f4f5aec3e46a27becd1a8e4cc5d6b7
22402028de8dda0bed3bc335ad4ab1c82c0d81d7
95765 F20101114_AABIOT cardonaponce_j_Page_016.jp2
1ea9f2d20fe5e2c218f799b0b06ace00
7d2b76f5c4bbfbc398fd6c9b2f69239622645f0d
836837 F20101114_AABIPJ cardonaponce_j_Page_058.jp2
31a3ac65e3fd64ead602d87a8647c329
9abcfcdb5c5bcefb549bd0cc9f6e6a1f3050ab06
118802 F20101114_AABIOU cardonaponce_j_Page_017.jp2
3b1e235f21cc245593391649d499a795
ec35fd6c6d274ad3bc2843c2ed571d15feebc466
1051922 F20101114_AABIPK cardonaponce_j_Page_059.jp2
3a0721ff189b707520008f481984c9bf
c8137204e78fa11fe378fb66b0c515ba1f00f392
989019 F20101114_AABIOV cardonaponce_j_Page_019.jp2
7e99263e4481e1d83bdbce544c9f9958
723b5dfe45f447fa68cb983c958045fe0ab575d5
133215 F20101114_AABIPL cardonaponce_j_Page_061.jp2
9229389f7004e5fcacb4ed623abc3cc3
b3d138eb53ed7c2be070a5366b9f8c548ad48e48
114407 F20101114_AABIOW cardonaponce_j_Page_024.jp2
dbae7b4dc7fe52ba2b2eae6b1878c791
36a94d8a026e5bd677caf8a0c6b71a5711689d0e
1051954 F20101114_AABIPM cardonaponce_j_Page_062.jp2
b71e44c9baa63228f78b1b73b0d21c89
3654da766b59adbb636a10580e8dd07b5a8a3c47
119348 F20101114_AABIOX cardonaponce_j_Page_026.jp2
9caf5526209c42a6b229b03ed351db60
08c0d2c0ba7342a7330166082b473c47c3950abf
77076 F20101114_AABIQA cardonaponce_j_Page_087.jp2
09dbb035841129fff2c244adce85b596
850c496bd9edbef641b4fad63d5b6e3af38df582
121268 F20101114_AABIPN cardonaponce_j_Page_064.jp2
1ffa79de964c5ef6319a3bc2e4f03a94
7649e5148e3210825e38d21a8dc5aec21616aef7
958342 F20101114_AABIOY cardonaponce_j_Page_028.jp2
4ea7ebb8655e756406bb996b66678b1c
1eaaa64e6983896d06a3aba7cdf03fbec0d9c9af
1051974 F20101114_AABIQB cardonaponce_j_Page_088.jp2
6020750601837c181a8a0caa4dd17aa7
e1783fd67362585468af076b01236ca2495c4169
902468 F20101114_AABIPO cardonaponce_j_Page_065.jp2
57428476fc4ddbdb10a3976ea456dd04
99e44a6b9ce5b8c8ff8718aa52e50b7b701ac70f
118396 F20101114_AABIOZ cardonaponce_j_Page_030.jp2
5fc470810855bd9838286b6c8556866a
68326b7ea3297b59c30c0b65b30f8a46c5b10012
85311 F20101114_AABIQC cardonaponce_j_Page_090.jp2
fcc1a1bca61c10f42c4dc74d08432f44
f7b814fc1d033c4dfb25ce7f9cb7a2de5bc81df5
116793 F20101114_AABIQD cardonaponce_j_Page_092.jp2
b06b4f5d0dcc16aa3c65f57828f2f859
1f0338bd5bf906bd81e823e943df5b81e6013bdf
66352 F20101114_AABIPP cardonaponce_j_Page_066.jp2
dda2d11bd032de1a79cb6c107658d7dc
ba9a7a175fc6c274ee3ac0ffbd3d8fbf252bb68c
363793 F20101114_AABIQE cardonaponce_j_Page_094.jp2
2f65e2d8b81fac37d6c25b77a7fce208
fba0cd70aceb2db75f77af7ce0dc2f2d66c31366
114072 F20101114_AABIPQ cardonaponce_j_Page_069.jp2
834fc05dbdec6027872c63b7b3f47f7e
3d368d7a53378b4f9b1d7a2091190f4149fadcc9
289323 F20101114_AABIQF cardonaponce_j_Page_095.jp2
6918eefe8eb1bace7b9a4bff2e4d7772
021e7041b8c47a7cbe574c96f5a9ddde72887433
64259 F20101114_AABIPR cardonaponce_j_Page_070.jp2
d92f7db680d098c38f39756260758136
7b061d5e1fe708cfe3d4f9b5218a5216027ec8dc
129576 F20101114_AABIQG cardonaponce_j_Page_099.jp2
6dcd975912b8230abe14cf6db4e440fa
9731c38d7b96e215aada3dc0d969d1d1a6599598
112573 F20101114_AABIPS cardonaponce_j_Page_072.jp2
cb04f81fe8fcf2b63214fa40d7cf5a13
0d4c290c73bff82c021bdf08aca4c1d968207edb
133453 F20101114_AABIQH cardonaponce_j_Page_101.jp2
7b57ab560b1f13e910d9af5d893a5008
0349a49b453e7234f10a017ec6d4f695d99fd992
111370 F20101114_AABIPT cardonaponce_j_Page_074.jp2
dfe48ba7cad178bb3e5d046fd5ecde83
d7ff9233aa5c861bb9635304865a3286d99bed7b
135079 F20101114_AABIQI cardonaponce_j_Page_103.jp2
e6d51325c072204fab94c305080b7a44
9db6fc1442cee2843a67d836769e000f92bdd957
817229 F20101114_AABIPU cardonaponce_j_Page_075.jp2
1c3e2cb4670ed762887e5c9e60b61790
9f68eae5092f7f3d295737831d50a3548f516ad7
134272 F20101114_AABIQJ cardonaponce_j_Page_105.jp2
51171f29a000b0e6eca9507919864f44
4f8c1889ec1d0d8260f3d1e014cbfe1711af6a3e
1051978 F20101114_AABIPV cardonaponce_j_Page_077.jp2
a401b75f6b504e8f1ac3ed6937d37c62
c2f973715008a336e680c7b4b47a8121f1f69f34
51649 F20101114_AABIQK cardonaponce_j_Page_106.jp2
17f97f0f7e302c6b0a2369f7452af49b
ff5d777f23e785c3e3aa8b359bb3b052d860ba43
120142 F20101114_AABIPW cardonaponce_j_Page_078.jp2
6a0e41725a7b874a6b0d9226e7866e73
ee64ff92d9d70e235584a7aa9fc7b22091a111f6
F20101114_AABIQL cardonaponce_j_Page_002.tif
9dd5aa7f79f3a0615b60bf640b55bf03
3da8d2b9ee8c5899c80cb91606c462a0cb19d038
720615 F20101114_AABIPX cardonaponce_j_Page_082.jp2
b38febf9b95827249491557030dc3a06
3afd8037db3217cceca646474ceeeafce6cb94ac
F20101114_AABIRA cardonaponce_j_Page_028.tif
93ecc420a40092699df5a42ada27b784
f9d38e89d66997239ea8dc3aec6b58d91f5bd1b4
F20101114_AABIQM cardonaponce_j_Page_005.tif
5d90d7fc20319be99436cf883a7ec8bd
023530de29325079599c924a0efb9ec0ebee086f
84935 F20101114_AABIPY cardonaponce_j_Page_084.jp2
5caca4eca961bffafebca5c4ed115d12
a3d0d8bb227f1388f65f1b5404d88b33a4840fc3
F20101114_AABIRB cardonaponce_j_Page_029.tif
7fab305b6a9d7b49ef71788824ede103
d3958e50f2aee6d83addfc34844e2390f5eb716a
F20101114_AABIQN cardonaponce_j_Page_007.tif
a9a1cca228bbb4686e6d297148f10050
1dedf380a9e1459ba7234df56e41bef77d4fb610
108710 F20101114_AABIPZ cardonaponce_j_Page_085.jp2
0fbbeae96a7d26f14a0ec4fa3a55e77c
7dfd4006904abf2c8294abe737aa90b185ff999a
F20101114_AABIRC cardonaponce_j_Page_032.tif
1955fd1a55bc3de17d0d6e765bff0c59
b3caf375ec429c227f5d20c598cd04a78b7b597b
F20101114_AABIQO cardonaponce_j_Page_009.tif
d0d03d211563f30d1e7d75ef179befbc
023106d2a439cecf07effbf8cf5ac115964ebe91
F20101114_AABIRD cardonaponce_j_Page_033.tif
d9d4c017eb0f1576eeae192be10539dd
3693359b8ea947bf26fd27fec810aa980415363f
F20101114_AABIQP cardonaponce_j_Page_012.tif
df2ea78b345fecc4f785d93491e6ddcb
0de6fc6f11f473fa4775f4bb63388e2571f71334
F20101114_AABIRE cardonaponce_j_Page_035.tif
d13ec7a8c447c48fd2ba709cc6866735
5564c74cc631c4fd9386d8cac31c841d096c706b
F20101114_AABIRF cardonaponce_j_Page_038.tif
dd95c623573ca06f6847f274728e6b4c
0439b6505dc9b1e02dc56892453f003e77fcaa4f
F20101114_AABIQQ cardonaponce_j_Page_013.tif
5e114bb90732e11b097d5e7328f62337
6234eaf93440923c200cc4d68644aa4f0dc63a92
F20101114_AABIRG cardonaponce_j_Page_044.tif
e21345cbc08a6a7ba13b629967da7343
9a9a1feff22e4d040c6e45e07a297a1853981ba5
F20101114_AABIQR cardonaponce_j_Page_014.tif
bb02be62232b47c10c4565f2474591d3
8158cf5d56a8c14a16f95a07ceb4f97fa2a8cef4
F20101114_AABIRH cardonaponce_j_Page_045.tif
0477fe4d0547675a943c9cc3db4df635
df700321c734217ff80e687e165624ea52f95555
F20101114_AABIQS cardonaponce_j_Page_017.tif
a71c2df25cab90cd34263ce60d357c9f
b4645f4df403222a2d259aa67c93a8b4b5870891
F20101114_AABIRI cardonaponce_j_Page_047.tif
e0e7d13d02df5de56f278704b35b6443
5f8651d943ddfcfa46f135a3ccc5c1e266002827
F20101114_AABIQT cardonaponce_j_Page_018.tif
df5d36a76eac88f665bb8d3e63a3268c
8a03faa03e0a73049305af227fa652581fa2ec48
F20101114_AABIRJ cardonaponce_j_Page_048.tif
f56d2131b04c90697945a6a2364f7382
4327c6e305098e03acbd3f34755ef68785158d38
F20101114_AABIQU cardonaponce_j_Page_019.tif
27895a91c9a13f7e4497342625e34f98
1512e17cc292a817ab6e010355e58bdf5d25f113
F20101114_AABIRK cardonaponce_j_Page_050.tif
fc5eba7d17d6ab4b66f6b159c22ae6f8
116ade4a155ff79b0fe555664ae66fce3864f795
F20101114_AABIQV cardonaponce_j_Page_020.tif
7112af120fc3fbd13849ffbd90ca5e0f
d0fae7fa3b74413023dc54d51e74cdd5eb2eef8d
F20101114_AABIRL cardonaponce_j_Page_052.tif
86612cfe017adc0c0d4614ee359e7061
a784c0552dc37e49c2c7ead4d9fa058085793f20
F20101114_AABIQW cardonaponce_j_Page_021.tif
aa6d88c4284acad1c7bc2b59bdcc81cb
7645a39c4a59682955907ea8424710ce88560735
F20101114_AABISA cardonaponce_j_Page_094.tif
d678c4567fd9e846d277832a39091f3c
7b6434f765912aad9b46fe1dc50b5d7d823161bd
F20101114_AABIRM cardonaponce_j_Page_053.tif
f89116980ed5e8660dd29e921d78b1eb
b4d0ceec62e6333ea6149e75a31aa553dab8c919
F20101114_AABIQX cardonaponce_j_Page_023.tif
99ea1571f87a2243588b3f77fd2e4ca3
59540a6c9158bfb64dd7035a24e4c7719c0e51d2
F20101114_AABISB cardonaponce_j_Page_096.tif
74f17bd1c705cb634bbb002a612fd4b5
8bd21b540432af17c4100c283b5cdce358af3b6d
F20101114_AABIRN cardonaponce_j_Page_055.tif
6b978806bdf2dd68c1e0fac8e90286f3
97599b636c1dc29748d7e2a4f4be37fd1b9e6005
F20101114_AABIQY cardonaponce_j_Page_024.tif
ffec7cdf4a416101a26ed3737ff5256b
84cad872d2efec31927855e3816bec2e614c3c52
F20101114_AABISC cardonaponce_j_Page_097.tif
c7fd4b1a3a558313ca745430546add8a
93c37b6c1d55db2c73141b044f8ec8d2751ae65d
F20101114_AABIRO cardonaponce_j_Page_058.tif
18ba35202ec13237c0b790ae47e7b510
08296748bd27c5cb65fbd136ace023fd57b5e5f3
F20101114_AABIQZ cardonaponce_j_Page_025.tif
3379a4a63ab86a6b987ed6a4c66e62df
acfad3f73c5ac81896a5f2a84b5d109bacd25b50
F20101114_AABISD cardonaponce_j_Page_098.tif
47faab8bf2095fef2d40fc472771115b
9d22487e3d8af04273e7be863a3457ea8c6f012a
F20101114_AABIRP cardonaponce_j_Page_061.tif
50dde1065a710745241179394ad4929a
c5dbf989d089f74c8724b33df44017204a89a2ec
F20101114_AABISE cardonaponce_j_Page_100.tif
76200f0ffd7933ea0f5dc486b09f8307
7a6089a592483001ab32f1c89830d40a9109a5aa
F20101114_AABIRQ cardonaponce_j_Page_063.tif
8bf42df98522c7a8d2e716affb6b03c9
a4f7e3ffd1f203f54801edfcadc8f5b3bf54da8e
F20101114_AABISF cardonaponce_j_Page_103.tif
57fb23e29677137b7d4ff246ebf3661b
b5e515b7b02645573cad8f5540047f8f5daaad73
1174 F20101114_AABISG cardonaponce_j_Page_002.pro
efe97bb5fbe98a29ef4cf7d66e3b9305
bcc2c66e946f1638510270d17dc4b77c021e668c
F20101114_AABIRR cardonaponce_j_Page_065.tif
140456d9acc5a3c2413e86373eede586
a9d7eff3d7dcda791a0694fb2a65bd7e0d46fbef
1746 F20101114_AABISH cardonaponce_j_Page_003.pro
3dac8d1020c024ba4a616b9e7f76fb22
3857d4ef26fd13e131e5f03d9c210e4688cdf0ab
F20101114_AABIRS cardonaponce_j_Page_068.tif
423022df3943293c097658508588cc9a
37afdf1a9ebae03c0e8c2bda8a3397b51c010797
32945 F20101114_AABISI cardonaponce_j_Page_004.pro
bb415b9727a7a099b170fa2777dc7130
d5fbb046241467fcd340744f1e1dc169060620b6
F20101114_AABIRT cardonaponce_j_Page_075.tif
80fc61de38ed561958fd78936159d581
4b219e7cb7467249301224c64d4550b12aa899c9
74340 F20101114_AABISJ cardonaponce_j_Page_005.pro
d679b61a99bcef64387a217e491cee1d
6486a4320c59536f5412ead579b021373086d098
F20101114_AABIRU cardonaponce_j_Page_077.tif
c0c4216d558ae4dec50a3fe1321bdc31
55562c0d1bcf4671fdf79caf9b93c83adaff1920
74435 F20101114_AABISK cardonaponce_j_Page_006.pro
5f5d977bd70531acd1a179c6b49268dc
624414098ae90d56c98ea20ec58554eac0a5149f
F20101114_AABIRV cardonaponce_j_Page_078.tif
ac4c21867504748621acd467695d6f8c
93528930b01f9646f782105209259a3b9ed8eec7
68356 F20101114_AABISL cardonaponce_j_Page_007.pro
d2fa1f47a95c0d9d960b12f06bd31518
5f9ba8d70c32bb4f720257f8a4573f3a70250b8a
F20101114_AABIRW cardonaponce_j_Page_081.tif
833acca0002c87bb237580ff04d36819
dec451ca4bdf0a1f16a9405a34873b985738c231
56751 F20101114_AABISM cardonaponce_j_Page_009.pro
b9eb360c3c3141b08d27fe62e2999c7a
bf40182a67b875a73782bbe4bf3c2a953dba8de6
F20101114_AABIRX cardonaponce_j_Page_087.tif
6f0c51418fd358e5cb680e77fa149e8f
acd1e50f6a73f49e734faae8f2d366a9c48f69fa
38466 F20101114_AABITA cardonaponce_j_Page_032.pro
d5bbab18129034ac085f48f158889582
09a56ced223cf49f1d3a61e312b5d9841f241616
48641 F20101114_AABISN cardonaponce_j_Page_010.pro
684c9abc6c4bc6c064cbeae57901ffd1
3298c1b4e9bb5396e280c064b186a6bea7945dd3
F20101114_AABIRY cardonaponce_j_Page_088.tif
ddd5a797a0afdda55ed8901b156cf3b3
fbcbaca93cf50d96fd6c6cac7f97308038416b55
33296 F20101114_AABITB cardonaponce_j_Page_033.pro
413c5f0d0139f0b862157869897e7397
eb08ebd851472a6509cf70f0fae44b886f7a00a0
F20101114_AABIRZ cardonaponce_j_Page_091.tif
f5b657e351de11ab30957229e784f5cc
c7ff4cee09c316aa7fe617de840f6beeb2362da4
39074 F20101114_AABITC cardonaponce_j_Page_034.pro
22e4df0bafb6d7d672efbc5ba134009e
70b09b8176d5e0cab290f17da84e291e734e2c8b
49536 F20101114_AABISO cardonaponce_j_Page_012.pro
addff042236a1bce7355b39a3eecaade
7c78cd35f7e05041d35a43ce3e6fd06b82dfbd82
53593 F20101114_AABITD cardonaponce_j_Page_035.pro
e05c8130f067d10a6e38566af5844e5c
76fe4e455b4b7c0caa3329abfafb04266cce9715
52594 F20101114_AABISP cardonaponce_j_Page_013.pro
b3cea1ef839b34c3aa97b154b843fbe1
fb5915f0ad3ebbdeb30441dadfe3ebebdf55180d
53071 F20101114_AABITE cardonaponce_j_Page_037.pro
fb23b1c7d2076b0ea21b9821c42d95fd
4814f101d3c3c7ed03fd67d089034ffe8e8fb424
43940 F20101114_AABISQ cardonaponce_j_Page_016.pro
1ddf0df20131a87cc0d9ab74202f1d5a
259c4be92e492cdbc853666c3dd0e49208be7507
37092 F20101114_AABITF cardonaponce_j_Page_039.pro
65fcdfb40dcb6732eb9d83d4d9decc61
e4ab99b0e9e962dd912aa044186623df83bc6a8f
54022 F20101114_AABISR cardonaponce_j_Page_017.pro
898a5e2c502114ae040c7681d5fdb3fc
c666d359c3f35795dc3f846fd4513de563563148
51220 F20101114_AABITG cardonaponce_j_Page_040.pro
dc2d76df40681da09ff8ec6ccdea177b
5905d3bb874874ea83113ea70b94a35d710c58c5
50077 F20101114_AABITH cardonaponce_j_Page_047.pro
9ccf6c5257efce632257767928f4f93d
48559a8111c02424225f7ce1d3eec210e0acb1e0
41618 F20101114_AABISS cardonaponce_j_Page_019.pro
cc67258aa5fa811b449119cc78586204
66b16a00f191c3b29e4c16c91b4a9bded558df78
53725 F20101114_AABITI cardonaponce_j_Page_049.pro
5fcf4c44f543d21b49b87088746581ba
524ef1debb7c75d78650874619e01fb626bf1e92
52413 F20101114_AABIST cardonaponce_j_Page_023.pro
dbad57435e96aa5489f7ee5c84290faa
7ceefe7d2b1bc59e0072414a20dd70eca6946480
37508 F20101114_AABITJ cardonaponce_j_Page_053.pro
e0ae8136e0d55b02e2ade2c9c0f6e48a
4a70672127316bb39569c0ab9b44b4c065b5fdbf
52090 F20101114_AABISU cardonaponce_j_Page_024.pro
8ea036f2273f53c9e2729050e1013b44
6ebd0cb09ca72310131b76046dc9c0901b69ccce
43790 F20101114_AABITK cardonaponce_j_Page_055.pro
913d23c7c4ab75bc1d46ebe5cbf5e556
79463218a51fa0015eb55133da86eab147367561
30879 F20101114_AABISV cardonaponce_j_Page_025.pro
18ddd9344889713a2b36ffb3b46494b3
46d8371fcf99fe21785b6cff31919c4bae44e140
38562 F20101114_AABITL cardonaponce_j_Page_056.pro
9d83ae0251701d0249b820deae28fc24
e2151a74875611fe2f8ea6593bca31b57d78876e
54030 F20101114_AABISW cardonaponce_j_Page_026.pro
efe72d80b9c8b6473c945cee34809fad
544fcfb6bcca2ce5157cbd9d22b8baa11ef9db2f
31812 F20101114_AABIUA cardonaponce_j_Page_087.pro
c5ba74a540dd52a15769248ff23f9993
856793a5b7ebbe55dc51d1c05b4ebd2ae0972676
53112 F20101114_AABITM cardonaponce_j_Page_057.pro
4fe813aefe894d93970efe9952bc3ab4
a5f371757058b4afd66bd9fe75542737fcf82e8f
40533 F20101114_AABISX cardonaponce_j_Page_028.pro
6af86a2c0ebcfc17bba2a41df95429ba
1a084f00def4d2584bd944c2aca137753de1cc6d
43582 F20101114_AABIUB cardonaponce_j_Page_090.pro
a380ee2a5c4d0f2a0cbbda56844f8dde
7a3e94e1c60cb174db6790cbb495ed774abca82b
34616 F20101114_AABITN cardonaponce_j_Page_058.pro
8f8408d67d759b8424c2272bed519dfe
4fb5fb7c8c05a13be9d21d455be23c4d375b9d9a
53556 F20101114_AABISY cardonaponce_j_Page_030.pro
0afd484132f26c7cca3b2fdc90ef2ed4
a34c7b692e0163447edde7ee7351b8c2d9533252
54230 F20101114_AABIUC cardonaponce_j_Page_091.pro
711f3815a368b3d4bf98509203f53626
3bd138ba9ec7674660a45d3411fb85aaf42d9abe
46856 F20101114_AABITO cardonaponce_j_Page_059.pro
9498c9a620c7b03619859fc80ba3b95b
9e5d4305821e59d7e0cc97dd91fad400e82ee016
31161 F20101114_AABISZ cardonaponce_j_Page_031.pro
958ff8c09129401b32660835235a07eb
13a365d35e8d7071c595aedc02f1f5773864be30
8543 F20101114_AABIUD cardonaponce_j_Page_095.pro
7368b2dae9b85f6d982356b0c2baa2b5
f437303ec036fc5c58997991580e39e3f4843581
53749 F20101114_AABITP cardonaponce_j_Page_060.pro
6ee4f3a4de9a49fe88b92948a16aa4e0
05aaec19fc4ef21f94cc9348413c238efca382a0
59710 F20101114_AABIUE cardonaponce_j_Page_099.pro
909a81faff8cacc516be542ac633e025
77ee3c1be79a25255f53bd8bb3d0a80229720354
65342 F20101114_AABITQ cardonaponce_j_Page_061.pro
677b676087713ea92384f8feb34b0fcc
f4134437f46da8f8e6a828c9cac24c7dc8f0adca
67202 F20101114_AABIUF cardonaponce_j_Page_102.pro
a02863482af27f616122a94ae6d3ce93
9b6449bb21c116f5d5bba862bde331680c14e29f
50498 F20101114_AABITR cardonaponce_j_Page_062.pro
b7ca831c0215f2257821a185b423c53c
8535197e369866900d465c3d87b904d7e96ad88c
21770 F20101114_AABIUG cardonaponce_j_Page_106.pro
84dd7a320777e998af18df95c9f13f24
39f92e8b9d4a0785574523b0e906321ba5da0255
56730 F20101114_AABITS cardonaponce_j_Page_063.pro
ed30d5b02054ff9dbb80daad0c453b9f
414e023a0dbc91528a4c9be9c7412d4dbc433102
17209 F20101114_AABJAA cardonaponce_j_Page_089.QC.jpg
6eaa8e99aba76e2d4a9eb3a758dfdb6a
0ec3a0d3fe56d30ada1fb923242c73954e0eadef
1350 F20101114_AABIUH cardonaponce_j_Page_004.txt
cc67c357585c516d3053cbc007803965
51b3a850c4f06896fee96318021c98823a9ee7d6
F20101114_AABJAB cardonaponce_j_Page_091thm.jpg
1753e093d21a473d4f9ad1aa053ff150
aabf2d2ee26b9b3449151e509de43fb135d67f3c
3252 F20101114_AABIUI cardonaponce_j_Page_005.txt
8186cc77b584ea8e834d600888ce683d
bdb170e094920eea0680304b018d324289511150
29547 F20101114_AABITT cardonaponce_j_Page_066.pro
9a0d62bf5c4dd6996dc8960dded8f352
89890ef22aabcdef9d719c20e010b0c8fa16644e
35194 F20101114_AABJAC cardonaponce_j_Page_091.QC.jpg
8861e3bb78959d0c8f8eefc359549ded
d802452cf529bced359af36cd1b70bc806356a67
3283 F20101114_AABIUJ cardonaponce_j_Page_006.txt
4fcc71f62387cbd6780e1d614d8d1556
5cff987e8f7cc5362fd61a4f92060ab411ee687c
53128 F20101114_AABITU cardonaponce_j_Page_069.pro
482b5671a369e87a94ddd384ff7504bc
5a644bee56d610b1fbfab125461ffb499768e216
35130 F20101114_AABJAD cardonaponce_j_Page_092.QC.jpg
3d8d6155e7bee4941ff0524eb1eb9ec0
ca52f5913b97992b56f0fda1f6f14e003aaca286
2798 F20101114_AABIUK cardonaponce_j_Page_008.txt
c603b72d4bdfee50f1b11086c31369a7
81131d49eb2c210a2db19eb1c5109f18dcb5e14e
22023 F20101114_AABITV cardonaponce_j_Page_070.pro
0c5d83dc7e1e81b329131aada57e3a80
d4747d28451a5d6c355990dac4ee14d77f4c0918
13400 F20101114_AABJAE cardonaponce_j_Page_095.QC.jpg
64114b66fef28dc0c610de64a57f5700
995e24a7d69433af6994d37a8f564b430cb6c102
2039 F20101114_AABIUL cardonaponce_j_Page_012.txt
c0d9af04e17439ec619b70072a10e657
4fb88e8343bb35d8b0aec9a67fc70fb684f0803b
56647 F20101114_AABITW cardonaponce_j_Page_073.pro
dafe9dcc4eb7e55e2f0bc14938f5cfce
5b45dbd287840e10f2f7ab5696e801ff1f8c3455
9035 F20101114_AABJAF cardonaponce_j_Page_096.QC.jpg
f48bc3119f759292a3f00c7397f8df3f
a52475f89bdcb6504658cc38c0dbebb4d17069ba
737 F20101114_AABIUM cardonaponce_j_Page_014.txt
df50c44a70401158621b11c587686fcb
4fe2f414d3b46a7ed60d427da8fd6b78510f6cc1
43426 F20101114_AABITX cardonaponce_j_Page_079.pro
1bf293d1e81a0f85b71a50e0b54ddd92
f11e7902f88f2a49816e51be77213aef85ca7f54
9856 F20101114_AABJAG cardonaponce_j_Page_098thm.jpg
bdcb77a6277145622bc551ff982b36d5
32ea5cf175bbc319e98db56f6feca0fb5f1c27e3
1708 F20101114_AABIVA cardonaponce_j_Page_034.txt
05714a8f1b8bbab04b79250d64e81614
39ed64ae008fa9ded24acfc704d1bf5c59041978
1747 F20101114_AABIUN cardonaponce_j_Page_016.txt
249a6dfdff65a9616a56f3148938663d
de522577cd40ce5d4319ba9f6310d2226e183a9b
44993 F20101114_AABITY cardonaponce_j_Page_081.pro
d309e575231c8bc099c46307a7bb08d0
0a73a7c0946bc9813518af36a672d9501d0d5a22
9522 F20101114_AABJAH cardonaponce_j_Page_100thm.jpg
fe1b5c9836fb53098ee61be984c35444
cd3dd90d2bd0a6e74a3267dfd29c6b11e4573e15
2099 F20101114_AABIVB cardonaponce_j_Page_036.txt
a4eed2198c92be1ff7374bb2eb63801a
5f304e7c0155ea49f6e77f1cb7aed2470dabb091
2156 F20101114_AABIUO cardonaponce_j_Page_017.txt
91286f13f85ab25632c08130b9801c3a
e8aa2dbf7d8579b0410c5282f92b63f2a7c34559
50025 F20101114_AABITZ cardonaponce_j_Page_085.pro
b23af5e9c4a96c4032fa25d4c0a2b72f
ad6be7bc02b600130e6921c70b99a970fcb06c64
38033 F20101114_AABJAI cardonaponce_j_Page_101.QC.jpg
bc8ad68b9f3fef2e1629405d718fa0f5
57cacd2008e0433ccb4d3846d130ee2af4324c53
2088 F20101114_AABIVC cardonaponce_j_Page_037.txt
bc30a4a0eb62f343414f2e95002e8dd3
247f015c965dabaa48c0581066fc01303f623db5
2005 F20101114_AABIUP cardonaponce_j_Page_018.txt
e5b547963324022d51d82c83ceac2eb3
f2b9f65c777351477aefaa53d148bc25784f2dac
9294 F20101114_AABJAJ cardonaponce_j_Page_104thm.jpg
2fb6b077da52c5d47952c9e94dafa7cd
f703ec7ec7cc1e3ad3fc9bb5b968d2ed630bf309
2030 F20101114_AABIVD cardonaponce_j_Page_040.txt
d35d49dc675bb01fe07deceb037fb902
1d4393783c86c6cfeb4c4f4b503d1e769ee03e5b
1691 F20101114_AABIUQ cardonaponce_j_Page_019.txt
fdaf21a2bc55eb0307d888ccac1f7052
353ee98cf3477af3075b11461d458eb88b48c009
38496 F20101114_AABJAK cardonaponce_j_Page_105.QC.jpg
0ff080b82c19f1e1475638bafee19e4d
871578cb27ad573c75c21be49a7f75d609cdc0f7
2038 F20101114_AABIVE cardonaponce_j_Page_043.txt
6f18676f370c7dfa41a055c040a8fe9b
f29f2d7d105e8bdf13d464ef78ff2baccf5365cf
1742 F20101114_AABIUR cardonaponce_j_Page_021.txt
f4cf2d308b02a40ecd3ea91f596c2270
ed3bffd9fa2067b9c70eab9e4b08163a60619bcf
4103 F20101114_AABJAL cardonaponce_j_Page_106thm.jpg
98218a067ae554ece74ed7c90fd8264b
77726e81e60e0904c9e9471d88c56fc2e25cde26
2101 F20101114_AABIVF cardonaponce_j_Page_046.txt
a98a83f7fa8446e051fb255850a420d5
b12d1aa65118cc0216f56fcf3c591c7e88c7d67c
2067 F20101114_AABIUS cardonaponce_j_Page_023.txt
19afcd14619695ed99e2b16b5799fb8e
26977ecceeaf992458c60d6eb75d950690313aa5
2108 F20101114_AABIVG cardonaponce_j_Page_049.txt
53cf3d84c4e18101e662cc33aeb14d0c
8900cd4c62783831c3b2fa1d82fb19061fa5fe37
2085 F20101114_AABIUT cardonaponce_j_Page_024.txt
a72f5b0d87139de66349920cb379f0d3
277115001ce81492ac0de836865aab00fcea8870
2041 F20101114_AABIVH cardonaponce_j_Page_050.txt
8ca563a8c0c0486cd5e416d4658c63a2
bbb49f53686ef1075b1765ff260e03dc026a2c36
2217 F20101114_AABIVI cardonaponce_j_Page_052.txt
5c838f566fd9bcae4ccd8add0f2d8e0e
cbac16ed0ac522c599d43694acd5eebbe29dace9
1498 F20101114_AABIUU cardonaponce_j_Page_027.txt
93bfd91c76b9d75f3762edb71daf2378
bc17b60ddda5ce38baa4b0b78fdb52918aea221d
1660 F20101114_AABIVJ cardonaponce_j_Page_053.txt
04395e0fe97e1b771214abed1d8a6d38
3d07df2434eb6fc0161b4d67e1d993806741bd04
F20101114_AABIUV cardonaponce_j_Page_029.txt
8a09a500c6c10d9c2667ad303a06d6cc
2488cc86a323d800b9cb4b80f0d1282ca3a1caa0
1895 F20101114_AABIVK cardonaponce_j_Page_055.txt
8d372236b771890f2c8b3eb6dbb99c78
c3f20eaa34c3947fde655515c66b7adc5b67dbe5
2111 F20101114_AABIUW cardonaponce_j_Page_030.txt
ce7ccb537e6475164c14e6a3c4e836f3
4082572b022996d75577f38cf88f406fcba906e1
1627 F20101114_AABIVL cardonaponce_j_Page_056.txt
fd575ae79f26feed9aca03ea90d9fdff
1af2385553e89d699c400d1264a5cfacb4e15722
1442 F20101114_AABIUX cardonaponce_j_Page_031.txt
6980d9997e17607ccc3ba939a73433d7
032386f8f6e3e87689f12d97c5be3a9828592d76
2173 F20101114_AABIWA cardonaponce_j_Page_092.txt
d6fb04c5f8ab28ecc2b745a118e1318d
eb5cd730b2c7affe97e997ddbdff4425491250a7
1943 F20101114_AABIVM cardonaponce_j_Page_059.txt
8c51c1e987e1dd0569cedc55896f6a3e
38544c322cce1f4a9aef2b7a6ae01cc3cae80473
1713 F20101114_AABIUY cardonaponce_j_Page_032.txt
7ddfe5b7cfeb58caf44ee567c6f2957c
6c3935a348d3d3130f325443c9d227af1ffd2d55
2482 F20101114_AABIWB cardonaponce_j_Page_097.txt
349f673dadf7b18e34ad85d8b7497f7c
6e6695d641896c52f396ed2ed00bb41cb6ce9d93
F20101114_AABIVN cardonaponce_j_Page_060.txt
95c7732d494fa72a61686f95c455fd2d
c9c8fcf3cfaaea3075329fb31ada3178dc8863b7
1562 F20101114_AABIUZ cardonaponce_j_Page_033.txt
1f8d29093d3f446546d6416d2abec161
79dd134ef8965970c60a2e627355cff264496fa3
2511 F20101114_AABIWC cardonaponce_j_Page_100.txt
e346e55225909c8373129c77c2872a04
7df062a7f4a9b44571bce4fd3d2ab253068104d7
2076 F20101114_AABIVO cardonaponce_j_Page_062.txt
bb4a391056f211c142f2ebfa83e8c89c
052058117cb983ed88bbb1909ed30628f913881f
2538 F20101114_AABIWD cardonaponce_j_Page_101.txt
5f9530f6b0c6d2af1a0d2064ad95fef3
69ae692bd9dd25df2921759a66a24db64ae5871f
2175 F20101114_AABIVP cardonaponce_j_Page_064.txt
1babfd48302f0d62f1794d1f9718a94d
9ae51401e51fec133660b116fcf04d6550d08361
2754 F20101114_AABIWE cardonaponce_j_Page_102.txt
61ac9e9a8ed88685ea01357520cd7e78
b002283719ddd985bd95a00e5b9f0cb48ec9c869
945 F20101114_AABIVQ cardonaponce_j_Page_070.txt
7815b74cd80cc198407cceda5b3e503b
a58a3ae493335d80f04f135a9949a6cf183fd4c9
2556 F20101114_AABIWF cardonaponce_j_Page_103.txt
555a4ba9e2f96c5c97177cfe9cac37bc
45fecc3409034bbcd1726c37c37b985f22f25cc9
F20101114_AABIVR cardonaponce_j_Page_072.txt
52cb9b59e8740169d45673d4f9505030
e621d2c07fe5ae3433be5a4ed8a34457c34f0921
2377 F20101114_AABIWG cardonaponce_j_Page_104.txt
bf6e5588d62fff6ca43c60c769499bbf
c20c323b4b60d39dde7e71de07ece54dad3e4a33
2406 F20101114_AABIVS cardonaponce_j_Page_078.txt
6ba3402e96ea046895c8662e7442cd70
4e8f5cdd6d2143c65d4bdb5a8cb04a7c4dac0919
634562 F20101114_AABIWH cardonaponce_j.pdf
803751df0e2057395402588e4f5d86c4
7ae522bfc8ccf04ad59e2a884eedc1bfacd6d6c1
1946 F20101114_AABIVT cardonaponce_j_Page_079.txt
5c97d3c29bd4d45daf245c653d703edd
5d5c1f109bfff2c66ae879ce23a4c87cc8ef123a
7399 F20101114_AABIWI cardonaponce_j_Page_032thm.jpg
fc4aefe3e69103b4f2d51ca018bc690d
f8163a005b063ecc1784765673c839e2ffa0905e
1916 F20101114_AABIVU cardonaponce_j_Page_081.txt
6d19fecc61115e3819975cf67240c12d
2af7e2adb8b27afa8640e545986ca98cd19abdcc
8913 F20101114_AABIWJ cardonaponce_j_Page_038thm.jpg
6dd9bc6ba8c31865aef2c2c10c71a674
43e79de8e5dd0fe7c6c068f0d559bd0e92ca6d3b
7119 F20101114_AABIWK cardonaponce_j_Page_027thm.jpg
cdf8ee545f66cb431450e802101579c8
74330725b347715e516bbc706a5ccd4c5c5e2cbe
1313 F20101114_AABIVV cardonaponce_j_Page_082.txt
057f2d44178bf9b15b7cf56303fb21cb
6c63d1558971aab510a495d2bc28cae2ed06fd99
38320 F20101114_AABIWL cardonaponce_j_Page_067.QC.jpg
f381417f6f75349fa7674d03b63d6068
328bfd4e95b47f68741d73f6e0600e83f8557f35
2450 F20101114_AABIVW cardonaponce_j_Page_083.txt
1d24dc73e1e1de63e5442d3d3121b78c
5c4e4415064bd79140e6ae1194d8c969c6298fa2
8950 F20101114_AABIXA cardonaponce_j_Page_043thm.jpg
5e2ec61a4dccb7befd698a379a21514c
fe6d8daaab592f58544fbc2ded7d18d5c289a3ae
8855 F20101114_AABIWM cardonaponce_j_Page_026thm.jpg
6c65c1764ef42d2dd6aac12d2cd357eb
e77671032244271203bf0b73130975e3e80cc149
2224 F20101114_AABIVX cardonaponce_j_Page_085.txt
b6dcd1d1e7949299b426aff1461b9503
a7cb3963732d6e1cc8dfefe728327725e1ffba67
9556 F20101114_AABIXB cardonaponce_j_Page_105thm.jpg
e52d705be38ec2470bd86065670fe730
3fc52eb9d2584debb0a05da2c843119ae611d36e
8625 F20101114_AABIWN cardonaponce_j_Page_048thm.jpg
14ca3b9987132aa272fa3ff0d8b5b148
89cac7e070833f9b2c3cd3532f4f0d4dc9467735
1455 F20101114_AABIVY cardonaponce_j_Page_087.txt
4fcd29b84bb8e75db231b1d19da74efc
3178977fed36d2a454c7180b9ad0c1734e6be7f5
37188 F20101114_AABIXC cardonaponce_j_Page_100.QC.jpg
0f4392627e488d85e0d614f1a6f986ce
b3390fbfee92445a0e6047912b54f3cc88a5e8e0
31170 F20101114_AABIWO cardonaponce_j_Page_034.QC.jpg
0271167d67eb9f4bad902174be01183a
68824011a0cbb26c7991a271aa8251350c085a18
971 F20101114_AABIVZ cardonaponce_j_Page_089.txt
49389fde4586b2a9acadac168e0cd9cd
4a9017d81783eb1ca196f54558f30cb01f1ea85d
6753 F20101114_AABIXD cardonaponce_j_Page_005thm.jpg
f2294107acfa1fa0552c17f664318f36
c57e1e6aaa33aea2a2ab6ae82628f2f60362184c
7264 F20101114_AABIWP cardonaponce_j_Page_019thm.jpg
486af971ca498430e3622b196f461cae
b26f3f2a49fb3c4f354007a3c82792478e2dc0fa
8560 F20101114_AABIXE cardonaponce_j_Page_062thm.jpg
23ed9d9c0a7e56f7ffad6f532cafbc02
6b4faa450d2df036d397a68bfcd3d46bce135ed0
36311 F20101114_AABIWQ cardonaponce_j_Page_046.QC.jpg
e17c6931ae0b2895dbdb4904e7713fd3
832ce48e8779c39ea5807ddcae020f7c7aa02de1
8954 F20101114_AABIXF cardonaponce_j_Page_052thm.jpg
9c1c767e9dc0c035ba9b2c6cfa22e96a
5c0ac1bf0c0858c4648218094e3f33496be99ca5
7777 F20101114_AABIWR cardonaponce_j_Page_071thm.jpg
3fc0c8399a080f78d007f21c8ee9ca75
5c96c100ee9c75d8a305ebe9434a4dce621d21ab
27862 F20101114_AABIXG cardonaponce_j_Page_053.QC.jpg
f55b4c7de13359e0e5ea35f86495596d
e0a028f0d588e58f794c5334c3707b6beb03aa12
28544 F20101114_AABIWS cardonaponce_j_Page_076.QC.jpg
54436d86ac2894ee0f52a4bb67fe6b9d
cd2c79506eaf2032250a1d76bf940e36c2f4046a
7313 F20101114_AABIXH cardonaponce_j_Page_076thm.jpg
f076c5a8afb74a4fe628df178e4f4834
d1aab01336252210b0aa3c773f93fd478772ceb3
8736 F20101114_AABIWT cardonaponce_j_Page_017thm.jpg
ded0f3864801b06e1e11f726598018c8
a7182d2cbbee46fa1427f3c0d290bb4983070226
26744 F20101114_AABIXI cardonaponce_j_Page_027.QC.jpg
d955ebc560800fe89d26d166122d24d1
d09278cf77972ebe69b90e03e3003e59691f0f14
16030 F20101114_AABIWU cardonaponce_j_Page_079.QC.jpg
7c3b223599280b81bafc973e1a3a46cd
e2f3bbcd4c1d1295bad5827d90b4939960c54b72
2139 F20101114_AABIAA cardonaponce_j_Page_035.txt
aeb5ead4c02b781dace9eae6f39e50cd
cb4355868819305e8f77315e67c8aa48d24eafae
F20101114_AABIXJ cardonaponce_j_Page_090thm.jpg
6abe6bb5f4d0be84366bdd9ee849399e
ce8b4f3da03d9fe66d45a2466713f6356438fa2c
39021 F20101114_AABIWV cardonaponce_j_Page_007.QC.jpg
78b2f57e6159f83a9226769d5ee0cc02
5314967ac72297d803beef3e1f3bec112f775c2a
F20101114_AABIAB cardonaponce_j_Page_060.tif
648376a824456c165fdd5869ced536ba
0a0aee44fd08b2340e9a725bbd61d8c86b700410
26268 F20101114_AABIXK cardonaponce_j_Page_058.QC.jpg
162e9486df42770fd19dbf190480a7c1
518e93cd7b3db049b19d3f69033f08e0c32d34e0
2258 F20101114_AABIAC cardonaponce_j_Page_063.txt
9a490be217050943bfec8d9c82c80c1f
dfc59447e7ce96490242bac23cbd8623d4c9328d
36553 F20101114_AABIWW cardonaponce_j_Page_042.QC.jpg
ccbf416315c222a368749226ceacc0e1
2392defd99b8d924b194e000e35393151e0e476f
7648 F20101114_AABIAD cardonaponce_j_Page_056thm.jpg
840edb341ca1a8bc75d2ba28d7a8523a
2ee885d6f91c1f6526400b3b132a2929833b1017
2756 F20101114_AABIXL cardonaponce_j_Page_093thm.jpg
5066d0a233a3d6e63624277051e74d2f
c087da69c97cddc7b0dbba60a45cf82751a46310
36764 F20101114_AABIWX cardonaponce_j_Page_097.QC.jpg
a1b0d73b490fc55f6b4d62d216f7e0f6
762e96caedaca6e5eee762a0a806b14b9ff95bee
606 F20101114_AABIAE cardonaponce_j_Page_093.txt
a0674f924f5c6165ca4f5dbeefe41258
815633345c2983b2e98d12e595087b2ee1e90402
32935 F20101114_AABIYA cardonaponce_j_Page_054.QC.jpg
f7f5dc0d512e00143ae5139876123d3f
3fb8d256c6aa4f6c72515558c1947de1faa2625e
9790 F20101114_AABIXM cardonaponce_j_Page_102thm.jpg
771c4341d3ce9f7303b0ea6679d01a32
65fb4af35344ddfa07534811844d399a4db94d68
8776 F20101114_AABIWY cardonaponce_j_Page_013thm.jpg
6e342583f14e8c7945860c0190d30972
9d0800c35362aeba8e9d8b46dc2179ac53c60132
F20101114_AABIAF cardonaponce_j_Page_071.tif
864bf5295d1b7fd434f88aeea45fce22
1de7e367cfcfed51f4945b5c746e3ee787c4b0c7
7498 F20101114_AABIYB cardonaponce_j_Page_053thm.jpg
b9cadfe98c5034bfcd6ee9ddee32bf82
6ff7e0fe8609fd4ae989ba85dee16a62773f54ee
8921 F20101114_AABIXN cardonaponce_j_Page_050thm.jpg
e956c95ef7fa5d07a5718db2980a5d30
fa86c19138830e4a5e0e0a6d28faa27ab73affde
31978 F20101114_AABIWZ cardonaponce_j_Page_010.QC.jpg
16a03817bde86e397586930b7a5e44cc
cb7c5d6ece171e7ce750af1d65f455fbad0546ca
F20101114_AABIAG cardonaponce_j_Page_022.tif
d589ef6f5cbf95135ff3a25254277162
ce087130deb42280722438d98f944ec7eb552010
36013 F20101114_AABIYC cardonaponce_j_Page_029.QC.jpg
3b6a6047a59091f04ca1ac2f01bd9e2d
bfa10b367a49b3000cf1668b71d9542d1f60d7e4
F20101114_AABIXO cardonaponce_j_Page_026.QC.jpg
8ad51cd99f8e2c5089eb38a3c84d0271
76d992d6f6ee650436312adf9e6e0e877b8918c8
8523 F20101114_AABIAH cardonaponce_j_Page_092thm.jpg
747043c8492f9cb02aaccc5a923ca28a
742b1cdc7cd91aa8f1cfaebfe5dbf49a7ee8bffd
15921 F20101114_AABIYD cardonaponce_j_Page_090.QC.jpg
0a5c2946b9bceb53075d11e75170ac72
5d91515145e087dd4023dae4ac1cbdc150ed67f4
24279 F20101114_AABIXP cardonaponce_j_Page_031.QC.jpg
ae78d72b91b5b83e907e3cc9e6f99104
3c3b40196a65722ea8404f84f72e05751a2836a3
2166 F20101114_AABIAI cardonaponce_j_Page_091.txt
cf4d162005f3777fad7168c9ef60cf80
1a322af289ad8e022355b7f159916280e74a3eb9
38648 F20101114_AABIYE cardonaponce_j_Page_102.QC.jpg
8f266ed7661310846886e2f49c2b0aa0
b0eb2221cb8cfb4d716d60b0e8c2b03c166072bb
8426 F20101114_AABIXQ cardonaponce_j_Page_012thm.jpg
7b06d1a91f468061a843cd5da7f06fa5
ebd186d38f52d6f9e6106768c8d34bff33bb4bb9
125950 F20101114_AABIAJ cardonaponce_j_Page_104.jp2
4cf1b4f2f773e7efdebf85b3dae64be9
74a03a0b7f62fbb2e1e113e8013b22b3513cea95
8840 F20101114_AABIYF cardonaponce_j_Page_042thm.jpg
3b84f7793e0abb81d41a8f33e1c38a66
4043087d6b9cb77b25d4324dc37a14e998c872e7
2682 F20101114_AABIXR cardonaponce_j_Page_096thm.jpg
1d5f5b0b476d1e2c61301d87a6b90109
88296afc6695963005ad70086916caf340366855
116318 F20101114_AABIAK cardonaponce_j_Page_013.jp2
83bff5397d4c4b25f8039e41cbaa3ad0
afe6407c336b5ce58f915f0ab8a0f0bf23e1ed64
29237 F20101114_AABIYG cardonaponce_j_Page_025.QC.jpg
e7a0397d513038acdcc717e303fc6d49
4f1454565bbba32ebcacd6f96584011e7f968fcd
8963 F20101114_AABIXS cardonaponce_j_Page_049thm.jpg
1ef55931c81d2867869f27b3a82bff25
9ec1703b7747abd819ceee7753280bd273b1c55d
34072 F20101114_AABIAL cardonaponce_j_Page_075.pro
9268dce5e684516edaa490b71a581de7
c923e8843ad3126094a933fda01bd12ddd745b1e
164051 F20101114_AABIYH UFE0021495_00001.xml
41aaa08aacf230e5457e24a084ce587d
bc6f85246595a672b1100f11e32636a87e136ed1
29509 F20101114_AABIXT cardonaponce_j_Page_028.QC.jpg
161f89085ed7431f020fdb12e8b94121
b438324bd1eca93807bc106803eb3cc441162d6b
F20101114_AABIBA cardonaponce_j_Page_016.tif
de70d10932526a61d340cad935243d5b
cbc998b66631f3d4d8ccdc012c3019df61392fd1
49995 F20101114_AABIAM cardonaponce_j_Page_020.pro
7ddc4bacecb15ec12291725765b6fea7
47ea6d247920b20885413bb6f6833cc8117fa204
8224 F20101114_AABIYI cardonaponce_j_Page_001.QC.jpg
87a2bcac8d594f6231aaf1876d6555f2
e6ebf0aa23b86b17cf5398e8a53908287bd820df
8981 F20101114_AABIXU cardonaponce_j_Page_067thm.jpg
00dd10c71b27464ac9595bf7e6c3d011
ce009dd0779dbaaeb10a37198f03e244189d6f82
7005 F20101114_AABIAN cardonaponce_j_Page_003.jp2
3d4202d97524e97da76d9b7ea105745b
aac90b77a05207e2fe1a7908491f2e933cdf5eda
702 F20101114_AABIYJ cardonaponce_j_Page_003thm.jpg
efe464fc25d10c5508e1668aad21dc27
14fc4ccd0aafc9f32baeb580187b92a714a21ad4
26692 F20101114_AABIXV cardonaponce_j_Page_075.QC.jpg
ecd36e3a3da0d9affed66bc7c185707a
b791ae8442fed348d30a9643f2f7d22e04eb3a49
53790 F20101114_AABIBB cardonaponce_j_Page_029.pro
192881b00d6e5c0e4b8ba6fc3bf582f7
c453df3187adb42328e9be4277b8aa5f20b2bad0
F20101114_AABIAO cardonaponce_j_Page_070.tif
89cc8a4257b4a1caffcc8ad6953a1fd5
d632bf095acd33bc490dee1bd06d8b5e3e0d202d
5909 F20101114_AABIYK cardonaponce_j_Page_004thm.jpg
3edfb0a0e67de2d64345eb00eb27305a
0cbd507d620b44cac7cab599229e58e44f8309d0
3396 F20101114_AABIXW cardonaponce_j_Page_014thm.jpg
93fea9f6354c6e5dd0446c0e4355fff4
8ab3257a733ca0d74c4b0262f24b16b51ff4693b
32371 F20101114_AABIBC cardonaponce_j_Page_059.QC.jpg
96b3eb6128e5702de2228b5ffcb1b3c5
0a4ac2d7b30d28530b9d6d80ea4c532cf395eadd
62277 F20101114_AABIAP cardonaponce_j_Page_101.pro
8e1bf9256756d50de71bdb1a17e94298
0151b8ddc4fed83b034315e7b374be97af2c4b7d
6852 F20101114_AABIYL cardonaponce_j_Page_006thm.jpg
ddb1f0f6878403583b455f398f5ede98
9e174c9038309bdba523a31dd6698ad45fbd3665
117809 F20101114_AABIBD cardonaponce_j_Page_051.jp2
473f662b0e3357c25fd57d597bcb269e
7173fe80ca44f98f7ac698987330e5e798b013d6
203129 F20101114_AABIAQ cardonaponce_j_Page_096.jp2
9c206218308bad9944e1a82e7192440b
9b0908cf8728de120a69093bfd2ab80d89e8df19
35957 F20101114_AABIZA cardonaponce_j_Page_036.QC.jpg
fe12964991391471c4cac77baa0e3bb1
2b89991f96891bcb647485cf0c1b192aa5716ba2
9443 F20101114_AABIYM cardonaponce_j_Page_007thm.jpg
5691812d5e44f20692d94c7a881468a7
30497cb5a1540d8dae0210292e823ed6e293bc23
34874 F20101114_AABIXX cardonaponce_j_Page_062.QC.jpg
e9d10b94aa5115a6f0219cbc1e935424
fd3c30b3e9715965677b09342e6d4d5e31b96998
1931 F20101114_AABIBE cardonaponce_j_Page_054.txt
5d5fe42015c529f03dc624bacdf4348f
28306196432813c48aec4c68c3e1a8f61bec0e0a
F20101114_AABIAR cardonaponce_j_Page_059.tif
5e4923d4338439985070c95b30cf2827
51372d8a236f81180bf68dcab0e8775bcec012bb
35392 F20101114_AABIZB cardonaponce_j_Page_038.QC.jpg
5b33dc54c59ed8875b0d2b3c40e3a756
d56073de219dfbf13af96b69e74231f613409415
8906 F20101114_AABIYN cardonaponce_j_Page_008thm.jpg
3ad4fd5ac6c456a798ff526431d13c78
46ae9f94a5406a4dbc22484d9f60c809dea7e70f
9632 F20101114_AABIXY cardonaponce_j_Page_103thm.jpg
57de72befa48656cf9bbfb8885891897
9a2fb2281cbefc7b502a6edb1c9bd41d289ffd71
32594 F20101114_AABIBF cardonaponce_j_Page_020.QC.jpg
4134d5471f4d91725a70fcb420be3bb2
0abc7de561b5df86ff1105fc3b08b97ec10579e1
37358 F20101114_AABIAS cardonaponce_j_Page_099.QC.jpg
b6954c80be520ff00020ab74936fa962
0ec3e6991a936052fd047e0de9d0b8c4f522a2ec
27859 F20101114_AABIZC cardonaponce_j_Page_039.QC.jpg
66971acafdb678baa43f778628d1d588
9122458f6fbca51ce7c1ac53ac4e5ec70fd53478
7647 F20101114_AABIYO cardonaponce_j_Page_009thm.jpg
740ef7f8a4538032d2706615cc9f2d08
f503f76cbd0ef8e2e9c87929626adff711fdc959
6570 F20101114_AABIXZ cardonaponce_j_Page_033thm.jpg
988efc17cc64cba79ccc9a9427aae8e0
7cd2462738821c82a805d71f6c111b9d1068125f
53614 F20101114_AABIBG cardonaponce_j_Page_092.pro
335526631535990be420ad8cdcede8de
11062b64bbdc3b5d843e9353b0e7f7b96dfcf113
1519 F20101114_AABIAT cardonaponce_j_Page_058.txt
6b06d78f5bffac72b2c214e170995677
7558d3d0c12531e08522123520bef0818d5a2f59
36731 F20101114_AABIZD cardonaponce_j_Page_045.QC.jpg
bcbf7dbf2aa1ea02af233776b71113d4
1327eedef7719597644af166c33a541b5cdd2795
30655 F20101114_AABIYP cardonaponce_j_Page_009.QC.jpg
cc5b61305966356f446f08232290d20c
c3bb20bd5d1f7d51a407d717d3e05b63738def39
8519 F20101114_AABIBH cardonaponce_j_Page_015thm.jpg
2b43f58aeeeffa68ff1e3e83bb5b4022
bbb77e48022e83469197124eb1e648e7e93c97a3
2384 F20101114_AABIAU cardonaponce_j_Page_051.txt
a29b12050b4fe5d80fb62231835d30d6
5fd3f0ff6b07a737ad3c1b714b6598fe83fc48da
8260 F20101114_AABIZE cardonaponce_j_Page_051thm.jpg
6833179e12f02b2a382e9152f210cdc2
f44e707e633a79fbec5eba93c1d40b42ef4e6fd4
33628 F20101114_AABIYQ cardonaponce_j_Page_012.QC.jpg
9b3298befd917584deb03c6454d9a8f6
b71d676afa77eaad5c13c844dc1220da1bc77a62
619 F20101114_AABIBI cardonaponce_j_Page_094.txt
ba73a86bd4ffaddb846ea3034e220f60
1f07cb1d9ce3ab93a2278fdbccde1d3a6cde2609
F20101114_AABIAV cardonaponce_j_Page_001.tif
82f2f47ad45b95f0dafb3d306f58ba8b
538b2e35df125dbd4de117ef0ad9b96a9f954394
33382 F20101114_AABIZF cardonaponce_j_Page_051.QC.jpg
fe83211a377e57626e07ebd26412e783
e3c0ad5840708df43ef675a01040890576712b12
34568 F20101114_AABIYR cardonaponce_j_Page_013.QC.jpg
b052eded760360e0669f1a6bbc190404
8852f8dc33f85a3b2157debe898592bac36f92dc
130007 F20101114_AABIBJ cardonaponce_j_Page_097.jp2
e3b1415cc03eca8e4588db103f19ea6a
372b45f034f4c4148c5026f8b5ff3367cae5490e
15237 F20101114_AABIAW cardonaponce_j_Page_093.pro
61f7d1b791fdf8179ea85064d6e3f2a0
e8f622fd55ffd898c3a0e92e6abf8d0595190ed1
38246 F20101114_AABIZG cardonaponce_j_Page_052.QC.jpg
51782b128fc42f1f6644bacbbb5fed5f
0a9f58319fb41879763aaae704bdf73a8d8afe2c
29149 F20101114_AABIYS cardonaponce_j_Page_016.QC.jpg
982362b175056465cebd695df71269a3
a56071a49214769461a0ba94cb121f9141668c4a
28538 F20101114_AABIBK cardonaponce_j_Page_032.QC.jpg
b5082f195c79c7031fc835a7b5f3003d
eee6ceff172f59c7eedb48d8d426870a4787acc3
82171 F20101114_AABIAX cardonaponce_j_Page_075.jpg
48ab61fbb3fd96aab24c95e05ee5e10a
c13ea6fd8dec825fcecf6a8e4cf945f351793a1c
8131 F20101114_AABIZH cardonaponce_j_Page_054thm.jpg
da2c5b7e2ff187293b0d37f3319ba554
552439f50f521324c3dd691547adf91618b3906d
30645 F20101114_AABIYT cardonaponce_j_Page_021.QC.jpg
fe632af7f276e828c24c1724bc34da8f
07e9bf795be1288dce9bf7e3ba2d72e5784041a0
8344 F20101114_AABICA cardonaponce_j_Page_024thm.jpg
eb461f2fc861dd66fd761ca72aa8d3c8
63dca1b0534eb514a0cb3f1d262e8f325d92d771
F20101114_AABIBL cardonaponce_j_Page_064.tif
7e7f50a3ba65a64b29e009499f9c2fd6
682f444f11220fafbf50be16e231dcf28a7af6bc
F20101114_AABIAY cardonaponce_j_Page_062.tif
e160a6237490f3e1bb19a33255e19588
3d9ab3e970da784eef4f7bbcf5ed6933cceb4594
30870 F20101114_AABIZI cardonaponce_j_Page_055.QC.jpg
6e9261578b9a4955c8e70ac73219c8fc
b075c6a9361948ef01bac52102dfeb3bc83be2b5
34058 F20101114_AABIYU cardonaponce_j_Page_022.QC.jpg
d8a8f83f925826dfce272e3ab6e38aa9
a7f38b45d97c99a8f8c62d1a4f46b725d45b897c
63004 F20101114_AABICB cardonaponce_j_Page_066.jpg
8a0bc4fb6b035df04afdd4c56889f726
01b1075e241f71d11696035ba8d4d1d66c8c8f6d
4147 F20101114_AABIBM cardonaponce_j_Page_095thm.jpg
c3e3bad59f5311196604e8c595c59277
ea01ef7e6a6a5fed231102e24741ce8c9b8ce8ae
33948 F20101114_AABIAZ cardonaponce_j_Page_043.QC.jpg
5fcdf32b56efdbc43a7ab23d2dd15622
adafe4b9a3055f63357dc06aba5afb876c84fcec
29438 F20101114_AABIZJ cardonaponce_j_Page_056.QC.jpg
d36349a22aa2b2edaaf09e34c9ad5434
04479cfa1f81da5e2755c653d9f5b5b64ff4c0e8
8727 F20101114_AABIYV cardonaponce_j_Page_023thm.jpg
b286f0e6932ddcdac5e67e10c1586cb1
981ee4c3c1cad7ea40904115542aeb50a1b30d6f
50579 F20101114_AABIBN cardonaponce_j_Page_018.pro
c35a6d8d9a73e6d2c94b8ab1115a66d8
4e52440f14cbc967ba1adbac8dfd34e52772f0e0
9131 F20101114_AABIZK cardonaponce_j_Page_060thm.jpg
d4e9860a8ee04eb79b6055f06194b04c
2c9624460e4e2fe0381d3188519e251a5debc690
35322 F20101114_AABIYW cardonaponce_j_Page_024.QC.jpg
2745a18355d9ec2dad7257ceaa51b0ab
ea7da2c21102565f4673d3c52653f48beeb0f155
101509 F20101114_AABICC cardonaponce_j_Page_020.jpg
e83ac29f6ec629e14e2f92fee25bf5d5
833c17ed052850b8e09518c6ebc14319902de776
111337 F20101114_AABIBO cardonaponce_j_Page_029.jpg
9f45e57f97f49a78d1106ae1c1c5a00f
10205ee6084139ad9241112832a747cfa315f9e7
8382 F20101114_AABIZL cardonaponce_j_Page_063thm.jpg
0a16245fb69f9cf093490bfbe575b879
b8c7e0cdc1096033d09ea0684b3527acd0e7e094
8836 F20101114_AABIYX cardonaponce_j_Page_029thm.jpg
305263113bf30d4a124a42978b14ab1c
6cb6a181908e416e4cca2ffa4efa7e0056f54ec8
104284 F20101114_AABICD cardonaponce_j_Page_068.jpg
b3ba825f42fc083dfc16bc81a8b08c8e
5148f08a24f3d4831e65493a2eceda0b1e4455bd
56944 F20101114_AABIBP cardonaponce_j_Page_078.pro
d688af350a0e83d32da06ee33cd81b2d
cbe2f769ac1c29f2f0e7af1d786c82da870e8f31
36634 F20101114_AABIZM cardonaponce_j_Page_064.QC.jpg
0623ad4c27a5b077e7a6b36275f95ee7
399fe9322631e584d7a57058de12a8eada45dac8
F20101114_AABICE cardonaponce_j_Page_006.tif
ba21c9d8f96d2c81f873672d65d462e0
d31bd0be025435e7c6bc54d363be6af02d033ebf
121 F20101114_AABIBQ cardonaponce_j_Page_003.txt
9d0a822406be6e425e471047fdc77621
483cf3b5ab8ac21d127cd3601fc60cc28c8a2445
35237 F20101114_AABIZN cardonaponce_j_Page_068.QC.jpg
14d1c659d143ff827ec7da0a0afe8995
f11db835fc69699afd7ad4d9ea5f138cba74d610
6298 F20101114_AABIYY cardonaponce_j_Page_031thm.jpg
4fb52040fe9ee6e41b1ed09dee903a76
13bda512488b6766f3399ea70fa172da9b515743
36682 F20101114_AABICF cardonaponce_j_Page_049.QC.jpg
051e1906694f5b864068e5c32a9be147
bc9399d070c999d55c9a561f7be1d68051ff8eea
53988 F20101114_AABIBR cardonaponce_j_Page_090.jpg
c178e26ae016f0565b255f86339f841f
8827e130dae991822e781557e288cd4e247289c4
8720 F20101114_AABIZO cardonaponce_j_Page_069thm.jpg
13125b6a01a9324250a6e64829f5ed74
1ac61b2dba30a63315869b8e552082cbc250dde1
25356 F20101114_AABIYZ cardonaponce_j_Page_033.QC.jpg
9b6afd8af51bd0c8a75e93f5fbdc5703
d1c937d1cd9d0e234ed23cb70478167cea2bdcbd
8834 F20101114_AABICG cardonaponce_j_Page_035thm.jpg
4b394d5f16e733ecfc131d147868f001
5d627f5d88cc8c1cdbd8468a1e6d0fa9c508f140
8958 F20101114_AABIBS cardonaponce_j_Page_045thm.jpg
ea5c67b448e10fa84b71f7fc688b7916
29d75e4490b3812963afe39c35f346bb3d401421
35446 F20101114_AABIZP cardonaponce_j_Page_069.QC.jpg
f4fed7eacc660564cafe12c4818297a6
37e91fb55b0b109158cd6e0d4cd77e9672263b58
80253 F20101114_AABICH cardonaponce_j_Page_027.jpg
efe56aa444a9fa3b6786927b818ee50d
7bfcbd29f0149a5b83494fb2bbdf0f2eba6575be
9386 F20101114_AABIBT cardonaponce_j_Page_061thm.jpg
84ab1445b88a42af74cfdeefb0c200b4
f73617c90af0b8b6fc312850a7b6ce06acf8cadb
28486 F20101114_AABIZQ cardonaponce_j_Page_071.QC.jpg
c6953d02c0fe2c75da64e225d7702604
15b4b45cda6f73d2606832ebf9e078fcb049b216
2305 F20101114_AABICI cardonaponce_j_Page_074.txt
5f25b0cf4d8f40dedb61a1b70c656d52
a85da7461447a44689f4eadc8dd997d3ef76b7cd
33921 F20101114_AABIBU cardonaponce_j_Page_077.QC.jpg
e282d68d2e2ec3d0d5767e57a9d38aee
906149d6f1e2bb79291bd676bce07996362b3eed
8979 F20101114_AABIZR cardonaponce_j_Page_073thm.jpg
6dbd93ea3a112d8c07206f7f83b2c03b
11bf5be69cc7b4ac4fd5d145070b9ba73fdc860d
6849 F20101114_AABICJ cardonaponce_j_Page_086thm.jpg
29192ea0e686be86250741bfb3af2cb9
0072b796d82f6b97d8c51d6b03471507520840bb
98662 F20101114_AABIBV cardonaponce_j_Page_071.jp2
6d1528bc998dbad13e2d4833c4400774
429e77f08e974f40ea17b90e14e348b949277a9f
37798 F20101114_AABIZS cardonaponce_j_Page_073.QC.jpg
d58415a34319082c8b36beba850fd55d
5cbad5e8a34ee36cb12888ab8f062a2cf46fe0bc
23672 F20101114_AABICK cardonaponce_j_Page_082.QC.jpg
87fb79576ad94056bdd558f9c53404e9
aa616dd938529506456d6d28c1ff531cf66ada28
24118 F20101114_AABIBW cardonaponce_j_Page_011.QC.jpg
2367c9e577a5b8b8b000082ce017d9f3
0cbf89dbfb6c3a22aa78d1ab0458805a9a91b639
8571 F20101114_AABIZT cardonaponce_j_Page_074thm.jpg
5b2f6efccc6f5ae3e27050e73badc270
13d3e740b3787a6025568f692548bbc2feb753ce
111013 F20101114_AABIDA cardonaponce_j_Page_017.jpg
309c6ee15e71c21140491f9022836206
50fc837c84284674726fbcc75bf12831c277b8c7
F20101114_AABICL cardonaponce_j_Page_074.tif
ca241c75b2f348072a1b315a365f6734
dc634a7852ae2aee9cd8e78cf66f967dab316e99
114346 F20101114_AABIBX cardonaponce_j_Page_048.jp2
1b448d49d1f9e9425ffa3920d8cf51fd
661d52dc6ca2c2c20f5b4afe98fecf79d74bcd04
6936 F20101114_AABIZU cardonaponce_j_Page_075thm.jpg
0b6c28d03e0213847cc6b1000c664f65
e95c103196763abab3f5c904a606c9eecbf9d0e0
104385 F20101114_AABIDB cardonaponce_j_Page_072.jpg
1f797451831123e71ffdfc28c0e7f838
3256cbd321eb36a1b4d886653f5be0533007c4b8
28809 F20101114_AABICM cardonaponce_j_Page_065.QC.jpg
5030e2d074689a18d4dfece8a1cf0b85
8c5a551908e3db311b50819f06c6878881f186a0
1623 F20101114_AABIBY cardonaponce_j_Page_003.QC.jpg
03b2b3db465ca114c489e9a631b3ea15
6de5d5053d1ee11315e87d4d75608dbf1355eb52
8682 F20101114_AABIZV cardonaponce_j_Page_077thm.jpg
0e431f6f3ad03187de86de89d52b176c
b00cd56c053158af65540af57333ad47fa96077b
1051973 F20101114_AABIDC cardonaponce_j_Page_005.jp2
30471119a09228bacf6490bfa3f77822
80c2c51c83a6be1fd93f7b4cd72f9dcd565efcb0
107739 F20101114_AABICN cardonaponce_j_Page_050.jpg
a570004b9992737c02b249bd63b6e0ea
161b8d1a3a4bb8df4b3e5dfc44e7520b1bcd762b
769466 F20101114_AABIBZ cardonaponce_j_Page_086.jp2
02ba77edd5a6c0f6bb4c46bad97f78f0
2c7ee2c3a95f2dac7a14b95f7488715257d76277
35069 F20101114_AABIZW cardonaponce_j_Page_078.QC.jpg
498fff0fcb8ef4bb8ba51aa47b6644fd
d5986ef08ef3dc382ed0fe84220d834f40e21ad6
33733 F20101114_AABICO cardonaponce_j_Page_063.QC.jpg
41688c734a50dff2da6a0def796db868
a76f1c4debcb43a03dcf51c858cb74719000f265
34818 F20101114_AABIZX cardonaponce_j_Page_083.QC.jpg
d2e43b3ac97804ea7cb89df556935c57
bdc1aef8799e47beaacd26d3dbe9d41a5adb44b5
54242 F20101114_AABIDD cardonaponce_j_Page_083.pro
e1f2dc220caaf2c3d5083242e90903d0
460fb5e47958d8efc7261483d70d3f5f26509afa
F20101114_AABICP cardonaponce_j_Page_015.tif
a6f08e4607e632de5b366306e1334df9
a1dfa0e8f8aec8bbf34f976a8428a9acb38860f5
7999 F20101114_AABIZY cardonaponce_j_Page_085thm.jpg
32bd156ae8cc2ab25a0f69925a61934a
bf8e6f0607451200ebed7a6b82ee409967cd7bed
2097 F20101114_AABIDE cardonaponce_j_Page_069.txt
8e7527937d27b4bb978c3764045c9878
51780e5fce12b69456dfe8697b7d31c515e8e644
F20101114_AABICQ cardonaponce_j_Page_093.tif
24fec8b638851194bea8d0160504645a
45968b5e3a90a332e4e33fd4bbcf19138a7f0dfb
53396 F20101114_AABIDF cardonaponce_j_Page_036.pro
3f03a57d57498c4fb262c9ae96f77f63
92dfe1797e6004e6adddbbc6809595c89a05a866
113781 F20101114_AABICR cardonaponce_j_Page_050.jp2
65df3f3b1e6f6ff5b6811662d1996883
cec1c3c63aea6ce4baf7dc453e76b33bc7c4c706
24689 F20101114_AABIZZ cardonaponce_j_Page_087.QC.jpg
a3adeb18e3f6f5f4ee045efea4d0a5bc
092f0bdc27bd6a6d267de96dcab55a22e3578fd1
52014 F20101114_AABIDG cardonaponce_j_Page_068.pro
edaf8392c57cbc19d5685a2feda43b05
6ba53373ec378b9744191854f9507428718e4336
115509 F20101114_AABHYA cardonaponce_j_Page_073.jpg
b5cf3c8500d19f5e7f3f65ef23ec31bb
d776e2c054ac6955eb862f7ebc992ae43e93788e
4874 F20101114_AABICS cardonaponce_j_Page_066thm.jpg
fc42c051af8200293a9cf7764db8b60d
e808b61d79c3d388f2bb5d16797904496a3f3907
34283 F20101114_AABIDH cardonaponce_j_Page_018.QC.jpg
814bafee34923d5b501fb93daeb953d9
cb2461a533278fa93f5938931e50aaf21a3f7380
8960 F20101114_AABHYB cardonaponce_j_Page_078thm.jpg
84e63c1ea44914c2157df7e03c3e00ef
6c51a31feecffd3e8a857d744ca40a23d4b158fa
65695 F20101114_AABICT cardonaponce_j_Page_098.pro
659807ff932e3908f041440829b3741a
d89e8dc2668b26ed2c9d171c482d41c77a98b618
8114 F20101114_AABIDI cardonaponce_j_Page_010thm.jpg
42ef48075d9f33a5571a9ceb5ed84ffd
845f8894ca97443c4d2433afdc13aadc0537f869
145735 F20101114_AABHYC cardonaponce_j_Page_007.jpg
c37187eb828d2427af896592f6142757
3a9088389f4d74755d4e42cbb2e3364f98e48448
62622 F20101114_AABICU cardonaponce_j_Page_103.pro
1a478f44b9facf947c427a772d45598b
bba3bdae869ca306f4cfd1457287f08233ef23ea
35440 F20101114_AABIDJ cardonaponce_j_Page_011.pro
e0c0990caa4067ae1512dd49784c9a5e
12b02b246296eadda1470fdf6e64e57ea82c0506
F20101114_AABHYD cardonaponce_j_Page_104.tif
a9949a680b095cb61e9cc7c55ff98736
ee67ae478a01e1e62ca0f053af2e7f712ebe9042
35494 F20101114_AABICV cardonaponce_j_Page_015.QC.jpg
34d7cec1a803b640ec9d5f8fba7f2561
77c1256b71cde902d40f5e63d0361c910bd2abf1
F20101114_AABIDK cardonaponce_j_Page_043.tif
290d3c7c9409ed1231f507b8c1e7dca3
c1c130017c200e5a21dd63c9d63eb438f7a8d314
1030957 F20101114_AABHYE cardonaponce_j_Page_034.jp2
6d155e55e832d41da3bbbd20612ed6b0
b033cf0aaeec5e833f247985eeb76b92f0fc2c7d
914810 F20101114_AABICW cardonaponce_j_Page_032.jp2
d57deedd6360f5c676b9bcb08bf9e966
b79c834b5bb7684d28315ca9749141bb12cb311e
17972 F20101114_AABIDL cardonaponce_j_Page_014.pro
4deb576eb309ce81458ba223bdddd468
4c77972b253321beb8d02d951025e4da11395400
33191 F20101114_AABHYF cardonaponce_j_Page_081.QC.jpg
7cc52c73ee5730260408f3cde39bcbb0
4c75981953c5b2c61e2a6fbbf43b0939aa59299b
8839 F20101114_AABICX cardonaponce_j_Page_046thm.jpg
db30ff332b1c82d41fb295709e3325db
a10f98bd97dd2470e0063ba65bd393ecdd9a96e0
1363 F20101114_AABIEA cardonaponce_j_Page_025.txt
4c8b1abd35a56e3d17bebd6314e0e7c7
060fdc1e8ec103bafe390a231184d1e25e150087
F20101114_AABIDM cardonaponce_j_Page_051.tif
b94db6f464191c4f7b99d6d01dce655f
49dfc0e89260a03b63e80d7d5281687ace205c18
F20101114_AABHYG cardonaponce_j_Page_010.tif
f76b02826d63617c2fcea3c5db5a1b22
8df84b6930953009b66c608ab5a8c245ebcbe0ee
7849 F20101114_AABICY cardonaponce_j_Page_034thm.jpg
55dba187bc176821ce6b6c7dd312032d
98d9a579927c78c11954c14eff22ff02d153e4db
2181 F20101114_AABIEB cardonaponce_j_Page_015.txt
aa51792ce3880cd7ee186509ecf5fc9e
9aac8b9cb394fdb240dc43be935c0851b7085a22
70560 F20101114_AABIDN cardonaponce_j_Page_004.jpg
0e1a4218a092b80f7b1e8acf44d04496
888dae5901a6eaf74d7d139852776b53c31ed23a
F20101114_AABHYH cardonaponce_j_Page_102.tif
3f16d8171bb36effb78002fb83017aa5
f8da57c96ae40acf8b28ebb5ca7c484dc1384fdf
116696 F20101114_AABICZ cardonaponce_j_Page_036.jp2
989f5340efdcc12f01174eb6db8f033b
e3a60a5b639a2cd397a0a032d674a0127afd4059
52527 F20101114_AABHXT cardonaponce_j_Page_089.jpg
039e01276c46f001a0787c177fbecb91
2fed7233d4c7a7ec98845a1bee391c85756c6a8f
1051927 F20101114_AABIEC cardonaponce_j_Page_081.jp2
bb5ef05b1c81150906212f92712fb3c5
33f90ed9138b5e3056f937d24805f56c70308808
1989 F20101114_AABIDO cardonaponce_j_Page_047.txt
d8d5c97410d77abf7841b395a8e66f96
eb74d35c2d758c2a1c6946451df813195615c3c1
110312 F20101114_AABHYI cardonaponce_j_Page_091.jpg
43a5660821f20eba8d08619765ea70a4
67d0302227c7dcd987b494cfbf5fe18b15d2f014
F20101114_AABHXU cardonaponce_j_Page_079.tif
8d4eba98d4dc5a59cbf1d635ee7671aa
a1a6715a942eca0b20981fbc5ea61cae8a76c075
92738 F20101114_AABIED cardonaponce_j_Page_071.jpg
30075e8d10352abf5ef4814b71b58a20
b5b168eecfc959f5bffec0da7d7b262d51e5605f
22755 F20101114_AABIDP cardonaponce_j_Page_004.QC.jpg
f718c951f3c6314625e9277547719846
0d7e787bd698599d005b8e53cac148694fbf327d
56719 F20101114_AABHYJ cardonaponce_j_Page_052.pro
d9f230963612018d0085b15029984a61
0f31ad8e0f952f660c4c2e58742608d3c0ea7c4d
8366 F20101114_AABHXV cardonaponce_j_Page_001.pro
3fa9bf3d8e11bb1d3c24b36aa27dd584
a275942c8d2e052c2b5ee533e60c08459857d77d
85332 F20101114_AABIDQ cardonaponce_j_Page_032.jpg
f03739848224215cb64d698edb45c504
267aa87066b963bbb53579d15ace470924dd08b7
38149 F20101114_AABHYK cardonaponce_j_Page_061.QC.jpg
e49aeeb544febd27bafd799df5d67cac
fb0165ed44cc395689e39fd6eadc453f13ef74cb
F20101114_AABHXW cardonaponce_j_Page_086.tif
725edb6ef4b09ed9bc7616f7cf80f582
e731c11c0b34b6b5f33c3a63509005b1a750be66
8763 F20101114_AABIEE cardonaponce_j_Page_040thm.jpg
db3e979b15ef9888296293248674d2db
e79a80a4b3a3d5bc888abda6c65cb115d177b101
35999 F20101114_AABIDR cardonaponce_j_Page_104.QC.jpg
4588c6fc6485d7db26a7945e26a8a6f2
0fd52a43c1aca21c83ca223c24322ce60c284daf
1948 F20101114_AABHYL cardonaponce_j_Page_090.txt
4d4cc73be1e6f9c28aef0098ac5fb8dd
cf8f1171170bfcf5ad2101e263c832b2b738b97a
F20101114_AABHXX cardonaponce_j_Page_033.jpg
79c15630ddf3451706b58614160cae72
9d6c30ba2524d06f31e8d9801087057949b8acf0
103270 F20101114_AABIEF cardonaponce_j_Page_085.jpg
8dfa2589352de9369e53c70db73342a9
6f691515dc4111107b423ee847be8e41319b0cee
7703 F20101114_AABIDS cardonaponce_j_Page_028thm.jpg
224bbdb548d3e8262c2c324b44a875c1
c2b1e9532a03f261418ea76c1a966e2b3a79e47d
36551 F20101114_AABHYM cardonaponce_j_Page_095.jpg
235e1890e8a611e23ee959a9e3e11045
df50ced409b043f02324a4d263a834e1c41d8ff7
54233 F20101114_AABIEG cardonaponce_j_Page_079.jpg
56ef6e338528e701adfc6b49f35b1db0
f337a42af6713c564a2e9ab67aeac5f928fc3dea
124075 F20101114_AABHZA cardonaponce_j_Page_052.jp2
20e196f35f744b4697f2235b144a0468
fa3fd87b50d5bd3b6213a64a4810bda4dc961725
489 F20101114_AABIDT cardonaponce_j_Page_001.txt
caae9693512e48876dda427a295531a7
ddeb414f39ebed7bd20255ed067bc24c805d559f
8714 F20101114_AABHYN cardonaponce_j_Page_068thm.jpg
f3174606b21bce8624f9ffb8fbba54a6
ab7e1118e46c406c75083da33ce1f30f071162d1
42668 F20101114_AABHXY cardonaponce_j_Page_084.pro
9f919c9261d050b509dce2cec93d734d
a49012d8e0a82bed954736cf6e45157042fa4bb0
117971 F20101114_AABIEH cardonaponce_j_Page_057.jp2
523401f020dbeba3ba2d92df1e81315a
16e44001d0c476932bbe165659516c173b71a8a9
1169 F20101114_AABHZB cardonaponce_j_Page_066.txt
12684e0a93c3ffaf43be73654c924013
ad4fdd960ca1c4a40c9c9336515d917d8d047358
113404 F20101114_AABIDU cardonaponce_j_Page_068.jp2
fbc5afc6b118abc665ea871cc3c622e5
23b064a1382ac48ed0caa763eec6c45594ac0961
35608 F20101114_AABHYO cardonaponce_j_Page_057.QC.jpg
f081facf93286229eea39553d57e6211
afde8d20bed3923a960827437760053bc896a38c
5815 F20101114_AABHXZ cardonaponce_j_Page_011thm.jpg
54fe43c2836cf0ec555abd1d2d2df9dc
497f9a94eb9bc122a1bad3ab8b6942ab0e274754
F20101114_AABIEI cardonaponce_j_Page_092.tif
e30fe314533ee70121c377b12bf3271e
f75ecb78dce49d079560bceafbbd1c809c3ebca1
8888 F20101114_AABHZC cardonaponce_j_Page_018thm.jpg
2f22ca3df2853d7a0740af2350c07bbd
8e51e53f37ff49e2d74ae91fbc6e22138227e2b4
11413 F20101114_AABIDV cardonaponce_j_Page_093.QC.jpg
a1b2d1295502c4d11f72e12a7ff59ed4
0d7468494045ab233222277ee7c0b41935875994
F20101114_AABHYP cardonaponce_j_Page_083.jp2
3756a0d00ce9a807da6e525af87cc09f
26649097186de497e623ae21f6eb9e1176c8bdf1
8310 F20101114_AABIEJ cardonaponce_j_Page_081thm.jpg
6082175d62e78cfbf3b2f26087f5924e
d8277750d9e586fd85ee72e3e048b81ce588b2a2
85303 F20101114_AABHZD cardonaponce_j_Page_079.jp2
7df485bca3ee4644b9b361525abd8657
58dd4c6b8f11a226be991609a1e491fe57c58e1b
112072 F20101114_AABHYQ cardonaponce_j_Page_043.jp2
13f4a406ef98d9afb55c26f5558bbfc9
5a1aa024577ab93cbad4f29b2ac4c39dd5f44edc
52071 F20101114_AABIEK cardonaponce_j_Page_048.pro
724808fef85a95ec37cda4b1a453b744
86b28ff01cc6b0b88eccc6c788114a2bfee319d4
38499 F20101114_AABHZE cardonaponce_j_Page_076.pro
f633c248bbf33bee1247bbe533275c97
f7578f09537c2d58464860210bcd41799b10f1cd
F20101114_AABIDW cardonaponce_j_Page_042.tif
a173eae65fb16fe6667590daa46e435f
ca7689b28aafac39831293908d0e3f98e33fd1e2
1997 F20101114_AABHYR cardonaponce_j_Page_022.txt
46ccb09fd62abcfefb346d5899c3e045
002583ef63e7b38f24c8fb91126a27b439f24c87
2257 F20101114_AABIFA cardonaponce_j_Page_067.txt
2af759170d2dc3b6cba93e21a8ae0c11
8901f88b9729d05730e497abd8b28bad5fcaab92
110438 F20101114_AABIEL cardonaponce_j_Page_060.jpg
9641b4b1392963175a2ea1f009697d0d
bc0169f14dfb347b5121b3ec5a6b6814c78cd627
F20101114_AABHZF cardonaponce_j_Page_085.tif
fd9d953825a5ae65bd38f31228a1c072
633df8ca329ace413c6b73b02bb26e9e841ea38c
1852 F20101114_AABIDX cardonaponce_j_Page_084.txt
03e6f34c357968847398b38a1f16294c
fe2e31606b25f2508b837bf8b4927cfa2b63aad6
2082 F20101114_AABHYS cardonaponce_j_Page_038.txt
514bf1aa0ddf2f7da7946f9f6b11bda2
2982f3a0d204d5751df2cd51815613e082895f2e
111501 F20101114_AABIFB cardonaponce_j_Page_022.jp2
147f2a98d48b34d810c705004b8c59ae
5c9f9fdb523a8ea20e3be5bed67d87059708d688
110114 F20101114_AABIEM cardonaponce_j_Page_088.jpg
1ab918ab926733dee869aa2de7af4bf7
d9b735e041ca9642961665f05337d0590c14f8d1
4180 F20101114_AABHZG cardonaponce_j_Page_094thm.jpg
831653d7449811289d0bb35965268cbb
b014c95881dec6b9631141cf5124b8fb3ecbaccf
F20101114_AABIDY cardonaponce_j_Page_003.tif
120f20413c8bba64e7d7f8145fbed021
bb69c4d203f8318e341dd31f0beb3813913a49dc
F20101114_AABHYT cardonaponce_j_Page_101.tif
09a98a4b265fb68272a8a628399a7cca
54025df285f3cbbbf9b5a3b9a4c04a191b589834
117580 F20101114_AABIFC cardonaponce_j_Page_091.jp2
e55d806dd19ed632a5959c64a5ba5f5a
bde33976a2ffa5f0f99e9e6cd572cad707b04f05
27504 F20101114_AABIEN cardonaponce_j_Page_005.QC.jpg
286eb8bc6ddfa12716fff5dc97c31217
c1eb56b0aa5d5dfe2972a51e074b08e9b5d5764c
2137 F20101114_AABHZH cardonaponce_j_Page_044.txt
e20090c0bfe99c0987c3d686138f9761
5057ddb7663b68b15769356d0b20a87444aacd52
52778 F20101114_AABIDZ cardonaponce_j_Page_038.pro
48e9f16e060fcecd3d79f6f457939f9b
75a2c2b047be112afcd57e1b6625e3a4a44a11b4
16009 F20101114_AABHYU cardonaponce_j_Page_084.QC.jpg
cec8c092176bd203cb1db217a0417f15
d66eb7bd60f0e416d9fe3577a055e87db6fb388b
9483 F20101114_AABIFD cardonaponce_j_Page_101thm.jpg
f5cb16ab69b2388f92c5fed6292affb4
7c254ecd68320fb486e3c11670b5d9fe591d3cfb
107386 F20101114_AABIEO cardonaponce_j_Page_020.jp2
40493d2dea98212b31fdb45c87f1e698
d4832d96983c777a32df55808c33a6941bcb5842
F20101114_AABHZI cardonaponce_j_Page_026.tif
8b3acbec95061b99be528e347dc5ccff
0b6cd77a5560b3db2e4223f8f2afad46b84183eb
35743 F20101114_AABHYV cardonaponce_j_Page_093.jp2
8da101401daca8245c84ee89bb6c9744
7773ce4b18d9a9de4560433e111beba4a16857c0
38484 F20101114_AABIFE cardonaponce_j_Page_065.pro
b50fb2fb08ff9e933437ffa541ca73f8
f8488add1e9590e2cc67d2a0f4b86b9780f8d133
2583 F20101114_AABIEP cardonaponce_j_Page_105.txt
bddb404750560356fd44209b2025dbd3
c0b202dafef0f3549cc933b2b99c240a565100eb
39322 F20101114_AABHZJ cardonaponce_j_Page_021.pro
c2ce3a9e4e4e3d6de128e7eab53286a8
68400603ab697fac7075f3c38841d017628bbc1f
90206 F20101114_AABHYW cardonaponce_j_Page_076.jp2
35d95822d6003ffff51b2857d1824485
2d03e29fd8f2d48528f115c693a589fc15d0a350
57747 F20101114_AABIEQ cardonaponce_j_Page_104.pro
9a36e5340732340f32c0eb2793d7601d
9f5f9f5d3513318e1558191c399bcfb61579d4eb
F20101114_AABHZK cardonaponce_j_Page_036.tif
0c6c797d699491cf81de5887371f738f
063b7196b8c213740b99f42c1a73f25ac1a1a580
14230 F20101114_AABHYX cardonaponce_j_Page_094.QC.jpg
d4cb192a14b57c773d067e914a399ef6
d80808e8ddd11b4c62af92f2d9787f0bf803a23b
130895 F20101114_AABIFF cardonaponce_j_Page_100.jp2
0895c7e6e8170aff00de456076180c7d
d8a948367846d01438b98251f7317fac50b75002
67772 F20101114_AABIER cardonaponce_j_Page_008.pro
82e24f248b727a5e4aedb53156a9ce62
5a46df5665050326a454658fdced4d5fd6480b6f
37014 F20101114_AABHZL cardonaponce_j_Page_008.QC.jpg
89ab84ae2252cda1fb51025cc1af2389
13d79e06e81ed8e7994ba6d9249b0b2882d349e2
7797 F20101114_AABHYY cardonaponce_j_Page_025thm.jpg
815f694055c2a6492334d0a0bf94f0c0
b0746dabb309c7a1aad8fdf78c5210a29b1edb38
47531 F20101114_AABIFG cardonaponce_j_Page_088.pro
94cfc62d3b7bc1234c35873bd121c561
b331e2de06d17856c818cf3a08906ad4b974806c
F20101114_AABIES cardonaponce_j_Page_040.tif
630b2b71dc61de327b176921840fa60e
09161e74a1903d3e97e359419de0709329bbe804
118759 F20101114_AABHZM cardonaponce_j_Page_029.jp2
086e5a30d7bd81d4c0f58ac241bf10ed
ed2cf3dfc0626580d9120f74e5a9ec5b6ccab0a2
108622 F20101114_AABIFH cardonaponce_j_Page_015.jpg
9b27c6c303ee4707f953893762a22fdc
f86d0a460d77a367e55c12dc944f9c57a87cd345
117362 F20101114_AABIET cardonaponce_j_Page_060.jp2
4683c72aedfe30d12b4a8c4763b1f29d
3092bcf56d8fd58acf6b48b046935908df70244f
859280 F20101114_AABHZN cardonaponce_j_Page_053.jp2
3a62574675b6a11c15db356d8cc2cc95
b56dd2d10903377537c286ba1dfcc97b9b9035ea
8379 F20101114_AABHYZ cardonaponce_j_Page_037thm.jpg
fa436be3169ca8b5b26f3aefb39332e5
890d38792c709c37b2f37c542b13840aa7fc27af
36032 F20101114_AABIFI cardonaponce_j_Page_060.QC.jpg
a6cd6a174713795799e8e7f6bfa234fc
04dde2556cfdd7572c5b357ba943878ddf634d98
6290 F20101114_AABIEU cardonaponce_j_Page_070thm.jpg
592668d172f77321299f6f62494aca89
6434e957e13f3c5a80550d33b89210135358de41
7156 F20101114_AABHZO cardonaponce_j_Page_058thm.jpg
124288b8072da517870e94163c31782f
9f2aa9a711441d228d7606a6d40a0f81b2dc711f
F20101114_AABIFJ cardonaponce_j_Page_042.txt
1fca4df9327900e14f825b755f851cd0
0fdba35fe3ad679d954a4572a2bb22d13da1d260
1894 F20101114_AABIEV cardonaponce_j_Page_071.txt
673293055b2958a82391c3ac337cbc89
c9de8dcbe6db94a8350d6fb374f4ea95eb570ed3
7923 F20101114_AABHZP cardonaponce_j_Page_055thm.jpg
929761089bf49074ee252c7aec482f8b
a478e0933dc21a79a1599b0e4a829bb71a9412a4
F20101114_AABIFK cardonaponce_j_Page_099.tif
d464f6d5cefbb333a52911774602a9a8
fc5634f0782a25673ec16e2813cba07d25e38184
F20101114_AABIEW cardonaponce_j_Page_056.tif
37d4f9a2e6cbf8eeba472334c804f943
892b86743194130b02c80423cfbf34aaa2f21351
50505 F20101114_AABHZQ cardonaponce_j_Page_022.pro
f0cfbcb05e2e12119207410b7f2cf733
8e36825b109a6f077ff4ea4b6ef77bfc5767063c
403 F20101114_AABIFL cardonaponce_j_Page_096.txt
f11b7e08176c135fcc455d0096b0350a
87c6ecd7d3ee0d6771696d5b78fa10692235a616
42938 F20101114_AABIEX cardonaponce_j_Page_094.jpg
97365e9e46459eddc4c9abd969f414ca
4efb86fe3255c3d08bfd233ad571e2b7fc76df5b
74237 F20101114_AABHZR cardonaponce_j_Page_087.jpg
174dac038d716c0ea1eaa570f18c2258
bdd581b3094f857d2fb81a4d07827a8913d692b6
1263 F20101114_AABIGA cardonaponce_j_Page_080.txt
41a8fc4d8650e39e03d3d421b796b614
fd6b46aa96e201271ed606ade0e8297d9dfec00e
61207 F20101114_AABIFM cardonaponce_j_Page_100.pro
9cbcafc4a680ecfeadbae4eb65cb98ac
709aa36a0caf3f211d4bfcf452aabb9617d82245
60007 F20101114_AABIEY cardonaponce_j_Page_097.pro
cff1c69fdcc47420b3ed58b31d0e20d3
987a77f98c83edc3a25531815e0624877972649a
7070 F20101114_AABHZS cardonaponce_j_Page_039thm.jpg
323de166a93fcc5d0f7a5dcf937c07b0
8996f096c9544bf999d369af9bec9e85a1a70a44
33281 F20101114_AABIGB cardonaponce_j_Page_088.QC.jpg
3b290e3ce2f3a760c2bd5ebee4bacbad
6eb0fd40bcfd374da277167917c10b0c6284aaa8
1476 F20101114_AABIFN cardonaponce_j_Page_086.txt
35e23beaa25bef1a69707b1faa8e8f54
d574af998199dea5338820aa8c19d5dd7310f070
36938 F20101114_AABIEZ cardonaponce_j_Page_017.QC.jpg
de629d70ae8f8ba2ff722f68ee13b12b
134ce6ac48a119566791a6a6623ef55e1dc2f1a0
88558 F20101114_AABHZT cardonaponce_j_Page_056.jpg
4f78dbead6d90c03da9ba12ef2747052
ba6ba1f49a40f79f73d4bbdf9861cd026da76ffb
43480 F20101114_AABIGC cardonaponce_j_Page_041.pro
e1b02ceda875547febb99ca875117af7
85b8a8d4cdedf7a634e648cff284c25170714e92
8258 F20101114_AABIFO cardonaponce_j_Page_020thm.jpg
19fd72a790ca8db37c61a8c5543086ab
c67ba36a7a598ea9f9b256b231a91591a87844c0
F20101114_AABHZU cardonaponce_j_Page_068.txt
c554c2c53d8a89de0567f8dfbe81aeef
53ea198867b0ae8f8be6f14c48ae4b9cfa60c910
2138 F20101114_AABIGD cardonaponce_j_Page_045.txt
bb7aa7eb14b9f8bafe3b4f1a58a5f203
8745e1d40e9b539d252555112998ecdf9624bab8
F20101114_AABIFP cardonaponce_j_Page_090.tif
24a35fae35a69adc2a0b600946a984fe
b873cdd0ba91f5e4a82c4c2712c60b1fd50cbcea
F20101114_AABHZV cardonaponce_j_Page_011.tif
3a6f4fbe41443ae637033bcfa66daf49
41b56cafa91203fd878676384681c807d45db037
30319 F20101114_AABIGE cardonaponce_j_Page_006.QC.jpg
3012aba2b8c897ad52fe4c5d27f368df
c0fd7a18a5aef491f92a1c2e9123607bcf74e9f9
F20101114_AABIFQ cardonaponce_j_Page_041.tif
5ee0583a884484e0229916fe70d175be
1c499992e6e9265ed6f8da73b09733be27be7502
F20101114_AABHZW cardonaponce_j_Page_072.tif
5391a273d7db7a7d841771d1f4274af4
9522e24f5e5207ef26c32141d65a83cee43bfe78
35615 F20101114_AABIGF cardonaponce_j_Page_037.QC.jpg
71c0d78e3865cf2a19f11142b332479f
3eb70eb3298534badb2841d0b1d3146ac7496d6d
4075 F20101114_AABIFR cardonaponce_j_Page_084thm.jpg
dd52c8fa19a62aacbc27245f4d887894
64a0c45a81aadec6f3350dd55d641d421a384ad5
1051970 F20101114_AABHZX cardonaponce_j_Page_007.jp2
4cd2f8074e5999a17f0a31ae949a9ec3
93049cb8cb6468cd1f295565fdd694dfae84e38b
8216 F20101114_AABIFS cardonaponce_j_Page_021thm.jpg
b35555618a4fcd5fe6dbbe69719e2072
97f7049790a76fbd92aaaa57c9c7f0d11c2aaf09
26388 F20101114_AABHZY cardonaponce_j_Page_001.jp2
018c859a0d393b2a3deb28e697532229
43d899acae09e5456387a0875413d9209ee45576
F20101114_AABIGG cardonaponce_j_Page_039.tif
07331558876c4272b88b1fe55a7d41fa
9bb1fcfa66a1e46643569a6dc7a708022c98ca2d
7183 F20101114_AABIFT cardonaponce_j_Page_041thm.jpg
0d42b28e3da84e457b9f46e63e31af9a
332eb8eb616f4279143e55a4bcc32d07aee9340f
50535 F20101114_AABHZZ cardonaponce_j_Page_072.pro
9c6cf05f956df819118651f2285415a3
f75f1e3ebe950ec9896bd34495751ac0f8f7e1a3
33089 F20101114_AABIGH cardonaponce_j_Page_074.QC.jpg
fd030d491b1d2cad625f8f4727397289
78f2c6647bd42bac4343e845aff26e558dbd85d3
50799 F20101114_AABIFU cardonaponce_j_Page_043.pro
2414b97c6b0324aa18ec1f901714a66d
e67667df6ec73164246d1a16bce6bf34fc8ab87d
F20101114_AABIGI cardonaponce_j_Page_027.tif
6e3169766e6f658bb30c4dfe91c57416
27f33105b433e2073285e51fe5ea75f7b6f54e10
F20101114_AABIFV cardonaponce_j_Page_049.tif
fe783abb269502952d9caffeccb67aa3
796fa7bf0fb67f1656b94c6fe59cfd51fe7f886f
7261 F20101114_AABIGJ cardonaponce_j_Page_016thm.jpg
14304241c7dd73518722cfc01554c91d
37d05ac7ca17f9387c6b242101aad35b990697ec
108196 F20101114_AABIFW cardonaponce_j_Page_054.jp2
51bb88dc0db287e610e9be99a2946e6e
93ae54fccdae68710cdffde920e0a8317ddcf8f6
47955 F20101114_AABIGK cardonaponce_j_Page_077.pro
facb2efe1274690a759a5e6776a982f9
a2cccedab84957280bdf211d5915120ea483e84d
103917 F20101114_AABIFX cardonaponce_j_Page_022.jpg
ae9fcd6356f9728cbd510197afcbdd55
dd19681ce1cc9759c621900460bf7b830ba319ad
6395 F20101114_AABIHA cardonaponce_j_Page_082thm.jpg
01804d781b7c280f7b4782c47aa7eb76
04556806d12245d42e10c53622aa093e1652797e
8744 F20101114_AABIGL cardonaponce_j_Page_044thm.jpg
dc904a36e2b33b04154c075c49c2e78d
58149f08f68f99cadcb30472317d1ae51ac1aa78
2669 F20101114_AABIFY cardonaponce_j_Page_098.txt
721ac7adfc7ee3f291a5849d7a8377cd
1e7110cd0d79bd55a82736f7ee582a7d8b0a4a69
25547 F20101114_AABIHB cardonaponce_j_Page_086.QC.jpg
fd2de6108f88c66d971000f833d1f1f2
a69398d7c62c4ace9d6c61cca0b4154b5eeb3701
57599 F20101114_AABIGM cardonaponce_j_Page_080.jpg
dd5096b7bc7c7bf13198490a73a06325
a6102f83b24e5e67de692dc47a28308ff8243903
54516 F20101114_AABIFZ cardonaponce_j_Page_045.pro
5f02d9b172dfec3e7dc8df09e00c368c
6639476f2b2f2a5b80853b0d63bf7469f7b3449a
61837 F20101114_AABIHC cardonaponce_j_Page_080.jp2
fc3bf21234289df8ede02272d9ecdb0f
3220db79d934df0e003969bd960b700ce548523b
16149 F20101114_AABIGN cardonaponce_j_Page_106.QC.jpg
d672eae7edfa9bdb4311551bb57540f6
6eb7048bac3d8175f133ba7977048b615d2c9dda
9549 F20101114_AABIHD cardonaponce_j_Page_099thm.jpg
9551150b43a346200dcf22b12a56b2f7
957d1de914303356d62e9d2e468a1ef459ef7e4d
55410 F20101114_AABIGO cardonaponce_j_Page_089.jp2
2500ea5e5975c453bbb2d4be04a97e0e
79c14da126265557af31e7408a7e0f85722d98a6
51969 F20101114_AABIHE cardonaponce_j_Page_050.pro
318dcccad8ee364bcdb254dce4c8992d
77528de5ef79b93f8fd071d83b379c18c359eb32
22135 F20101114_AABIGP cardonaponce_j_Page_070.QC.jpg
9c65309b59236ebb277715169159ad81
ab58536f33383f7842c89290b9ca9bc10ecaea0d