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Flavor Stability and Off-Flavors in Thermally Processed Orange Juice

Permanent Link: http://ufdc.ufl.edu/UFE0021072/00001

Material Information

Title: Flavor Stability and Off-Flavors in Thermally Processed Orange Juice
Physical Description: 1 online resource (111 p.)
Language: english
Creator: Dreher, James G
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: chromatography, flavor, juice, olfactometry, orange, storage, thiamin
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The aroma active components of thermally processed orange juice were determined and compared between orange juices of good and poor quality. A general loss of fresh aldehydes including hexanal, heptanal, octanal, nonanal and decenal coupled with the occurrence of potent off-flavor compounds 4-vinylguaiacol and methional contributed to the differences seen between the good and poor quality juice. Of significance, the widely reported orange juice storage off-flavor compound a-terpineol was found in greater concentration than previously reported but without aroma activity. The aroma active components of orange juice were noted to change over time during storage at 35?C. Difference in aroma active compounds at 4?C and 35?C were seen, with a loss and/or diminishing impact of fresh aroma active compounds including (Z)-3-hexenal, octanal, (Z)-4-octenal and (E)-2-octenal. Differences were noted between glass and PET containers, with PET performing worse with the formation of off-flavor compounds eugenol, sotolon, 4-mercapto-4-methyl-2-pentanone, 2-methyl-3-furanthiol and higher aroma intensities of the well documented storage off-flavor 4-vinylguaiacol. Through a model orange juice solution, thiamin, the second most abundant water-soluble vitamin in orange juice, was determined to be the precursor for the off-flavor compound 2-methyl-3-furanthiol (MFT) and its very potent dimer, bis(2-methyl-3-furyl) disulfide (MFT-MFT). Both MFT and MFT-MFT impart a meaty aroma and are documented in meat flavor systems. Recent studies document them as off-flavors in stored orange juice. MFT and MFT-MFT increased in concentration over time at real world storage conditions of 35?C. Results of this study show the importance of balance in flavor composition and how packaging and storage can affect the quality of orange juice. Producers can take steps to add back the fresh aroma active compounds lost during processing, while designing the packaging to minimize storage off-flavors.
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 James G Dreher.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Rouseff, Russell L.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021072:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021072/00001

Material Information

Title: Flavor Stability and Off-Flavors in Thermally Processed Orange Juice
Physical Description: 1 online resource (111 p.)
Language: english
Creator: Dreher, James G
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: chromatography, flavor, juice, olfactometry, orange, storage, thiamin
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The aroma active components of thermally processed orange juice were determined and compared between orange juices of good and poor quality. A general loss of fresh aldehydes including hexanal, heptanal, octanal, nonanal and decenal coupled with the occurrence of potent off-flavor compounds 4-vinylguaiacol and methional contributed to the differences seen between the good and poor quality juice. Of significance, the widely reported orange juice storage off-flavor compound a-terpineol was found in greater concentration than previously reported but without aroma activity. The aroma active components of orange juice were noted to change over time during storage at 35?C. Difference in aroma active compounds at 4?C and 35?C were seen, with a loss and/or diminishing impact of fresh aroma active compounds including (Z)-3-hexenal, octanal, (Z)-4-octenal and (E)-2-octenal. Differences were noted between glass and PET containers, with PET performing worse with the formation of off-flavor compounds eugenol, sotolon, 4-mercapto-4-methyl-2-pentanone, 2-methyl-3-furanthiol and higher aroma intensities of the well documented storage off-flavor 4-vinylguaiacol. Through a model orange juice solution, thiamin, the second most abundant water-soluble vitamin in orange juice, was determined to be the precursor for the off-flavor compound 2-methyl-3-furanthiol (MFT) and its very potent dimer, bis(2-methyl-3-furyl) disulfide (MFT-MFT). Both MFT and MFT-MFT impart a meaty aroma and are documented in meat flavor systems. Recent studies document them as off-flavors in stored orange juice. MFT and MFT-MFT increased in concentration over time at real world storage conditions of 35?C. Results of this study show the importance of balance in flavor composition and how packaging and storage can affect the quality of orange juice. Producers can take steps to add back the fresh aroma active compounds lost during processing, while designing the packaging to minimize storage off-flavors.
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 James G Dreher.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Rouseff, Russell L.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021072:00001


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FLAVOR STABILITY AND OFF-FLAVORS IN THERMALLY PROCESSED ORANGE JUICE By J. GLEN DREHER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007 1

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2007 J. Glen Dreher 2

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To my wife, son and parents who have give n me unending love, support and encouragement 3

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ACKNOWLEDGMENTS I would like to thank my committee members Dr. Charles Sims, Dr. Renee Goodrich, Dr. Ron Schmidt and Dr. David Powell for their supp ort and guidance throug hout this project. I would also like to thank Dr. An son Moye and Dr. Kenneth Berger who were original members of my committee and have since retire d. I especially would like to thank my major advisor, Dr. Russell Rouseff for his mentoring, and most of all, for his continuing support and encouragement. I have learned a lot from him not only about flavor chemistry but also perseverance and dedication. I would like to thank the United States-Isr ael Binational Agricultural Research and Development Fund (BARD) for their financial s upport and O-I Analytical for the use of the PFPD. I would like to thank everyone who participated as GC-O pa nelists including Dr. Kanjana Mahattanatawee, Aslaug Hognadoittir, and Dr. Jian ming Lin as well as Jack Smoot, Kelly Evans and Dr. Filomena Valim for all thei r support while working in the lab. I would like to thank my family for sticking with me and supporting me to finish my goals, especially my wife, Renee, for her unending enco uragement. Finally, I would like to thank God for giving me the strength and gui dance to complete this task. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................8 LIST OF FIGURES .........................................................................................................................9 ABSTRACT ...................................................................................................................................10 1 INTRODUCTION................................................................................................................. .12 2 REVIEW OF LITERATURE.................................................................................................15 Orange Juice ...........................................................................................................................15 Orange Juice Flavor and Processing .......................................................................................16 Flavor Production ...................................................................................................................18 Terpene Glycosides .........................................................................................................19 Shikimic Acid pathway ...................................................................................................20 Maillard Reaction ............................................................................................................21 Strecker Degradation .......................................................................................................22 Microbial .........................................................................................................................22 Packaging ........................................................................................................................23 Gas Chromatography-Olfactometry .......................................................................................25 Extraction Methods .................................................................................................................29 Thiamin as a Source of Potent Sulfur Aroma Compounds .....................................................30 2-methyl-3-furanthiol ......................................................................................................30 Bis(2-methyl-3-furyl) disulfide .......................................................................................31 Thiamin Degradation Pathway ........................................................................................31 Alternate Pathways for the Production of 2-M ethyl-3-furanthiol ...................................32 3 AN AROMA COMPARISON BETWEEN ORANGE JUICES OF DIFFERING ORGANOLEPTIC QUALITIES............................................................................................35 Introduction .............................................................................................................................35 Materials and Methods ...........................................................................................................36 Survey of Commercial orange juice ................................................................................36 Chemicals ........................................................................................................................37 Sample Preparation ..........................................................................................................37 Gas Chromatography-olfactometry Conditions ..............................................................38 Time-intensity Analysis ...................................................................................................39 Sulfur Analysis ................................................................................................................39 Results and Discussion ...........................................................................................................39 -Terpineol, Furaneol, and 4-vinylguaiacol ....................................................................41 -Terpineol ......................................................................................................................42 4-Vinylguaiacol ...............................................................................................................44 5

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Methional .........................................................................................................................45 Conclusions .............................................................................................................................46 4 ORANGE JUICE FLAVOR STORAGE STUDY: DIFFERENCES BETWEEN GLASS AND PET PACKAGING OV ER TIME AND TEMPERATURE...........................54 Introduction .............................................................................................................................54 Materials and Methods ...........................................................................................................56 Chemicals ........................................................................................................................56 Orange Juice .....................................................................................................................57 Visual and Organoleptic Evaluation ................................................................................57 Sample Preparation ..........................................................................................................57 Gas chromatography-olfactometry Cnditions .................................................................58 GC-olfactometry ..............................................................................................................58 Gas Chromatography-mass spectrometry (GC-MS) .......................................................59 Results and Discussion ...........................................................................................................60 Aroma Changes over time ...............................................................................................61 Off-Flavor Compounds ...................................................................................................61 Methional .................................................................................................................62 Furaneol and 4-vinylguaiacol ...................................................................................62 2-Methyl-3-furanthiol and bis( 2-methyl-3-furyl) disulfide ......................................62 M-cresol ...................................................................................................................63 Sulfur Compounds ...................................................................................................63 Carvone ....................................................................................................................64 Vanillin .....................................................................................................................64 Changes in Fresh Juice Compounds ................................................................................65 (Z)-3-Hexenal ...........................................................................................................65 Linalool ....................................................................................................................65 Ethyl butyrate ...........................................................................................................65 Octanal .....................................................................................................................66 Acetic and butanoic acids .........................................................................................66 Trans-4,5-epoxy-(E)-2-decenal ................................................................................67 Container Comparison ......................................................................................................67 Conclusions .............................................................................................................................67 5 GC-OLFACTOMETRIC CHARACTERIZATION OF AROMA VOLATILES FROM THE THERMAL DEGRADATION OF THIA MIN IN MODEL ORANGE JUICE............78 Introduction .............................................................................................................................78 Materials and Methods ...........................................................................................................79 Preparation of M odel orange juice solutions ................................................................. .80 Sample Preparation ..........................................................................................................80 Gas Chromatography pulse flame photometric detector (GC-PFPD) .............................80 Quantitative Analysis .......................................................................................................81 Gas Chromatography ........................................................................................................81 GColfactometry ...............................................................................................................81 Gas Chromatography mass Spectrometry (GC MS) .......................................................82 6

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7 Injector Decomposition Study .........................................................................................83 Microbiological Analysis ................................................................................................83 Results and Discussion ...........................................................................................................83 Day 7 and 42 Aromagrams ..............................................................................................84 Aroma Volatile identifications ........................................................................................85 Quantification of MFT and MFT MFT ...........................................................................88 Thiamin as a Source of MFT and MFT MFT in Citrus Juices ........................................89 Possible GC Injector Thermal Artifacts ..........................................................................90 Possible M icrobiolog ical Artifacts ..................................................................................91 Conclusions .............................................................................................................................91 6 CONCLUSIONS ....................................................................................................................97 LIST OF REFERENCES ...............................................................................................................99 BIOGRAPHICAL SKETCH .......................................................................................................111

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LIST OF TABLES Table page 3-1 Summary of aroma active compounds found in good and poor quality juice. .......................47 4-1 Aroma active compounds in orange ju ice stored at 4 and 35C over 112 days. ....................69 4-2 Comparison of total overall aroma in tensity under various package, time and temperature conditions. ......................................................................................................73 5-1 Aroma active compounds detected in model orange juice solution .......................................93 8

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LIST OF FIGURES Figure page 2-1 Pathways for -terpineol formation from linalool and (+)-limonene ....................................34 2-2 Thiamin thermal degradation pathways A =thiamin hydrochloride, B = pyrimidine moiety, C = thiazoles moiety, D = diaminopyrimidine, E = formic acid, and F = 5hydroxy-3-mercapto-2-pentanone. .....................................................................................34 3-1 Normalized aroma peak intensity comp arison of good and poor quality orange juice. .........49 3-2 Aldehyde comparison between good and poor quality orange juice. .....................................50 3-3 Comparison of known off-fla vor components in orange juice. ..............................................51 3-4 Possible pathway formations of -terpineol ...........................................................................51 3-5 Individual response chromatogram of -terpineol GC/FID aromagram overlay. ..................52 3-6 GC-O aroma threshold determination of -terpineol. ............................................................52 3-7 Methional formation through St recker degradation of methionine ........................................53 4-1 Aroma comparison of day 0 and 112 (35C) in glass packaging. ..........................................73 4-2 Aroma comparison of day 0 and 112 (35C) in polyethylene tere pthalate packaging. ..........74 4-4 Aroma comparison of orange juice stored at 4 and 35 for 112 days in polyethylene terepthalate. ........................................................................................................................76 5-1 SPME headspace samples of GC-O aromagrams comparing day 7 and 42, where peak intensities were inverted for day 42 data. ..........................................................................93 5-2 Structures of select aroma active sulfur compounds detected in the model orange juice solution. ..............................................................................................................................94 5-3 Comparison between PFPD chromatogram and corresponding aromagram from a model orange juice solution stored for 7 days at 35C. ...............................................................95 9

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Abstract of Dissertation Pr esented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy FLAVOR STABILITY AND OFF-FLAVORS IN THERMALLY PROCESSED ORANGE JUICE By J. Glen Dreher December 2007 Chair: Russell Rouseff Major: Food Science and Human Nutrition The aroma active components of thermally processed orange juice were determined and compared between orange juices of above a nd below average quality. A loss of aldehydes including hexanal, heptanal a nd octanal; imparting aromas such as floral, green and citrus coupled with the occurrence of potent off-fl avor compounds 4-vinylguaiacol and methional contributed to the differences seen between th e above and below average quality juices. Of significance, the widely reported orange juice storage off-flavor compound -terpineol was found in greater concentration than previ ously reported but w ithout aroma activity. The aroma active components of orange juic e were noted to change over time during storage at 35C. Difference in aroma active compounds at 4C a nd 35C were seen, with a loss and/or diminishing impact of aroma active co mpounds that contribute to good quality orange juice flavor including (Z)-3-hexe nal, octanal, (Z)-4-octenal and (E)-2-octenal. Qualitative differences were noted between glass and PET containers, with orange juice stored in PET forming off-flavor compounds including eugenol, sotolon, 4-mercapto-4-methyl-2-pentanone, 2methyl-3-furanthiol as well as higher aroma intens ities of the well documented storage off-flavor 4-vinylguaiacol. 10

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Through a model orange juice solution, thiami n, the second most abundant water-soluble vitamin in orange juice, was determined to be the precursor for the off-flavor compound 2methyl-3-furanthiol (MFT) and it s very potent dimer, bis(2-me thyl-3-furyl) disulfide (MFTMFT). Both MFT and MFT-MFT impart a meaty aroma have recently been documented as offflavors in stored orange juice. MFT and its dimer increased in concentration over time at storage conditions of 35C. The results of this study show the importan ce of balance in flavor composition and how packaging and storage can affect the quality of orange juice. Producers can take steps to add back the specific fresh aroma active compounds lost during processing, while designing the packaging to minimize storage off-flavors and limiting off-flavor compounds through fortification. 11

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12 CHAPTER 1 INTRODUCTION Orange production has an enormous impact on the world and U.S. economy both as fresh fruit and juice. The total dollar amount spent in the US in 1999 was approximately $1.7 billion on fresh orange and juice combined (2007). Citrus is valued for its bala nce of sweet and sour tastes as well as distinctive aroma. Although the orange has its highest monetary value when sold as fresh fruit, over 90 percent of orange production in Florida is for juice processing (Chadwell et al., 2006). The flavor of orange juice is complex and the difference between a good and poor quality juice starts with the initial flavor quality of th e orange. The ripening process for an orange is non-climacteric, ripening only occurs while on the tree (Alonso et al., 1995). During nonclimacteric maturation, respiration remains level, decay is rapid and no definitive abscission time exists; whereas climacteric fruit such as banana s have an increased respiration during maturation and a definitive abscission time. For this reason, oranges are picked for the optimal Brix (primarily sugars) to acid ratio. As the orange matures, the acidity decreases while the Brix, or soluble solids, increases. Alt hough citrus is a non-climacteric fr uit, peel color may be altered after picking through controlled atmosphere storage. Stewart and Wheaton (1972) found carotenoid accumulation in Robinson tangerine to increase in the pres ence of ethylene at 10 g/mL, with degreening occurr ing after 1 week followed by carotenoid development from yellow to orange in weeks 2 and 3. The study also reported that carotenoid development is best at lower degreening temperatures and is inhibited at temperatures above 30C. The proximate analysis of orange juice is 11.27 Brix, 0.67% citric acid, 12% pulp (volume by centrifuge) and 0.0123% o il (v/v) (Balaban et al., 1991). As with most foods, the smallest component of the total, oils/aromas, contributes the most impact to the overall flavor of

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the fruit. The Brix/acid ratio is important, but the arom a composition can profoundly impact juice quality because much of what humans percei ve as flavor is really produced from aroma components. Aroma active volatiles are sec ondary metabolites formed during maturation and are concentrated in the oil glands in the peel as well as in the juice vesicles. Orange juice flavor is not only produced duri ng fresh fruit maturation but is also affected by subsequent processing and storage of the finished juice. The main factor which alters flavors during processing is heat. Thermal processing is necessary to create a stable product; however, heat can also alter the volatile composition by reducing some of the in itial flavor volatiles through reactions as well as produce off-flavors from non volatile precursors. Aroma composition will continue to change during storage because of certain chemical reactions. The extent of these chemical changes will be dependent on storage time and temperature. Packaging material can also affect juice flavor. Materi als such as low and high density polyethylene and polyethylene terephthalate can cause flavor scalping or addition of compounds to the juice through migration especially with the major oran ge juice volatile (+)-limonene (Kutty et al., 1994; Lune et al., 1997; van Willige et al., 2003; Fauconnier et al., 2001). There were three objectives in this study. The first objective was a comparison between orange juices of differing qualities, determin ing differences in volatile compound composition and concentrations to identify which com ponents correlate with good quality and which components correlate with poor quality. Secondl y, orange juice aroma impact compounds were determined in a time/temperature/packaging stud y to determine the effects of storage time and temperature as well as packaging materials. Fi nally, a model orange juice system was employed to determine possible formation pathways of the off-flavor aroma compounds 2-methyl-3furanthiol and bis(2-methyl-3-fur yl) disulfide that were detected in the first two studies. 13

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By determining the difference between a poor and good quality orange juice as well as flavor changes associated with different pack aging materials during st orage, a processor can tailor the add back flavor package or alter packaging material to improve juice quality. A real world application of my final m odel orange juice study solution w ould be the confirmation of the source of a potent off-flavor and the informati on necessary to alter processing, packaging or storage so as to provide the highest quality of orange juice to the consumer. 14

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15 CHAPTER 2 REVIEW OF LITERATURE Orange Juice Sweet oranges, Citrus sinensis have long been prized as a fresh fruit and as juice. As a fresh fruit, the orange ranks thir d behind bananas and apples in c onsumption per year in the U.S. (USDA, 2006a). As a juice, oranges rank number one, with Americans drinking 2.5 times more orange juice than the second-ranke d apple juice (Pollack et al., 2003) An 8oz serving of orange juice contains 100% of the daily value (d.v) of Vitamin C, 20% of the d.v. for folic acid, 15% of the d.v. for potassium and 10% of the d.v. for thiamin. Oranges are the most important fruit in the citrus family, comprisi ng roughly 65% of the worlds estimated citrus crop. Prior to the 2004/ 2005 season, the United States has been traditionally the second largest producer of citrus behind Brazil. Due to hurricane damage, the United States is currently the third larges t citrus producer behi nd Brazil and China. Approximately 68% of citrus produced in the Unite d States is processed in to juice, but 95 96% of Floridas orange crop in us ed for juice (USDA, 2006b). The different cultivars of oranges are split into three categories by the ripening season: early, mid, and late. Early cult ivars reach maturity before D ecember and include the Hamlin, Parson Brown and navel orange s. Mid-season cultivars reach maturity between December and March and include Pineapple, Queen, Sunsta r, Gardner and Midsweet cultivars. The late season fruit peak from March to June, with the main cultivar being Valencia. The navel orange is prized for fresh fruit consumption as th ey can develop a bitter note when processed into juice. The Valencia is the primary sweet orange cultivar grown in Florida and the world and is mainly processed into juice (Williamson and Jackson, 1993).

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Orange Juice Flavor and Processing There are four main categories in which orange juice can exist: fresh squeezed orange juice, frozen concentrate orange juice (FCOJ) not-from concentrate orange juice (NFC) and orange juice from concentrate (RECON). The firs t group, fresh squeezed, is highly valued for its fresh flavor and natural quality. The lack of heat treatment sets this group apart from the others (Schmidt et al., 2005). However, because the ju ice does not have any heat treatment, its shelflife is limited to a few days. Fresh squeezed juice is an important part of the European market (2006a). Frozen concentrate orange juice is concentrated by thermal processing, during which water and volatile flavors are removed. The flavor vapors are cooled and reclaimed in one of the first stage condensers and fractiona ted into oil and aqueous phases. A flavor system comprised of portions of the captured essence is then added back to the concentrated juice to restore some of the lost flavor. Not-from concentrate orange juice comprises the largest single segment in the United States, as it was responsible for 49% of the total orange juice mark et in the 2004-2005 season (2006b). NFC is pasteurized but not concentrated or frozen. NFC is the closest thermally treated juice to fresh squeezed in terms of flavor. Orange juice from concentrate is FCOJ that has been commercially reconstituted to single strength orange juice. The advantage of reconstituting FCOJ commercially is reduction in transportation cost to the producer. However, th e main disadvantage from a flavor standpoint is that RECON receives a second heat treatment when it is repackaged, causing more flavor loss and degradation. From a flavor standpoint, RECON is the furthest away from the fresh squeezed juice that is prized for its flavor. 16

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Of the four types of processed juice, the two largest groups consis t of NFC and FCOJ. The standards of identity for these types of ju ices are set in the Code of Federal Regulation (CFR) Title 21. The USDA has set standards for grading orange juice within the 47 Federal Register (FR) (USDA, 1983). The orange juice is separated into grades A, B and substandard within the types of orange juice. The main f actors affecting the quality grade include color, defects, and flavor. Other fact ors are specific to the type of juice and include appearance, reconstitution and coagulation. Th e color is scored as compared to USDA Orange Juice Color Standards with a max score of 40 points, with Grade A having a minimum of 36 score points. Defects include juice cells, pulp, seeds or portion of seeds, specks, particles of membrane, core, peel, or any other distinctive feat ures that adversely affect the appearance or drinking quality of the orange juice. Defects are scored on a scale with max points of 20. Grade A orange juice is considered practically free of defects with a minimum score of 18. Flavor is evaluated and scored on a scale with a maximum of 40 points and separated into three categories: very good flavor, good flavor and poor fla vor. Grade A orange juice has very good flavor with a minimum of 36 points and defined as fine, di stinct, and substantially typical of orange juice extracted from fresh mature sweet oranges and is free from off flavors of any kind. Grade B orange juice meets the good flavor standards, ranging from 32 35 points, and is sim ilar to the flavor of juice extracted from fresh mature sweet oranges but may be slightly affected by processing, packaging, or storage conditions. Poor flavor ora nge juice would score less than 32 points and is defined to fail to meet the requirements set for good flavor. As defined, poor flavor juice would be categorized as substandard orange juice. The main difference between NFC and FCOJ is the concentration step in FCOJ. FCOJ takes orange juice through a seri es of concentration steps taki ng the juice from approximately 17

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11.0 Brix to 65 Brix. There are advantages of FCOJ over NFC. The FCOJ process will strip off-flavors and excess oil in the evaporator. Th e evaporator cannot be used for NFC production; therefore a softer extraction is used to prevent excess oil add ition. The softer squeeze might result in lower juice yields as compared to FC OJ. One way to remove excess peel oil is to employ centrifuges, thereby allowing maximum yield. Grade A orange juice has a maximum limit of 0.035% by volume of recoverable oil (U SDA, 1983). By being below this level, essential oil flavor systems can be added. Not-from concentrate orange juice u ndergoes a pasteurization step to reduce microorganisms and to inactivate enzymes. The ma in enzyme in orange juice is pectinesterase, PE. PE activity is a major concern in the citrus industry. PE is naturally present in the peel, rag and pulp and is released during ex traction and finishing. PE leads to cloud loss in single-strength juice and gelation in concentrate. The thermal pro cess needed to inactivate PE is higher than that needed for microbial purposes. A recent trend in the United States has seen the consumption of NFC increase from 183.1 million SSE gallons in 1990 to 629.9 million SSE gallons in 2000. This has in turn increased the amount of Floridas or ange crop going to NFC to approxi mately 50% in the 1998-1999 season (Spreen and Muraro, 2000). Flavor Production Off-flavor production in or ange juice can be caused by many different pathways. Sources can include enzymatic off-flavors, microbial off-flavors, packaging, processing, and storage off-flavors. Storage off-flavors will be discussed in detail, examining the following possible pathways: precursor development, Shickimic acid pathway, Maillard reaction and Strecker degradation. Flavor precursors are flavorless compounds that produce flavor compounds in consequence of enzymatic or chem ical reactions that occur during maturation 18

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(usually enzymatic driven) or processing (usually chemically driven). Process flavors can positive or negative depending on the food matrix and desired goal, such as in the formation of garlic odor from flavorless precursor allin to th e garlic odor alliein. In grapefruit juice one reaction includes the formation of a characteristic grapefruit aroma of 1-pmenthene-8-thiol from limonene by the acid catalyzed addition of hydr ogen sulfide across the external double bond. Lin et al. (2002) found 1-p-menthe ne-8-thiol present in concentr ated grapefruit juice but not fresh juice and suggesting that this character impact compound might be a reaction product of thermally treated juice. The (R )-(+)-enantiomer of the 1-p-menthene-8-thiol is one of the most potent naturally occurrin g volatiles with a detection threshol d of 0.02 g/L (Leffingwell, 2002). Another citrus flavor precursor example is the breakdown of carotenoids, large C40, tetraterpenoid compounds such as -carotene into the smaller (C13) -ionone (dried, fruit woody aroma). Kanasawud and Crouzet st udied the thermal degradation of -carotene in an aqueous medium and identified -ionone as a volatile degradation pr oduct, showing an increase in concentration of -ionone with an increase in temper ature (Kanasawud and Crouzet, 1990). Terpene glycosides Another important type of fruit flavor precu rsors includes terpene glycosides. In this process, volatile terpene and norisoprenoid compounds are cleaved from nonvolatile terpene glycosides via enzymatic or acidic hydrolysis. Te rpene glycoside reactions have been studied in many fruits including the peach, yellow plum and apricot (Krammer et al., 1991) and grapes (Maicas and Mateo, 2005). Phos phate ester reactions are an in vivo source of terpenoid compounds. One example is the formation of geranyl pyrophosphate (PP), neryl-PP and dimethyl-allyl-PP from enzymatic breakdow n of mevalonic acid-PP (Lindsay, 1985). 19

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Terpene alcohols can also be formed through acid catalyzed hydrations. A reported off-flavor compound in orange juice is -terpineol (Rymal et al., 1968; Tatum et al., 1975). -Terpineol has a floral, lilac-like aroma, but when added to orange juice a stale, musty or piney aroma has been reported (Tatum et al., 1975). Haleva-Toledo et al. (1999) demonstrat e the pathways of the precursors, linalool and (+)-limonene, present in citrus juice, that can undergo acid catalyzed hydration to form -terpineol (Figure 2-1). The conversion of linalool to -terpineol is much faster than the reaction with (+)-limonene. However, it was noted that with the high concentration of (+)-limone ne in citrus juice, -terpineol production is due to both linalool and (+)-limonene equally. Perez-Lopez et al. (2006), show production of -terpineol increases after pasteurization of mandarin jui ce with a simultaneous decomp osition of linalool and (+)limonene. Measurement of linalool, (+)-limonene, -terpineol and terpinen-4-ol were suggested as a tool to monitor the qua lity of the mandarin juice. Shikimic acid pathway The shikimic acid pathway starts a series of reactions that can lead to several different classes of flavor compounds. Shikimic acid can produce other precursors such as cinnamic acid and ferulic acid which can lead to potent arom a compounds such as eugenol, 4-vinylguaiacol and vanillin (Lindsay, 1985). 4-Vinyl guaiacol is described as possessing a peppery/spicy aroma and is considered a major off-flavor. In orange ju ice it imparts an old/rotten fruit aroma (Tatum et al., 1975; Peleg et al., 1992; Naim et al., 1988). Vanillin has also been noted in orange, tangerine, lemon, lime and grapefruit juices (Go odner et al., 2000). The shikimic acid pathway also plays an important role in flavor production of wines. Lop ez et al. (2004), studied the aroma compounds from mild acid hydrolysates in Spanish wine grapes. The author found the shikimic 20

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acid pathway produced important flavor components in the flavor of red wine such as phenolic compounds guaiacol, 4-vinyl phenol and isoeugenol as well as vanillin. Maillard reaction The Maillard reaction, also known as nonenzymatic browning, is a very significant source of flavors in cooked foods. Depending on the food, Maillard r eaction flavors can be deemed positive or negative. Maillard reaction flavors in food systems such as meat (Mottram and Leseigneur, 1990), coffee (Montavon et al., 2003), cocoa (Countet et al., 2002) and bread (Kimpe and Keppens, 1996) are highly important and beneficial. On the ot her hand, the Maillard reaction is responsible for off-flavors in food sy stems like fruit juices and also produce pigments which darkened juice color (Tatum et al., 1975; Haleva-Toledo et al., 1997). The Maillard reaction take s place between free amino groups from amino acids and reducing sugars. Reaction products are dependent on not only the starting reducing sugars and amino acids but are also dependent on time, temp erature, water activity and pH of the system. As with most chemical reactions, the Mailla rd reaction rate increases with increasing temperature. Color formation is much greater in the Maillard reaction wh en the pH is above 7. However, at lower pH compounds such as furfural and some sulfur compounds are preferentially formed (Mottram, 1994; Mottram and Whitfie ld, 1994; Mottram and Leseigneur, 1990). Compounds created from the Ma illard reaction are classified into three groups: 1) Sugar dehydration/fragmentation pr oducts including furans, pyrone s, cyclopentenes, carbonyl compounds and acids 2) Amino acid degradation products including aldeh ydes, sufur compounds (e.g. hydrogen sulfide and methan ethiol) and nitrogen compounds (e.g. ammonia and amines) 3) Volatiles produced by further in teractions: pyrroles, pyridines pyrazines, imidazoles, oxoles, thiazoles, thiophenes, diand trithiolanes, diand trithianes, furanthiols and compounds from aldol condensations (Mottram, 1994). 21

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As previously mentioned, Maillard reaction products can be consider ed negative in fruit juices. One of the main off-flavor compounds in orange juice is 2,5dimethyl-4-hydroxy-3(2H)furanone sometimes called Furaneol or DMHF, which has been well documented to increase with increasing storage time and temperature in orange juice (Tatum et al., 1975). HalevaToledo et al. (1997) determined the production of Fu raneol in orange juic e is via the Maillard reaction between rhamnose and arginine in the presen ce of the acidic matrices of ascorbic acid in orange juice. Strecker degradation A closely related reaction to the Maillard reaction is Strecker degradation. In Strecker degradation, the reaction is the oxidative deamina tion and decarboxylation of -amino acids with a dicarbonyl compound (Mottram, 1994). One main difference between Strecker degradation and the Maillard reaction is the lack of browning products produ ced in Strecker degradation. Strecker degradations produce amino acid aldeh ydes with one less carbon including pyrazines, oxazoles and thiazoles as well as producing -amino carbonyls. Strecker degradation produces the potent methional with a potato-like arom a from the odorless amino acid, methionine. Methional has been noted in diverse matr ices including coffee (Czerny and Grosch, 2000), cooked mussels (Le Guen et al., 2000), cheese (Milo and Reineccius, 1997), aged beer (da Costa et al., 2004) and cashew apple nectar (Valim et al., 2003). Methional is an off-flavor in citrus juice as has been found in grapef ruit juice (Buettner and Schieb erle, 1999; Lin et al., 2002) and orange juice (Buettner and Schieberle, 2001a; Bezman et al., 2001). Microbial Another possible source of of f-flavor compounds in orange juice is from microbial contamination. Alicyclobacillus strains were studied as a source of medicinal off notes in orange 22

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juice (Gocmen et al., 2005). Thr ee medicinal aromas were identifie d and attributed to guaiacol, 2,6-dibromophenol and 2,6-dichlo rophenol in orange juice inoculated and incubated with different Alicyclobacillus strains. Packaging An important variable in maintaining the in itial orange juice fla vor is packaging. A variety of packages are available, including cans, glass, corrugate, plastics and laminates. An ideal package would contain th e juice and provide an inert system allowing no interaction between the package, the juice and the outside envi ronment. Glass containers are considered as close to a totally inert package as possible; however the weight of glass containers is a disadvantage in terms of transportation costs. Packaging materials must be evaluated on the basis of cost, weight and ability to protect the product. Scalping of flavors into the packaging and migration of flavors from the package into the product are two variables that must be considered. Tetra Brik (Duerr et al., 1981; Marin et al., 1992) as well as low density polyethylene (LDPE) (Kutty et al., 1994) have been shown to readily scalp (+)-limonene in orange juice. Van Lune et al., examined the adsorpti on of organic compounds in polyethylene terephthalate (PET) and polyeth ylene naphthalate (PEN) materi al (Lune et al., 1997). The premise of the study examined the importance of ab sorption of chemicals into plastic bottles and how the chemicals would effect recycling and re use by the consumer. If a consumer reuses a plastic container, absorbed compounds may be pres ent before refilling, causing the possibility of migration into the product. The migration can add non-typical volatiles to the product thus producing off-flavors. Absorpti on of methanol and toluene was reported to increase with an increase in temperature and is also affected by the compositi on of the plastic container. 23

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Fauconnier et al. (2001) studi ed migration from high densit y polyethylene (HDPE) into various liquids including hexa ne, ethanol, lemon terpenes and their emulsions. A phenolic compound was shown to migrate from the HDPE in to each test liquid and was most likely attributed to an antioxidant a dditive. The organoleptic effect of the migration, however, was not examined. Orange juice aroma compounds were compared over time by Berlinet et al. (2005) using glass and various PET containers Of note, the study determined no statistical difference in aroma composition between the packaging types. Aroma composition was determined to be affected by storage over time by reactions within the juice matrix. The researchers suggest the inherent acidic matrix of the orange juice produced acid-catalyzed reactions which lead to a loss of aldehydes, ketones, esters, aliphatic alcohols and terpene alc ohols; while increasing levels of 4-vinylguaiacol and furfural. Van Willige et al. (2003) compared the absorp tion of orange jui ce flavor compounds in LDPE, polycarbonate (PC) and PET containers. Polyethylene terephthalate and PC containers showed only small decreases in limonene, myrcen e and decanal through absorption; while LDPE had a more significant loss of limonene and a smaller decrease in myrcene, valencene, pinene and decanal. Organoleptic evaluation through duplicate triangle testing did not show a significant difference between packages at up to 29 days of dark storage at 20C. Glass, monolayer PET and multilayer PET package effects on orange juice quality and shelf life was recently studied by Ros-Chumillas et al. (2007). Ascorbic acid, vitamin C, was evaluated as a measure of shelf life with a minimum amount of 200g/mL. The monolayer PET had a significantly lower shelf life at 4C, with ascorbic acid dropping below 200g/mL at 180 days where the multilayer PET and glass were approximately 300g/mL levels at 300 days. 24

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They concluded that the shelf life of the monolayer PET orange juice can be extended through use of oxygen scavengers, nitrogen headspace and aluminum foil seals in the closure. Gas Chromatography-Olfactometry The use of gas chromatography-olfactometry (GC-O) is a technique where the gas chromatograph separates aroma mixtures into individual components and the human nose is used as a detector. Modern GC-O instruments use both human and inst rumental detectors by splitting the GC effluent between the sni ffing port and an instrumental dete ctor such as flame ionization detection (FID), mass spectrometer (MS), or pulse d flame photometric detection (PFPD). GC-O is used to determine which of the volatile comp ounds in a food matrix have aroma activity and thus contribute towards the overall aroma of the sample. The primary advantage for using a human assessor as a detector is the sensitivity and selectivity of the human nose. The human nose can detect some volatiles at extremely low concentrations such as bis(2-methyl-3-furyl) disulfide at a thre shold level of 8.9 x 10 -11 nM (Buttery et al., 1984). This is significant as the nose is often more sensitive to some aromaactive compounds than the best instrumental det ector. The concept of aroma value has been developed to determine if a volatile has aroma activity when direct aroma measurement is not possible or to determine relative aroma strength. Aroma value (sometimes called odor activity value, OAV) is defined by the ratio of the conc entration of an aroma active compound divided by its detection threshold. Aroma values assigned to a compound in a given matrix will therefore determine if and by how much the conc entration exceeds it threshold value (Mistry et al., 1997). How the threshold for a given aroma activ e compound is calculated can cause a large variance in the reported threshold. The interac tion between a compound and its matrices has an effect on the threshold. For example, an arom a active compound will have a different threshold 25

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if measured in air, water or oil. Generally, a volatiles threshold will be higher in a food matrix compared to water because the matrix interacts w ith the volatile to a greater degree than water. Plotto et al., (2004) determined the aroma and fl avor thresholds for key components in orange juice using orange pump out (concen trated orange juice whose volatiles have not been restored). They have reported odor thresholds up to 200 times higher in an orange juic e matrix as compared to published thresholds in water. GC-O has been used to characterize the odor ants in a variety of matrices from coffee (Holscher and Steinhart, 1995; Akiyama et al., 2002) to wine (Chisholm et al., 1995; Cullere et al., 2004) to orange juice (Marin et al., 1992; Rouseff et al., 2 001a; Schieberle and Buettner, 2001) to orange essence oil (Hognadottir and Ro useff, 2003). Determining which compounds in a matrix have aroma activity can impact current industrial practices. For example, traditionally the sesquiterpene valencene is used as an indicator of quality in orange peel oils. However, Valencene has been recently shown to not have aroma activity at concentrations typically found in orange oil (Elston et al., 2005). Early GC-O devices had two main limitations: nasal discomfort caused by hot dry carrier gas and the lack of sensitivity of the chemical detector as co mpared to the human nose (Acree and Barnard, 1994). Dravnieks (1971) enhanced the GC-O technique by using humidified air in combination with the effluent. Another limitatio n of GC-O is evaluati ng individual components outside of the original matrix (M istry et al., 1997). GC-O does not take in effect the contribution of the solubility of the aroma active compounds within the matrix or the interaction of the aroma active compounds with nonvolatile com ponents within the matrix. GC-O methods can be categorized into th ree groups: dilution analysis techniques including combined hedonic and response measur ements (Charm) and aroma extract dilution 26

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analysis (AEDA), time-intensity techniques su ch as OSME, and frequency of detection techniques including global analysis. Each tech nique has advantages and disadvantages that will be discussed. Dilution techniques operate by sn iffing the effluent of an extr act in a series of dilutions, usually in a series of 1:2 or 1:3 dilutions (Acr ee and Barnard, 1994). Charm analysis (Acree et al., 1984) constructs a combined response from several experiment s where the concentration of the aroma active compound is directly proportional to the sniffed peak area. Thus a compound that is detected after more dilutions is consider ed to be more potent than those compounds which can be no longer detected after a few dilutions. The relationship between intensity response and concentration is spelled out in Stevens Law: I = k(C-T) n where I is intensity, k and n are constants based on the type of compound, C is concentration, and T is th reshold (Stevens, 1960). For aroma, Stevens applies different values to the exponent from 0.55 for coffee odor to 0.6 for heptane (Stevens, 1961). Charm has been used to study anosmia. Charm values are reportedly proportional to the amount of stimulus while inversely proportional to the individual subjects threshold limit (Marin et al., 1988). AEDA is a dilution technique similar to Charm, where the flavor dilution, FD, values are comparable to Ch arm values. However, the main difference being that AEDA only determines dilution intensity us ed when calculating FD factor whereas Charm also takes a compounds elution dura tion into effect (Mistry et al., 1997). Another advantage of AEDA is that it does not require specialized so ftware as in the case of Charm. The main disadvantage to both dilution techniques is the nu mber of chromatographic runs needed to find the largest dilution for all compounds in the sample. Time-intensity techniques are similar to Charm as a compounds intensity and elution duration are determined without dilution. The original time-inte nsity technique is called Osme, 27

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developed by da Silva et al. (1994). In Osme the assessor continuously rates the intensity of aromas using a sliding scale from 0 being no detec tion to 7 being moderate to 15 being extreme. The assessor is simultaneously rating the intensity and characterizing the aroma. Panelists need to be trained to use the equipment as well as develop a common sensory language for descriptors. Aroma active peaks have to be dete cted at least 50% of the time by panelists in order to be considered aroma ac tive. A combined panelist Osme gram is then constructed. An advantage of Osme over Charm or AEDA is that no dilutions are made and therefore the number of chromatographic runs is reduced. The main disadvantage of Osme is the aforementioned training for panelists. Frequency of detection methods are simila r to time-intensity techniques however the number of panelists is increased while the traini ng per panelist is decreased or in many cases, eliminated. One main difference between fre quency of detection methods and other GC-O methods is the aroma peak intensity is based on th e frequency of detection and not related to the perceived intensity of the compound. One main disadvantage of this method is the number of panelists needed, ideally 8 -10 (Pollien et al., 1997). Frequency of detection has been used to characterize odorants in cooked mussels (Le Guen et al., 2000), red wine vi negar (Charles et al., 2000), Iberian ham (Carrapiso et al., 2002), French fries (van Loon et al., 2005), leeks (Nielsen and Poll, 2004), and fresh and smoke d salmon (Varlet et al., 2006). Frequency of detection has also been used in comparing odorants in orange juice of different cultivars, including blond and blood types (Arena et al., 2006). The study found difference between blood types (Moro and Taro cco) and blond types (Washington navel and Valencia late). One of the most intense aroma active compounds found in the blood types, 28

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methyl butanoate, was not found in the blond cul tivars. Conversely, linal ool, was only reported in blond cultivars Extraction Methods Most sample matrices are not able to be di rectly injected onto a gas chromatograph. The object then lies to extract the volatile components from the sample and be able to represent the original matrix. The two main types of extractio ns are solvent extraction such as liquid-liquid and direct headspace adsorption of the volatiles onto a solid phase such as Solid Phase Micro Extraction (SPME). The solvent used for extraction is dependent on the nature of the food matrix. Organic solvents are usually used in a matrix that is lipid free and includes matrices such as fruit, berries, and alcoholic beverages. A separa te preparatory procedure is needed to separate lipids from an organic solvent extraction. When extracting lipids there is no one sta ndard procedure and the method and solvent is again dependent on the food matrix (Marinetti, 1962). Often a combination of different solvents will give the be st results. One such matrix that often uses a combination of solvents is citrus juices, wher e a common extraction method is with a mixture of pentane and diethyl ether (Tonder et al., 1998; Lin et al., 2002; Bazemore et al., 2003). Liquid-liquid extractions can give different results compared to SPME. SPME fibers have been shown to selectivel y absorb volatile compounds through competition (Roberts et al., 2000). For example, Ebeler found in brandy th e polydimethylsiloxane SPME extraction was more selective for esters and acids than liquid-li quid extractions (Ebeler et al., 2000). In citrus, SPME is more selective for terpenoid compounds as compared to liq uid-liquid extractions (Rouseff et al., 2001a). A SPME fiber (carboxinpolydimethylsiloxane) headspace analysis of heated orange juice resulted in 86% of the total FID peak area from 3 terpene compounds (limonene, myrcene, and -pinene) as compared to 24% in a liquid-liquid extr action of pentane29

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ether. Rega, et al. (2003) worked to optimize a SPME method for use in orange juice, examining fiber coatings, exposure time and sample equi libration time. However, the optimized SPME conditions were skewed to minimize extraction of unpleasant odors and ar e therefore not fully representative of the juice. A recent study (Jordan et al., 2005) co mpared polydimethylsiloxane (PDMS) and polyacrylate (PA) SPME fibers in orange juice at different stages in pr ocessing (fresh juice, deaeration and pasteurization. The deaerated pro cess, as compared to fresh juice showed the greatest processing difference. Both fibers ha d similar results for alcohols and terpenes. However, a statistically significant change in aldehydes and esters was noted only with the PA fiber. The researchers concluded that the PA fiber is more suitable for use in studying processing affects on orange juice. Thiamin as a Source of Pote nt Sulfur Aroma Compounds Thiamin (vitamin B 1 ) is the second most abundant watersoluble vitamin in orange juice, and is a more concentrated source than many fo ods that are better known sources of vitamin B 1 such as whole wheat bread (Nagy and Attaway, 1980; Ting and Rouseff, 1981). Thiamin is readily degraded by thermal treatment, producin g potent sulfur compounds with meaty and roasted notes. This reaction is important in many food systems, producing flavor impact compounds typical in meat and breads. 2-methyl-3-furanthiol 2-Methyl-3-furanthiol, MFT, is a significant th ermal degradation product of thiamin. This potent sulfur compound gives an intense savory, meaty aroma. This compound is well known in meat flavor systems (Mottram, 1991; Grosch and Zeiler-Hilgart, 1992; Kerscher and Grosch, 1998) and has a low aroma threshold of 6.14 x 10 -8 mM/L water (Munch and Schieberle, 1998). MFT has been found in a number of different fl avor systems, including coffee (Hofmann and 30

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Schieberle, 2002; Tressl and S ilwar, 1981), cooked brown rice (Jezussek et al., 2002), beer (Lermusieau et al., 2001), reconstituted grapefruit juice (Lin et al., 2002) and as an off-flavor in orange juice (Bezman et al., 2001). Bis(2-methyl-3-furyl) disulfide Thiols are known to readily oxidize into thei r corresponding disulfid e. Hofmann et al., 1996 (1996) studied the oxidative stab ility of odor active thiols. Results show that after 10 days of storage at 6C, 53% of a dilute ethereal MF T solution was oxidized to its dimer, bis(2-methyl3-furyl) disulfide, MFT-MFT. Bis(2-methyl-3-f uryl) disulfide has also been reported in meat flavor systems (Evers et al., 1976; Farmer and Mo ttram, 1990). Bis(2-methyl-3-furyl) disulfide, portraying a savory, meaty aroma is responsible for the most potent food aroma to date, having an odor threshold of 8.9 x 10 -11 mM water (Buttery et al., 1984). The same study also determined MFT-MFT to be responsible for the characteristic odor of vitamin B 1 Thiamin degradation pathway The thermal degradation pathway, determined by van der Linde and coworkers (1979), involves the rupturing of the C-N bond between th e pyrmidine and thiazoles moieties of thiamin by a hydroxyl ion attack (Figure 22). The thiazole moiety (III) then degrades to form other potent aroma-active thiazoles such as 4,5-dimethy lthiazole (roasted meat) and 4-methylthiazole (green hazelnut). However, from an aroma perspective, the hydr olysis of the thiazole ring in the thiamin hydrochloride (Figure 2-2) leads to a key aroma intermediate, 5-hydroxy-3-mercapto-2pentanone (VI). This intermediate produces ma ny aroma active thiophenes and furans, including MFT (van der Linde et al., 1979; Gunter t et al., 1990; Guntert et al., 1992). 31

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Alternate pathways for the produc tion of 2-methyl-3-furanthiol Another pathway for the production of MFT is through the Maillard reaction. Meynier et al. (1995) observed the formation of MFT in a cysteine/ribose model system where the MFT formation was greatly increased at a lower pH of 4.5 with almost a 2.5 fold increase from pH 5.0 and a 10 fold increase from pH 6.0. Whitfield et al. (1999), studi ed the reaction between 4-hydroxy-5-methyl-3(2H)-furanone (norfuraneol) and cysteine or hydrogen sulfide. MFT was found in both the norfuraneol/cysteine and norfuraneol/hydrogen sulfide sy stems at similar concentrations The author suggests that this points to only hydrogen sulfide being necess ary and not needing othe r cysteine degradation compounds. Cerny et al. (2003), fu rther investigated the possible source MF T from norfuraneol a model system of cysteine ribose and norfuraneol. A 13 C 5 -labeled ribose and norfuraneol were reacted with cysteine. The resu lting MFT contained some of the 13 C-label 93% of the time, suggesting that the more probable sour ce being the cysteine/ribose reaction. A study by Bolton et al. (1994) combined thiami n and cysteine in model systems. Four model systems were examined for MFT formati on using combinations of thiamin, cysteine, labeled cysteine and D-xylose at a pH range of 5.5 to 5.8. Of interest, the only model system that MFT was not detected in was the only sy stem without thiamin addition, suggesting the primary mechanism for the formation of MFT, under the conditions of the model system, involves thiamin degradation. In the two model sy stems using labeled cysteine, only a net 8% of the MFT contained the labeled sulfur, 34 S, from cysteine as compared to the unlabeled cysteine model solution. Of note, much of the thiamin degradation studies have been carri ed out at elevated temperatures on meat systems rather than explor ing thiamin degradation in other matrices such as orange juice that would not receive the elevat ed temperatures as compared to the cooking of 32

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meat. Ramaswamy et al. (1990) determined the ki netics of thiamin degradation in an aqueous solution at temperatures ranging from 110C to 150C to be first order reactions. Van der Linde et al. (1979) determined that MFT is a pr oduct of 5-hydroxy-3-mercapto-2-pentanone from a breakdown of thiamin at 130C in an aqueous system. Hartman and co-workers (1984b) studi ed the effect of water activity, a w in a model meat system containing thiamin, with heat treatment at 135C for 30 minutes. Results show a higher a w produced more boiled meat-like arom a such as MFT while the lower a w system produced more roasted meat-like aromas including 2-met hylthiophene with a roast beef aroma. Meynier and Mottroam (1995) st udied pH effect in model meat systems with thermal reactions at 140C. The study determined a cy steine model system at a lower pH of 4.5 produced the highest amount of MFT. One study does look at MFT at a lower temp erature of 6C (Hofmann et al., 1996), with the purpose of determining the oxidative stabilit y of odor-active thiols including MFT. MFT was shown to have the highest concentrati on over the 10 day storage in n-pentane and dichloromethane where the concentration read ily decreased in a diethyl ether system. Conversely, MFT-MFT showed the highest formation ra te in diethyl ether, with very little being formed in a dichloromethane or n-pentane system. 33

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34 OH OH d-Limonene -Terpineol +HOH -H+ H+, -HOH Linalool + + H+, HOH Figure 2-1. Pathways for -terpineol formation from linalool and (+)-limonene (Haleva-Toledo et al., 1999). N N N+S OH NH2 N N NH2 OH N S OH OHH3O+ N N NH S OH NH2 CHO O SH OH H3O+ HCOOH N N NH2 NH2 Cl+ (A) (B) (C) (D) + + (E) (F) Figure 2-2. Thiamin thermal degradation pathwa ys. A =thiamin hydrochloride, B = pyrimidine moiety, C = thiazoles moiety, D = diaminopyrimidine, E = formic acid, and F = 5hydroxy-3-mercapto-2-pentanone. Adapted from (van der Linde et al., 1979; Guntert et al., 1990; Mottram, 1991).

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CHAPTER 3 AN AROMA COMPARISON BETWEEN ORANGE JUICES OF DIFFERING ORGANOLEPTIC QUALITIES Introduction Orange juice is ranked number one in fruit juice consumption in Am erica (Pollack et al., 2003). One of the major attributes consumers are looking for is flavor. Considerable research has been spent examining the volatile components that are responsible for the desired aroma and flavor in orange juice. Much of this research has involved the use of thermally abusive storage studies to determine changes in volatile content and formation of off-flavor compounds. The assumption being that elevated thermal temperat ures will produce a larger quantity of storage off-flavors in a shorter period of time. Thermal abuse studies will also produce storage offflavors in higher concentrations making volatile iden tification easier. Tatum et al. (1975) stored single-strength canned orange juice at 35C for up to 12 weeks and identified ten degradation compounds. Of the degradation compounds, thre e exhibited negative aroma impact in the orange juice: -terpineol, 2,5-dimethyl-3(2H)-furanone (Furaneol or DMHF) and 4vinylguaiacol. These three compounds were determined to be above their taste thresholds; and when added to a control orange juice imparted a characteristic aroma of heat-abused juice. Moshonas and Shaw (1989) noticed an increase of -terpineol during storage. Tonder et al. (1998) studied stored reconst ituted orange juice for up to 12 months at 20C. Earlier studies, (Walsh et al., 1997; Peleg et al., 1992; Naim et al., 1997) show minimal formation of both 4vinyl guaiacol and Furaneol at temperatures under 30C. Chemical reaction rates are known to increase with a rise in temperature. This is explained through the Arrhenius equation and the relationship between temperature and the rate at which a reaction takes place. The relations hip is explained in the following equation: 35

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k = Ae -Ea/RT where k is the rate constant, A is the frequency f actor (specific to a particular reaction), e is the math quantity or exponent, Ea is the activation energy or mi nimum energy required for the reaction, R is the gas constant and T is temper ature in K. Through this equation, either a temperature increase or a decrease in Ea results in an increase in reaction rate. In orange juice, an increased reaction rate would derive from temp erature as a decrease in Ea being would need a catalyst which would not normally be present in juice. A general rule of thumb for reactions around ambient temperature states that for every 10C increase in temperature a reaction rate doubles. However, in a complex matrix such as orange juice, the reaction rates of competing reactions can differ considerably. The dominant reaction at a temperature of 40 to 50C may not be the dominant reaction at a lowe r temperature range of 4 to 20C. The dominant reactions that produce specific off-flavors at higher storage temp eratures may not be the same reactions that produce off-flavors that de velop at lower storage temperatures. Therefore, the reactions that produce flavor changes under typical industrial storage c onditions may not be the same as those which occur under an accelerated storage study. The purpose of my study was to evaluate flavor differences in products obtained from supermarke ts without subjecting the samples to additional thermal abuse and determine which aroma active compounds differentiate between poor quality and good quality flavor. Materials and Methods Survey of commercial orange juice Juices for this survey were collected from local supermarkets and consisted of orange juice reconstituted from concentrate produced in Florida. All juices were within the product expiration dates and contained the Florida Seal of Approval on the container. The juices were formed a market basket survey of orange juic e, categorizing each ju ice into one of three 36

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categories: above average, av erage, and below average flavor quality based on an informal organoleptic panel. One above average juice and one below av erage juice were chosen to compare the extremes between the categories. The above average quality RECON juice was purchased refrigerated in a gable-top carton; wh ile the below average flavor quality juice was a canned RECON juice packaged purchased at ambient temperature. Both juices were chilled for sensory evaluation. Chemicals The following chemicals were obtained commer cially from Aldrich (Milwaukee, WI): 1octen-3-one, 4-hydroxy-2,5-dimethyl -3(2H)-furanone, vanillin, (E,E)-2,4-decadienal, (E)-2undecenal, (E)-2-nonenal, methional, (Z)-4-decenal, 4-Vinyl-guaicol, hexanal, octanal, nonanal, decanal, linalool. The following chemicals were obt ained as gifts from S unPure (Lakeland, FL): myrcene, limonene, 1,8-cineol e, geraniol, geranial and -sinensal. 3a,4,5,7a-tetrahydro-3,6dimethyl-2(3H)-benzofuranone (wine lactone), was a gift from Professor Dr. G. Helchmen at the University of Heidelberg, Heidelberg, Germany. -Damascenone was obtained from Givaudan. (Z)-2-nonenal was found as an impurity in (E)-2-nonenal at approximately 5-10%, while (E,Z)2,4-decadienal and trans-4,5-epoxy-E-2-decenal wa s found in an oxidized sample of (E,E)-2,4decadienal. Sample Preparation Extraction of volatiles was done in a simila r method to Parliment (1986) as modified by Klim and Nagy (1992) and Jella and coworkers ( 1998). Liquid-liquid extracts were obtained using 1:1 pentane: diethyl ether. 10 mL of 1:1 pentane: diethyl ether was added to 10 mL of single strength orange juice from concentrate and vigorously mixed by fo rcing between syringes connected with a three-way valve. After mixing, samples were centrifuged at 3000 g for 10 37

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minutes. The sample was re-extracted with an additional 10 mL of 1:1 pentane: diethyl ether and re-centrifuged. Solvent layers we re combined, and then dried ove r anhydrous sodium sulfate. 25 L of 4000 g/mL 2-heptadacanone in 1:1 pentan e: diethyl ether was added as an internal standard. Samples were concentrated to 100 L under a gentle stream of dry N 2 and stored in a septum-sealed vial in a freezer at 15C until later analysis. Gas chromatography-olfactometry conditions A HP-5890A GC (Agilent Technologies, Palo Alto, CA) with a standard FED detector was used to separate the orange juice volatiles with the following fused silica capillary columns: DB-Wax (30 m 0.32 mm id, film thickness 0.5 m) and DB-1 (30m 0.32 mm id, film thickness 0.5 m). Column oven temperature was programmed from 40 to 240C at a linear rate of 7C/min with no hold. Column injection volume was 0.5 L and splitless. Injector and detector temperatures were 225C and 275C, respectively. A Gerstel (Baltimore, MD) column splitter was used to split the effluent with a ratio of 2:1 between the olfactometry and FID detectors respectively. The olfactometer used in this study is similar to that desc ribed by Acree (Acree et al., 1984). The hot effluent from the capillary column was comb ined with a large stream of humidified air in a 1 cm diameter stainless tube. The air was purified by passing through activated charcoal, Drierite, and molecular siev e 5A (Alltech, Deerfield, IL). The purified air was then humidified by bubbling through a temperature controlled, water filled round-bottomed flask. Airflow to the stainless t ube was adjusted to 11L/min. Pane lists sniffed the effluent as it passed through the stainless steel tubing and rated the intensity of the volatiles on a 10 cm linear potentiostat (0-1.0 V output). A panelist rated the intensity on a 0 15 scale with being no aroma detected, a moderate intense aroma and a highly intense aroma. Data was then collected and recorded using Chrom Perfect Software. 38

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Time-intensity analysis The olfactometry panel consisted of two to four trained panelists, 1 male and 3 females between the ages of 21-40. Panelis ts were trained in a manner similar to Rouseff and co-workers (2001b), with a standard solution of 11 com pounds typically found in citrus juice (ethyl butanoate, cis-3-hexenol, trans-2-hexenal, pinene, myrcene, linalool, citronellol, carvone, terpin-4-ol, geranial, and neral). The standard mixture helped train panelists in a time-intensity scale, optimum positioning, and brea thing techniques. Panelists al so were trained by evaluating at least 10 commercial orange juice flavor extracts in order to gain experience and consistency. Panelists were not used for this study until they demonstrated the ability to replicate aroma intensity responses in the practice juice extracts. Pa nelists ran each experimental sample in duplicate and summary reports were generated for each aromagram. Only peaks detected at least 50% of the time were included in this study. Results from each panelists aromagram were normalized with their own maximum peak inte nsity (set to 100) be fore being averaged. Sulfur analysis Methional concentrations were determined using a Sievers chemiluminescence detector (Boulder, CO) attached to a HP-5890 Series II ga s chromatograph (Agilent Technologies, Palo Alto, CA). A Gerstel (Baltimore, MD) CIS-3 temperature programmable injector was employed to minimize thermal artifacts that could be genera ted in the injector port. Injector temperature was 40C increasing at 20C/sec to 150C after injection. The same column and temperature program described chromatogra phic conditions were used. Results and Discussion Table 3-1 summarizes the normalized panel re sponses for the aroma impact compounds found in the two commercial from concentrate ora nge juices. Of note, a one word descriptor consensus for a compounds aroma is not always reached. For example, the panelists describe 39

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hexanal as green and bitter in Table 3-1. This shows the challenge in determining if a descriptor by one panelist is the same com pound described differen tly by another panelist. Use of standard chemicals is necessary to cr eate a common lexicon and understand how aroma compounds can be perceived and described differently by panelists. A total of 42 aroma impact components were detected between the two juices. The good quality juice had a total of 37 aroma impact co mponents while the poor quality juice had 26. Of these components, 21 were found in both juices and 20 components were detected in the good quality juice but not the poor quality juice. It should be noted th at several of the components that were not detected in the poor qual ity juice were detected by an individual panelist, but failed to meet the 50% response criteria. This suggests th at these components may be present in the poor quality juice, but at a concentra tion that is just below the pane ls aroma threshold. Eight aroma active components were detected in the poor qual ity juice but not the good quality juice. The aroma active compounds with the greatest impact, by aroma peak area, in the poor quality orange juice were vanillin, Furaneol, and 4-vinylguaiacol and (Z)-2-nonenal. As shown in Figure 3-1, a significant different, p<0.05, exists between the aroma intensities of the above average and below averag e juice, with higher normalized aroma intensity in the above average quality juice. Differences between the juices might be attributed to the procedures used in the process of reconstituting the juices from concentrate. During the concentration process, water is evaporated from the juice, as well as most of the volatile fractions. These volatiles must then be restored to the concentrate juice before packaging for the consumer. The restoration of th e juice volatiles can be an ex pensive process and some juice manufactures may use a less expens ive flavor package that may not restore the concentrate to its original full flavor. 40

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The difference may also be attributed to the quality of the oranges used in production and the processing itself. The quality of the orange directly affects the quality outcome of the juice the consumer purchases; and detrimental proces ses including possible excess thermal treatment can cause off-flavor production that will still re main in the juice even with the use of a high quality add-back flavor package. From Figure 3-2, it is noticed the above averag e quality juice has a three fold increase in aldehydes aroma activity as compared to the poor quality juice. Of the 17 aldehydes found between the two juices, the below average quality juice contained 7 (hexan al, heptanal, octanal, (Z)-2-nonenal, (Z)-4-decenal, geranial and trans-4,5-epoxy-E-2-decenal) with a diminished aroma response as compared to the above averag e quality juice. Eight aldehydes (nonanal, (E)2-octenal, (E,E)-2,4-heptadienal, decanal, undecenal, (E,Z)-2,4-decadienal, (E,E)-2,4-decadienal and -sinensal) were only detected in the good qual ity juice. Two aldehydes were noted only in the below average quality juice, methional (potent off-flavor) and (E)-2-undecenal. Buettner and Schieberle (2001a) noted di fferences in aroma active compounds when comparing freshly squeezed to reconstituted orange juice, with the main differences being attributed to higher Flavor Dilution (FD) fact ors of acetaldehyde, (Z)-3-hexenal in the fresh juice, while the reconstituted juice had higher FD factors of the terp enoid compounds (limonene, -pinene and linalool) as well as 3-is opropyl-2-methoxypyrazine and vanillin. -Terpineol, Furaneol, and 4-vinylguaiacol Much research on off-flavors in orange juice has focused on -terpineol, Furaneol and 4vinylguaiacol. Furaneol is thought to be responsib le for the pineapple-like aroma of aged orange juice (Tatum et al., 1975). It is considered as one of the major flavor impact compounds in both pineapple and strawberries (Pickenhagen et al ., 1981). As seen in Table 3-1, its aroma is 41

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described as cotton candy or caramel and it impart s a sweet aroma that altars the flavor balance in orange juice, causing an off-flavor which ma ny assessors find unacceptable in an orange juice matrix. The perceived cotton candy aroma, a lthough pleasant on its own, does not contribute a desired flavor in orange juice. Even though concentrations of 4-vinylguaiacol, methional and vanillin were profoundly different (Figure 3-3), it can be seen that Furaneol aroma intensity concentrations were similar for both the good and poor quality juice. However, with less aroma impact compounds in the poor quality juice, especi ally aldehydes and esters, Furaneol may have a greater relative impact on juice quality. -Terpineol -Terpineol concentrations are known to increase with increased storage time and elevated storage temperature (Rymal et al ., 1968), therefore, th e concentration of -terpineol has been proposed as a marker for thermally abused citrus juices (A skar et al., 1973b). -Terpineol, Figure 3-4, has been shown to be produced by d-limonene, through acid catalyzed hydration, as well as through linalool degradation. However, in orange juice, -terpineol is mainly produced through linalool degradation (Ask ar et al., 1973a; Haleva-Tole do et al., 1999). The sensory contribution of -terpineol is also unclear. Tatum and coworkers (1975) described -terpineol as imparting a musty, stale, or piney aroma when added to fresh juice. However, other references (Arctander, 1969) list -terpineol as having a delicate floral and lilac aroma. It is not uncommon for slight differences in aroma descriptors for a specific compound in literature; it is unusual to see the kind of range noted for -terpineol. Some aroma-active compounds depict different aromas based on the concentration of the compound. For example, -terpineol when present in low concentrations can be described 42

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as in Arctander with a delicate floral aroma as compared to a piney, musty aroma when present in higher concentration. In this study, there was a total lack of aroma activity for -terpineol in either juice. As seen in Figure 3-5, there is no aroma activity in the region of -terpineol. The peak for linalool has an aroma peak superimposed over the FID peak, indicating the FID peak responsible for linalool also has aroma activity. Conseque ntly, the lack of an aroma response by -terpineol by any assessor in either juice suggests that its arom a threshold in orange juice is much higher than its threshold in water. This also suggests that -terpineol is not an off-flavor and therefore not responsible for the poor quality juice flavor found in orange ju ices prepared and stored under commercial conditions. Tatu m and coworkers (1975) found -terpineol to cause a significant difference (p < 0.001 and p < 0.05) in orange jui ce at levels of 2.0 and 2.5 g/mL respectively. This study found -terpineol at a level of 2.16g/ mL with no aroma activity. Tonder and coworkers (1998) compared fr eshly reconstituted orange juice with reconstituted orange juice stored for 9-12 months at 20C. -Terpineol was detected at 0.33 and 1.15 g/mL in freshly reconstitute d and stored juice re spectively. However, they reported an olfactory response for -terpineol only in the stored juice, but this was not confirmed using a second GC column as generally required. In addition, the levels of -terpineol were not statistical ly different. Our current study differs, in that -terpineol displayed no aroma activity al though being present at a concentration almost twice as great (2.16 g/mL) as the Tonder study. The aroma threshold of -terpineol using GC-O was de termined in the current study through a series of standards and three assessors. All three respondent s first noticed aroma activity at 0.217g/100mL or 2170 g/mL, (Figure 3-6). At this level, all three assessors were 43

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only able to note a just noticeable difference as an aroma descriptor. The aroma descriptor for terpineol from concentrations of 0.249g/100mL to 0.900g/100mL were all musty. As seen in Figure 3-6, the aroma intensities of each assessor tend to follow a sigmoidal path as concentration increases. This sigmoidal relationship is e xpected of flavors, as reported originally by Beidler (1954). Beidler explains that when a fla vor stimulus reaches a saturation level, the magnitude of the response will hold constant. Beidler also mentions a minimum threshold level that must be obtained before a re sponse is noted, being defined as the response is slightly greater than a given limiting value. 4-Vinylguaiacol 4-Vinylguaiacol is commonly accepted as the single most detrimental compound in orange juice and has a sensory impact that is qu ite negative. Previous studies show that this compound is formed at storage temperatures above 30C (Peleg et al., 1992; Naim et al., 1997; Walsh et al., 1997; Marcotte et al., 1998). Th is study shows (Figure 33) that 4-vinylguaiacol was only detected in the below average quality juice. It should also be noted that 4vinylguaiacol had the second highest aroma intensity in the below average quality juice (Figure 3-1). Vanillin exhibited the highe st aroma intensity in the below average quality juice but may not be as important in the complete juice matrix. Other investigators (Goodner et al., 2000) have noted a relatively high vanillin response with GC-O, however did not find a correlation of vanillin and flavor score in NFC grapefruit juice. Possible expl anations mentioned for the lack of correlation include: the assessor inflating the inte nsity score due to vanillins distinct aroma or a possible interaction with anothe r compound/s in the juice matrix (antagonistic or synergistic). A more likely explanation is that the solvent extraction of the aroma volatiles overemphasizes this compound which has a relatively low vapor pressure. Therefore, 4-vinylguaiacol is the 44

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single most important aroma contributor to the poor quality juice compared with the relative aroma intensities of other aroma impact compounds However, it should also be noted that good quality juice was characterized by the absence of this compound. Methional Methional (3-(methylthio)-propanal) is a high ly potent sulfur cont aining aldehyde whose presence appears to be profoundly negative. Meth ional is formed through a Strecker degradation pathway from methionine, Figure 3-7. As seen in Table 3-1, methional imparts a cooked potato aroma. Research has reported it as producing si gnificant off-flavors in stored orange juice (Bezman et al., 2001), wine (Escudero et al., 2000), cooked mussels (Le Guen et al., 2000), cooked spinach (Masanetz et al., 1998), chedda r cheese (Milo and Reineccius, 1997), and beer (Anderson and Howard, 1974). As shown in Figur e 3-1, the aroma of methional was detected only in the below average quality juice at a mid level range. Its aroma intensity was approximately one third that of 4-vinylguaiacol, one of the more nega tive off-flavor compounds found in stored juice. To quantify the level of this potent sulfur com pound, the extract obtained for GC-O analysis was analyzed for sulfur usi ng a chemiluminescence det ector. It was found at a level of 30 g/L in the poor quality juice. The pub lished threshold for methional is matrix dependent and ranges from 1.6 g/L in beer (Jansen et al., 1971), to 0.2 g/L in tomato (Buttery et al., 1971). The level detected in this study in the poor qual ity reconstituted orange juice considerably exceeds these thresholds, thus conf irming the GC-O observations. An interesting note, Buettner and Schieberle (2001a) detected methional in freshly hand-squeezed juice and reconstituted juice at th e same FD factor of 64. This differs from our results, as methional was only found in the below average quality reconsti tuted orange juice and not the above average quality reconstituted orange juice. 45

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Buettner and Schieberle agai n detected methional in hand squeezed Valencia late and Navel orange juice at concentrations 0.4 g/kg and 0.3 g/kg and FD factors of 64 and 32, respectively (Buettner and Schi eberle, 2001b). The odor activity values (OAV), or ratio of concentration to odor threshold in water, both orthonasally and retr onasally, were also calculated for methional. Orthonasally, both Valencia Late and Navel juices reported low OAV values of <1, while showing 10 and 8 respec tively by retronasal ev aluation. The low OAVs for methional in the fresh juices show that its contribution to the overall aroma of the juice is low. Conclusions The diminished and or lack of aroma res ponse of aldehydes such as hexanal, heptanal, octanal, nonanal, (E)-2-octenal, unde canal and in the below average quality juice as compared to the above average quality juice seems to have played a major role in the overall quality assessment of the juice. This is also combined with the occurrence of off-flavor compounds such as methional and 4-vinylguaiacol that were not found in the above aver age quality juice. Consequently, the lack and or diminishment of certain aldehydes most likely compounded the impact of methional and 4-vinylgu aiacol. Of note in this stud y, the often cited orange juice storage off-flavor -terpineol (Tatum et al., 1975) was s hown to be above previously reported concentrations but without aroma activity. 46

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Table 3-1. Summary of aroma active com pounds found in good and poor quality juice. LRI (DB-Wax) Below Average Quality Juice Above Average Quality Juice Descriptor Tentative ID 1098 1098 Green/bitter Hexanal 1160 1167 Grapefruit/musty Myrcene 1202 1209 Lemon/floral Heptanal 1208 Citrus/sweet Limonene 1223 Citrus/minty Limonene/1,8cineole 1251 Cooked/fermented beans Unknown 1299 1299 Citrus/grapefruit Octanal 1309 1309 Mushroom 1-Octen-3-one 1350 Fermented bean/musty Unknown 1378 1379 Green/musty (E)-3-Hexenol 1400 Oily/bitter Nonanal 1438 Minty/floral (E)-2-Octenal 1451 Sour Acetic acid 1463 Potato Methional 1494 Fatty/oily (E,E)-2,4Heptadienal 1505 Fatty/oily Decenal 1515 1515 Green/pungent (Z)-2-Nonenal 1546 1541 Green/pungent (Z)-4-Decenal 1552 1547 Green/floral Linalool 1595 Floral/minty Undecenal 1683 Beef/musty Unknown 1736 Overripe/oats Unknown 1741 1748 Sweet/honey Geranial 1760 Burnt/pepper Unknown 1758 Sweet/citrus (E)-2-Undecenal 1772 Burnt cooked food/spicy (E,Z)-2,4Decadienal 1820 Smoky/pepper (E,E)-2,4Decadienal 1836 1832 Tobacco/sweet Damascenone 1855 1855 Fruity/floral Geraniol 1882 Fermented juice/herbal Unknown 1954 Roses -Ionone 1981 1983 Bread/cooked rice Unknown 2020 2016 Green/paint Trans-4,5-epoxy-E2-decenal 47

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Table 3-1. Continued LRI (DB-Wax) Poor Quality Juice Good Quality Juice Descriptor Tentative ID 2036 Candy/burnt sugar Unknown 2056 Spicy/roasty Unknown 2089 Overripe Unknown 2169 2164 Sweet/baked grain Unknown 2184 2178 Honey/burnt candy Eugenol 2212 Spicy/burnt sugar 4-Vinylguaiacol 2212 Pepper Unknown 2242 Fresh -Sinensal 2269 2263 Spicy/dill Wine lactone 2413 Floral/sweet (Z)-Methyljasmonate 2611 2623 Vanilla Vanillin 48

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0 20 40 60 80hexanal heptanal 1-octen-3-one (E)-3-hexenol (E)-2-octenal methional decanal (Z)-4-Decenal undecanal (E,Z)-2,4-decadienal -damascenone trans-4,5-epoxy-2E-decenal Furaneol eugenol wine lactone vanillinNormalized Aroma Peak Intensity Good Quality OJ Poor Quality OJ myrcene limonene/1,8-cineole octanal nonanal (acetic acid (E,E)-2,4-heptadienal (Z)-2-nonenal linalool geranial (E)-2-undecenal (E,E)-2,4-decadienal geraniol -ionone 4-vinylguaiacol -sinensal (Z)-methyl jasmonate Figure 3-1. Normalized aroma peak intensity co mparison of good and poor quality orange juice. 49

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(Z)-4-Decenal Geranial Undecanal (E)-2-Undecenal (Z)-2-Nonenal Decanal (E,E)-2,4-Heptadienal Methional (E)-2-Octenal Nonanal Octanal Hexanal Heptanal (E,Z)-2,4-Decadienal (E,E)-2,4-Ddecadienal Trans-4,5-epoxy-2E-decenal b-SinensalNormalized Peak Aroma Intensity Poor quality OJ Good quality OJ Figure 3-2. Aldehyde comparison between good and poor quality orange juice. 50

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0 10 20 30 40 50 60 70 80 Furaneol4-VinylguaiacolMethionalVanillinNormalized Aroma Peak Intensity Poor Quality OJ Good Quality OJ Figure 3-3. Comparison of known off-flavor components in orange juice. OH OH H+. HOH d-Limonene -Terpineol +HOH -H+ H+, -HOH Linalool Figure 3-4. Possible pathway formations of -terpineol (Haleva-Toledo et al., 1999) 51

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Valencene 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 Linalool -Terpineol FID Response Aroma Response Z-4-Decenal GeranialRetention Time (min)(E,Z)-2.4-Decadienal (E,E)-2.4-Decadienal -Damascenone Valencene 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 Linalool -Terpineol -Terpineol FID Response Aroma Response Z-4-Decenal GeranialRetention Time (min)(E,Z)-2.4-Decadienal (E,E)-2.4-Decadienal -Damascenone Figure 3-5. Individual re sponse chromatogram of -terpineol GC/FID aromagram overlay. 0.0 50.0 100.0 150.0 200.0 250.0 300.0 350.0 400.0 0.00000.10000.20000.30000.40000.50000.60000.70000.80000.90001.0000g a-Terpineol in 100mL MeOHAroma Intensity Respons e Assessor 1 Assessor 2 Assessor 3 Just Noticeable Difference 0.0 50.0 100.0 150.0 200.0 250.0 300.0 350.0 400.0 0.00000.10000.20000.30000.40000.50000.60000.70000.80000.90001.0000g a-Terpineol in 100mL MeOHAroma Intensity Respons e Assessor 1 Assessor 2 Assessor 3 Just Noticeable Difference Figure 3-6. GC-O aroma th reshold determination of -terpineol. 52

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53 R1 R2 O O O O N S -CO2N O O S R2 R1 O H H N H S O H R1 R2 O S R1 R2 NH2 OH R1 R2 NH2 O + Methionine dicarbonyl compound -H2O H2O + Methional Aminoketone Figure 3-7. Methional formation through Strecker degradation of methionine (Mottram and Wedzicha, 2002).

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CHAPTER 4 ORANGE JUICE FLAVOR STORAGE STUD Y: DIFFERENCES BETWEEN GLASS AND PET PACKAGING OVER TI ME AND TEMPERATURE Introduction Past studies have looked at th e effect of plastic polymers on orange juice flavor. Duerr et al. studied the effects of Tetra Brik, polyethylen e lined cartons on reconstituted orange juice and found a 40% decrease in (+)-limonene in 6 days as compared to 10% decrease in glass (Duerr et al., 1981). (+)-Limonene is a known precursor to a widely reported off-flavor compound in orange juice, -terpineol (Tatum et al., 1975; Haleva-T oledo et al., 1999). Duerr reported a linear increase in -terpineol formed from (+)-limonene that was greater in glass as compared to the Tetra Brik. The rate of formation of -terpineol was more rela tive to temperature as compared to initial limonene c oncentration. Marin et al. repo rted (1992) (+)-limonene producing only trace aroma activity and not contributing much aroma to orange juice. They concluded that its adsorption into polyethylene may be considered positive. Marin et al. also studied the effects of low density polyethylene (LDPE)/Surlyn Brik -Pak on the aroma volatiles of orange juice (Marin et al., 1992) and noted 70% of the (+)limonene was scalped by th e Brik-Pak within 24 hours at 25C. Kutty et al. investigated the oxidation of (+)-limonene in the presence of Low Density Polyethylene (LDPE). (+)-Limone ne was readily absorbed by the LDPE, with 95% absorption into the polymer in week 0. Higher amounts of headspace oxygen remained in LDPE samples by week 10 compared to the control, with 95% and 83% headspace oxygen respectively, indicating higher (+)-limonene oxidation in the control. In bo th the control and LDPE samples, degradation products of oxidi zed limonene were found, includ ing linalool, limonene oxide, 54

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terpineol, carveol and carvone. Carveol has been reported as an off-flavor in orange juice (Ahmed et al., 1978). A recent study by Berlinet et al. (2005) compared the volatile aroma compounds of orange juice in glass and polyeth ylene terephthalate, PET, over fi ve months storage. The study showed no statistical difference between volatiles in glass or PET, but rath er a similar decrease in aldehydes, ketones, esters, a liphatic alcohols, sequiterpene a nd monoterpene alcohols, and an increase in 4-vinylguaicol and furfural. Overal l, no difference in aroma composition was noted with PET. Polyethylene terephthalate bot tles are commonly used in beve rage applications because of their relatively good barrier against flavor and gas permeation, due to biaxial molecular orientation (Lune et al., 1997). In order to hasten results, heat treatment and or accelerated stor age studies are a common method used for determining orange juice aroma impact compounds. Tatum et al. (1975) studied canned orange juice over 12 weeks at 35C, and pr oposed the three most detrimental storage offflavor compounds as 4-vinylguaiacol, -terpineol and Furaneol. Addition of 4-vinylguaiacol to fresh juice noted an old fruit/rotten flavor. -Terpineol imparted a stal e, musty or piney note; while Furaneol added a pineapple-like aroma. A ll are considered unfavorab le in orange juice. Bazemore et al. (1999) treated orange juice with extreme heat at 96C for 60 seconds and analyzed the volatile composition. The ten most impactful aroma compounds include: ethyl butanoate, myrcene, (E)-2-nonenal, decanal, octa nal, terpin-4-ol, (Z)-3-hexenal and three unknowns (imparting a metallic, vinyl and nutty notes). Compounds of interest formed after heat treatment includes 4-vinylguaiacol. 55

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Peterson et al. (1998) compared normal storage conditions of 5 and 20C to accelerated conditions at 30, 40 and 50C. Findings show 6 month/20C samples correlated with either 13 day samples at 40C or 5 days at 50C. Peterson et al. showed a decrease in linalool and octanal while an increase in -terpineol, comparable with results from Tatum (1975). The major purpose of this study was to examine storage off-flavor production under refrigerated conditions at 4C as compared to el evated thermal conditions of 35C. Additionally, the effect of packaging was examined. Stor age off-flavor production in PET and glass containers were compared to determine if increa sed levels might be observed in juices from the more gas permeable PET containers. Materials and Methods Chemicals The following chemicals were obtained commer cially from Aldrich (Milwaukee, WI): 1octen-3-one, 4-hydroxy-2,5-dimethyl-3(2H)-furanone (Furaneol), vanillin, (E,E)-2,4-decadienal, (E)-2-undecenal, (E)-2-nonenal, methional, (Z)-4 -decenal, 4-Vinyl-guaicol, hexanal, octanal, nonanal, decanal, linalool. The following chem icals were obtained as gifts from SunPure (Lakeland, FL): myrcene, limonene, 1,8-cineole, geraniol, geranial and -sinensal. 3a,4,5,7atetrahydro-3,6-dimethyl-2(3H)-benzofuranone (wine lactone), was a gift from Professor Dr. G. Helchmen at the University of Heidelberg, Heidelberg, Germany. -Damascenone was obtained from Givaudan. (Z)-2-nonenal was found as an impurity in (E)-2-nonenal at approximately 510%, while (E,Z)-2,4-decadienal and trans-4,5epoxy-E-2-decenal was found in an oxidized sample of (E,E)-2,4-decadienal. 56

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Orange Juice The commercial orange juice from concentrat e used in this study was obtained from a Florida manufacturer in 16 fluid ounce (473mL) glass containers with metal closures. The PET containers were also obtained from the same Fl orida manufacturer with polypropylene closures. Half of the orange juice from concentrate was then transferred to 16 fluid ounce PET containers by way of sterile transfer. The PET containers were dipped in a 190F water bath, drained and filled with the orange juice from the glass co ntainer. All samples were then stored at temperatures of 4, 25, and 35C for up to 16 week s. Samples were frozen until analysis at 38C. Visual and organoleptic evaluation Samples were evaluated at days 7, 14 28, 56, 84 and 112. Visually, samples were evaluated against a reference of orange juic e for noticeable color change. After visual evaluation, samples were compared informally by organoleptic evaluation against the reference at ambient temperature. The informal organole ptic evaluation determined if the sample would still be considered acceptable for a consumer against the reference. Sample preparation Extraction of volatiles was done in a simila r method to Parliament (1986) and modified by Klim and Nagy (1992) and Jella and coworkers (1998). Liquid-liquid ex tracts were obtained using 1:1 pentane: diethyl ether. 10 mL of 1:1 pentane: diethyl ether was added to 10 mL of single strength orange juice from concentrate and vigorously mixed by fo rcing between syringes connected with a three-way valve. After mixing, samples were centrifuged at 3000 g for 10 minutes. The solvent layer was re-extracted with an additional 10 mL of 1:1 pentane: diethyl ether and re-centrifuged. Solvent layers were combined, and then dried over anhydrous sodium sulfate. 25 L of 4000 g/mL 2-heptadacanone in 1:1 pent ane: diethyl ether was added as an 57

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internal standard. Sample s were concentrated to 100 L under a gentle stream of dry N 2 and stored in a septum sealed vial in a freezer until later analysis. Gas chromatography-olfactometry conditions A HP-5890A GC (Agilent Technologies, Palo Alto, CA) with a standard FID detector was used to separate the orange juice extracts with the following fused silica capillary columns: DB-Wax (30 m 0.32 mm id, film thickness 0.5 m) and DB-5 (30m 0.32 mm id, film thickness 0.5 m). Column oven temperature was programmed from 40 to 240C at a linear rate of 7C/min with no hold. Column injection volume was 0.5 L and splitless. Injector and detector temperatures were 225C and 275C respectively. A Gerstel (Baltimore, MD) column splitter was used to split the effluent with a ratio of 2:1 between the olfactometry and FID detectors respectively. The olfactometer used in this study is similar to that desc ribed by Acree (Acree et al., 1984). The hot effluent from the capillary column was comb ined with a large stream of humidified air in a 1 cm diameter stainless tube. The air was purified by passing through activated charcoal, Drierite, and molecular siev e 5A (Alltech, Deerfield, IL). The purified air was then humidified by bubbling through a temperature controlled, water filled round-bottomed flask. Air flow to the stainless tube was adjusted to 1.1L/min. Pa nelists sniffed the effluent as it passed through the stainless steel tubing and rated the intensity of the volatiles on a 10 cm linear potentiostat (0-1.0 V output). Data was then collected and recorded using Chrom Perfect Software. Samples were evaluated at days 0, 7, 14, 38, 56, 84 and 112. GC-olfactometry GCO equipment and conditions were identical to those described in earlier studies (Bazemore et al., 1999). The olfactometry panel c onsisted of two trained panelists, 1 male and 1 female, between 25 and 30 yrs old. Panelists were trained in a manner similar to Rouseff and co58

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workers (Rouseff et al., 2001b), using a standard solution of 11 compounds typically found in citrus juice (ethyl butanoate, cis-3-hexenol, trans-2-hexenal, pinene, myrcene, linalool, citronellol, carvone, terpin-4-ol, ge ranial, and neral). The standard mixture helped train panelists in a time-intensity scale, optimum positioning, and breathing techniques. Panelists also were trained by evaluating at least 10 commercial orange juice flavor extracts in order to gain experience and consistency. Panelists were not us ed for this study until they demonstrated the ability to replicate aroma intens ity responses in the practice jui ce extracts. Panelists ran each experimental sample in duplicate and summary re ports were generated for each aromagram. Only peaks detected at least 50% of the time were included in this study. Results from each panelists aromagram were norma lized with their own maximum peak intensity (set to 100) before being averaged. Gas chromatography-mass spectrometry (GC-MS) Sample separation was performed on a Finni gan GCQ Plus system (Finnigan Corp., San Jose, CA), using a J&W Scientific DB-5 co lumn (60m, 0.25 mm i.d., 0.2 5 m film thickness (Folsom, CA)). The MS was operated under pos itive ion electron impact conditions: ionization energy, 70 eV; mass range, 40 300 amu; scan rate, 2 scans/s; el ectron multiplier voltage, 1050 V. Transfer line temperature was 275 C. Initial column oven temperature was 40 C and increased at 7 C/min to a final temperature of 275 C. Injector temperature was 250 C. Helium was used as the carrier gas at a linear ve locity of 32 cm/s. When searchable spectra could not be obtained for compounds of interest because of low signal-to-noise ratio, chromatograms of selected masses were reconstructed from the MS data matrix. These selected ion chromatograms (SIC) employed at least three unique m / z values from the mass spectrum of standards were used as 59

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identification aides. Whenever possible, the molecular ion (M + ) was chosen as one of the three m / z values. Results and Discussion In addition to chromatographic analysis, samp les were evaluated visually for color and organoleptically for overall qualitative acceptance. Samples changed color as time progressed under heated conditions. Samples, both glass and PET, stored at 25 and 35C were noted with increased brown hues as storage time increased, whereas 4C samples did not visually darken during storage. This implies that non-enzyma tic browning occurred due to increased storage temperature. In a related manner, 25 and 35C were deemed unacceptable organoleptically after 112 days storage, imparting brown/cooked notes. 4 samples were still acce ptable but had lost a significant amount of fresh notes. Table 4-1, shows the results for the arom a active compounds detected over the 16 week storage. In all, 67 different co mpounds were detected by GC-O in the orange juices. Juice at time zero produced 37 aroma active compounds. Th e number of compounds increased to 41 and 46 respectively for glass and PET packages after 112 days of storage at 35C. The most potent aroma active compounds, as measured by normalized peak intensity, in the day 0 sample include the following in decr easing intensity: vani llin, 4-vinylguaiacol, 4mercapto-4-methyl-2-pentanone, 1-octen-3-one, wine lactone, decanal, Furaneol, ethyl vanillin and linalool. The glass, day 112, 35C sample m easures the following as the strongest aroma active compounds in decreasing intensity: vanilli n, Furaneol, ethyl vani llin, wine lactone, 4vinylguaiacol and linalool. The PET, day 112, 35C sample includes the following as the highest aroma active intensities: 4-vinylguaiacol, vanillin, ethyl va nillin, Furaneol, wine lactone, 2-methyl-3-furanthiol, linalool and butanoic acid. Of note, all three sets contain the known orange juice off-flavor component s of Furaneol and 4-vinylguaiaco l. However, with diminished 60

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aroma peak intensities at day 112 as compared to day 0, the occurrence of Furaneol and 4vinylguaiacol more profoundly impacted the overall aroma. The impact of vanillin and wine lactone are also considered as some of the mo st impactful aroma contributors by Buettner and Schieberle in reconstituted orange juice (Buettner and Schieberle, 2001a). Aroma changes over time Measuring aroma change over time, five com pounds were noted at time zero that were completely lost by olfactometry in either 4 or 35C regardless of package. Of these compounds, 2 were identified as (E,E)-2,4-heptadienal imparting a pungent/oily aroma and undecanal imparting a musty aroma. Three unknown peak s imparted skunky, musty and grain notes. The overall number of compounds was lowest in day 0 juice with 37 aroma active compounds; however, overall aroma activity, as measured by th e sum of normalized aroma peaks, was greater at day 0 as compared to day 112 samples, Table 4-2. This differe nce is most evident comparing juice stored in PET at 35C with a 28% loss over 112 days storage. The diminished aroma activity can also be seen in Figures 4-1 and 4-2 comparing day 0 and day 112 aromagrams of glass and PET respectively. The decrease in arom a activity at day 112 is significantly different from that at day 0 at p<0.01. The condition with the highest number of aroma active compounds is day 112 PET stored at 35C. When considering temperature, both PET and glass packages had more aroma active compounds at the higher temperature of 35C as compared to 4C. Off-Flavor Compounds Of note, known off-note compounds in orange juice were observe d starting at day 0, including methional (18), Furaneol (50), 4-vinylguaiacol (57). -Terpineol, as discussed in more detail in chapter 3, was not noted as aroma active in this study at any te mperature or packaging conditions, showing that its concentr ation is below its aroma threshold. 61

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Methional Methional (18) has been reported as an off fl avor in orange juice (Bezman et al., 2001), grapefruit oil (Lin and Rouseff, 2001) and in grapefruit juice (Lin et al., 2002), imparting a cooked potato note. As mentioned in chapter 3, methional is a product of Strecker degradation of the amino acid methionine. When comparing th e aroma intensities of methional, the highest level is in day 0 juice. However, with overa ll diminished aroma intensity at day 112 as compared to day 0, methional likely plays a greater role in the overall characteristic of the stored juice. The highest aroma intensity occurrence at da y 0 is notable as compared to the orange juice studied in chapter 3, where methional was onl y not found in the good quality orange juice. Furaneol and 4-vinylguaiacol Furaneol (50) and 4-vinylguaiacol (57) have long been noted as an off flavor in orange juice (Tatum et al., 1975). As shown in Table 4-1, both Furaneol and 4-vinylguaiacol are one of the few compounds that start and remain at a high aroma impact through storage. The constant intense aroma activity of Furaneol also agrees with the findings in chapter 3, where Furaneol showed high aroma activity in both the good and poor quality juice. However, 4-vinylguaiacol was only noted in the poor quality juice; where it was noted at a high aroma intensity starting a day 0 in this study. Surprisingly, Buettner and Schieberle did not note either compound in their reconstituted orange jui ce, which would correspond to the day 0 sample in this study (Buettner and Schieberle, 2001a). 2-Methyl-3-furanthiol and bis( 2-methyl-3-furyl) disulfide The potent storage off-note 2-methyl-3-furanth iol (11), discussed in detail in chapter 5, was found under PET packaging at day 112. 2-Met hyl-3-furanthiol is a degradation product of thiamin that imparts a meaty to grainy aroma and has a low aroma threshold of 6.14 x10 -8 mM in water (Munch and Schieberle, 1998). It has been reported as an offnote in grapefruit (Lin et al., 62

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2002) and orange juice (Bezman et al., 2001). Add itionally, the dimer of 2-methyl-3-furanthiol, bis(2-methyl-3-furyl) disulfide (46), was also noted in glass cond itions on day 112 at 4C and 35C as well as PET conditions at 35C. Bis(2methyl-3-furyl) disulfid e is the most potent aroma compound observed in foods to date at 8.9 x10 -11 mM in water (Buttery et al., 1984). With bis(2-methyl-3-furyl) di sulfide being found in both gl ass and PET at day 112, it is surprising that its monomer is only found in PET. One explanation is 2-methyl-3-furanthiol is present but at levels below its aroma threshold (a s compared to its more potent dimer). Another explanation is the dimerization during storage in glass is more complete than in PET. M-cresol M-Cresol (52) imparted a manure aroma and was present at day 112 in glass and PET. m-Cresol occurred at the highest normalized aroma intensity in PET at 35C. It was also found at PET day 112 at 4C where at the same day a nd temperature was not in glass. Hognadoittir and Rouseff (2003) reported m-cresol in orange essence oil for the first time. Sulfur compounds Compound (14), 4-mercapto-4-methyl-2-penta none is a characteristic aroma compound in grapefruit (Buettner and Schieberle, 1999; Lin et al., 2002). It wa s found at day 0 at its highest aroma impact level and disappears in the glass packagi ng at day 112, while decreasing by approximately 50% in PET. However, with the overall decrease of arom a activity as shown in figure 4-2, 4-mercapto-4-methyl-2-pentanone plays an important role in th e overall quality of the juice. 4-mercapto-4-methyl-2-pentanone gives a pleasant grapefruit aroma when in very small concentrations. At higher con centrations, the compound is commonl y described as sulfury or cat urine. Another probable sulfur containing com pound is the unknown (21), described as having a beefy or savory aroma. It is only present at the day 112, 35C storage conditions. Of note, as 63

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with the general trend, the com pound has a higher aroma intensity level in the PET packaging as compared to glass. Carvone The main constituent of orange oil is th e terpene compound limonene. Limonene has a very prominent FID peak on the FID chromatogram, but does not produce a major aroma impact. As seen in Table 4-1, on a wax and DB-5 colu mn it coellutes with the minty 1,8-cineole. Limonene, however can under go oxidation to form carvone, a reported off-flavor in orange juice (Papken et al., 1999; Buettner a nd Schieberle, 2001a). In this study, carvone (32), imparting a sweet/ licorice aroma is not noted in day 0 juice but is form ed during storage. This agrees with (Buettner and Schieberle, 2001a ) where the comparison of aroma active compounds in fresh squeezed orange juice and reconstituted orange juice found carvone only in the reconstituted juice. The aroma descriptor of carvone in this study of sweet/licorice differs from that used by Buettner as caraway-like. Buettner and Schieberle suggest the carvone found in their study is the (S)-enatiomer, with the caraway-like aroma, wher e (R)-carvone has a minty aroma. The minty descriptor is more in line with the sweet/licorice aroma describe d in this study. Tonder et al. (Tonder et al., 1998) also noticed a higher aroma intensity of carvone in stored orange juice. Vanillin Vanillin (67) stays at a consistent aroma in tensity level in this study, ranging from 9.4 to 10.6. Buettner and Schieberle report a large incr ease in FD factors of 32 to 1024 in fresh juice compared to reconstituted juice. The high FD f actor in the reconstituted juice agrees with the high normalized intensity level for vanil lin under all conditions in this study. 64

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Changes in Fresh Juice Compounds (Z)-3-Hexenal One noteworthy difference when comparing juic e at 4C and 35 is the loss of key fresh aromas. One such compound is (Z)-3-hexenal (5). As can be seen in Figure 4-3 comparing the aroma activity of 4 and 35C in glass at day 112, (Z)-3-hexenal is not present in the higher temperature sample. This phenomenon is also no ted by Buettner and Schieberle (2001a), where (Z)-3-hexenal has a FD factor of 512 in fresh squeezed juice and was not detected in reconstituted juice. Linalool Linalool (23), imparting a fl oral, lemon-like aroma, is considered a positive compound in orange juice. Linalool remained at a constant aroma intensity level across time, temperature and containers; ranging from a normalized aroma inte nsity of 7.4 to 8.9. Buettner and Schieberle report a large difference between fresh and rec onstituted juice, with FD factors of 16 and 512 respectively (Buettner and Schieber le, 2001a). The likely explanation in this latter case is that the flavoring added to restore lost juice arom a volatiles contained an excess of linalool, a relatively inexpensive aroma volatile. Ethyl butyrate Ethyl butyrate (3) imparts a fruity aroma was found starting at day 0 and diminished over time. Ethyl butyrate is noted in orange juice in literature (Marin et al., 1992; Buettner and Schieberle, 2001a; Tonder et al., 1998). The aroma values noted in this study agree with Tonder et al. (1998), who reported aroma values decreasi ng from 180 to 76 in fresh reconstituted and stored juice respectively. Buettn er and Schieberle (2001a) report ethyl butyrate at a FD factor of 1024 in fresh squeezed juice and 2048 in reconstituted orange juice. 65

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Octanal Another key aroma loss is that of octanal (8 ), with a lemon/green aroma. Octanal is present at day 0 and at juices st ored at 4C for both glass and P ET but observed in juices stored at 35C, Figures 4-3 and 4-4. Tonder et al. (1998) found similar results with octanal being present in freshly reconstituted concentrate but no t stored juice. Peters on and Tonder (Petersen et al., 1998) also report an appr oximate 50% loss of octanal after 12 days at 30C. A similar diminishing of (Z)-4-octenal (12) is noted in gl ass with a total absence in 35C stored juice. However, (Z)-4-ocental is present in 35C day 112 PET samples, although at a slightly lower aroma intensity. Acetic and butanoic acids Two compounds that were not present in the juice at day 0 are acetic acid (17) and butanoic acid (27). Acetic acid is present in glass and PET packages at both 4 and 35C conditions at day 112. The aroma intensity increa ses slightly between temperature for both glass and PET, with the highest amount being noticed in the 35C PET condition. Butanoic acid is reported only at the 35C conditions for glass and P ET. Again the highest intensity is reported in PET with an intensity of 7.8 as compared to 2.8 for glass. The observanc e of these compounds is also noted by Tonder et al. (1998), with both buta noic and acetic acids being present in freshly reconstituted juice and r econstituted juice stored for 9 12 months at 20C. Both acetic and butanoic acid had a higher aroma in tensity in stored orange jui ce. Buettner and Schieberle (2001a) reported acetic acid in both fresh and reconstituted orange juice, with slightly higher FD factor in the reconstituted jui ce (32 compared to 16). No buta noic acid was reported in their study. 66

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Trans-4,5-epoxy-(E)-2-decenal Trans-4,5-epoxy-(E)-2-decenal (47) imparting a spicy aroma is found only at 35C conditions in this study. Buettn er and Schieberle (2001a) report the compound to have a higher FD factor in fresh juice as compared to recons tituted juice (128 and 16 FD factors respectively). This differs from this study as trans-4,5epoxy-(E)-2-decenal was not found at day 0, which would be the closest variable with Bu ettner and Schieberles fresh juice. Container comparison Figure 4-5 displays a comparison between jui ces stored in glass and PET containers at day 112, 35C. Most compounds are found in both packages. However, there were some differences. Five compounds were detected in glass but not PET. These compounds impart the following aromas: orange/fruity (1 ), sour/estery (28), burnt/unrip e (guaiacol) (40), green (43) and smoky/soapy (64). Compounds ( 40) and (64) are considered o ff-flavors in orange juice. The PET samples contain the following 11 compounds that are not in th e glass 35C, day 112 conditions: grainy/savory (2-methyl -3-furanthiol) (11), grainy ((Z )-4-octenal) (12), cat urine (4mercapto-4-methyl-2-pentanone) (14), floral/car amel (25), rose/sour (citronellol) (35), green/plant (41), burnt sugar (eugenol) (55), spicy/cooked (s otolon) (56), gr een banana ( undecalactone) (58), pepper (60) and herbal/wee ds (65). Of these compounds (11), (14), (41),(55), (56) and (60) are considered negative characteristics in orange juice. Conclusions Aroma active compounds change over time and most importantly, temperature. The total number of aroma active compounds and the normali zed aroma intensity between 4C and 35 in glass were comparable. However, a loss of important compounds such as (Z)-3-hexenal (green banana) and octanal (lemon, green ) and a decrease in (Z)-3-he xenol (green, citrusy), (E)-2ocenal (sour green), (Z)-4decenal (woody, sharp green) and -ionone (roses) occurred. 67

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Concurrently, negative compounds were formed including butanoic acid, (E,E)-2,4-nonadienal (fatty, grainy), trans-4,5-epoxy-(E)-2-decen al (spicy) and m-cresol (manure). Differences exist when comparing glass a nd PET containers at 35C day 112. PET has higher total normalized aroma intensity at 248 comp ared to glass at 192 as seen in Table 4-2; however the difference is not statistically di fferent, p>0.10. Main differences include the following negative compounds found in PET and not glass: 2-methyl-3-furanthiol, eugenol, sotolon and 4-mercapto-4-methyl-2-pentanone as well as higher normalized intensities for butanoic acid, trans-4,5-epoxy-(E)-2-decenal and 4-vinylguaiacol. Through the differences above, glass has shown to be a better containe r for orange juice by minimizing the number of off-flavor compounds created during storage. 68

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Table 4-1. Aroma active compounds in orange juice stored at 4 and 35C over 112 days. LRI Descriptor Day 0 Aroma Intensity* Day 112 Glass Aroma Intensity* Day 112 PET Aroma Intensity* No. Tentative ID DBWax ZB5 4C 35C 4C 35C 1 Unknown 982 Orange, fruity n/a 3.3 3.0 0.9 n/a 2 -Pinene 1030 935 Citrusy n/a 2.4 2.4 n/a 2.9 3 Ethyl butyrate 1040 795 Fruity 5.1 2.5 2.9 3.9 2.3 4 Unknown 1099 Skunky, earthy 8.5 n/a n/a n/a n/a 5 (Z)-3-Hexenal 1150 780 Green banana n/a 2.8 n/a n/a n/a 6 Myrcene 1168 990 Musty, geranium 7.4 4.8 5.1 5.4 3.8 7 Limonene/1,8cineole 1208 1032 Licorice, minty 8.3 3.0 2.8 4.6 4.1 8 Octanal 1297 1002 Lemon, sharp green 7.7 3.6 n/a 6.1 n/a 9 1-Octen-3-one 1307 977 Mushroom 9.5 4.6 3.4 4.1 4.2 10 Unknown 1310 Cooked rice 7.8 n/a n/a n/a n/a 11 2-Methyl-3furanthiol 1317 874 Grainy, savory n/a n/a n/a 4.4 8.3 12 (Z)-4-Octenal 1345 Grainy 7.1 3.0 n/a 3.8 2.9 13 Unknown 1377 Musty green, rubbery 7.3 7.8 4.7 5.9 5.4 14 4-Mercapto-4methyl-2pentanone 1390 943 Cat urine 9.6 n/a n/a 5.3 5.5 15 (Z)-3-Hexenol 1398 Green, citrusy n/a 4.0 3.3 3.8 2.9 16 (E)-2-Octenal 1438 1058 Sour green 4.9 4.9 3.7 3.2 2.9 17 Acetic acid 1447 Sour, vinegar n/a 2.5 2.9 3.1 5.0 18 Methional 1464 908 Potato 8.7 3.1 4.4 6.2 6.5 19 (E,E)-2,4Heptadienal 1501 1022 Pungent, oily 7.0 n/a n/a n/a n/a 20 Decanal 1511 1207 Woody, green 9.4 6.6 6.2 6.5 7.3 21 Unknown 1523 Beefy, savory n/a n/a 3.3 n/a 5.7 69

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22 (Z)-4-Decenal 1541 1198 Woody, sharp green 7.6 5.9 4.9 4.5 5.2 23 Linalool 1548 1101 Lemony, floral 8.9 8.6 7.4 8.4 8.3 24 (E,Z)-2,6Nonadienal 1593 1161 Cucumber n/a n/a n/a 4.7 n/a 25 Unknown 1601 Floral, caramel n/a n/a n/a n/a 4.0 26 Undecanal 1623 1277 Musty, moldy 4.6 n/a n/a n/a n/a 27 Butanoic acid 1625 817 Sour butter, manure n/a n/a 2.9 n/a 7.8 28 Unknown 1669 Sour, estery n/a n/a 2.8 n/a n/a 29 (E,E)-2,4Nonadienal 1701 1209 Fatty, grainy n/a n/a 2.1 3.4 n/a 30 Unknown 1728 Moldy, rubber 6.0 n/a n/a n/a n/a 31 Unknown 1734 Woody, sweet grain 5.1 6.5 7.1 5.2 5.4 32 Carvone 1748 1252 Licorice, sweet n/a 3.7 2.8 n/a 3.0 33 Unknown 1758 Grain, musty 5.5 n/a n/a n/a n/a 34 (E,Z)-2,4Decadienal 1772 1297 Grainy, sour 7.8 4.0 3.2 n/a 2.5 35 Citronellol 1773 Rose, sour n/a n/a n/a n/a 2.5 36 Unknown 1815 Sweet dough 5.6 n/a n/a 4.0 n/a 37 (E,E)-2,4Decadienal 1820 1327 Fatty green, cucumber n/a 4.4 4.2 3.2 3.4 38 Damascenone 1834 1393 Tobacco, apple juice 6.8 5.2 4.8 5.4 6.0 39 Geraniol 1852 1258 Rose, floral 6.0 n/a 3.2 6.2 6.1 40 Guaiacol 1863 1087 Burnt, unripe 4.6 2.7 4.5 n/a n/a 41 Unknown 1867 Green, plant n/a n/a n/a n/a 4.6 42 Unknown 1883 Sweet grain, toasted n/a 7.3 5.8 n/a 3.0 70

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oats 43 Unknown 1905 Green n/a n/a 2.7 n/a n/a 44 -Ionone 1956 1491 Raspberry, roses 5.1 8.2 6.5 6.1 5.3 45 Unknown 1965 Moldy 4.0 n/a n/a n/a n/a 46 Bis(2-methyl3-furyl) disulfide 1980 1543 Spicy, grainy n/a 3.8 4.5 n/a 4.1 47 Trans-4,5epoxy-(E)-2decenal 1996 1384 Spicy, syrup n/a n/a 3.6 n/a 7.1 48 Unknown 2008 Dusty n/a n/a n/a 4.1 n/a 49 Unknown 2016 Woody, floral n/a 3.6 4.6 3.3 2.9 50 Furaneol 2041 1061 Cotton candy 9.3 8.5 10.2 9.1 10.4 51 Unknown 2061 Spicy, meaty 6.7 2.6 4.3 n/a 4.6 52 m-Cresol 2088 1087 Manure n/a n/a 3.9 4.0 6.4 53 Unknown 2121 Licorice, rubbery n/a 3.0 n/a n/a n/a 54 Unknown 2161 Burnt bread 5.6 n/a n/a 5.4 n/a 55 Eugenol 2174 1352 Burnt sugar n/a 3.4 n/a 5.5 4.1 56 Sotolon 2180 Cooked, spicy n/a n/a n/a n/a 4.1 57 4Vinylguaiacol 2205 1323 Spicy, cloves 10.5 8.2 8.0 10.2 11.7 58 Undecalactone 2238 Green banana 7.3 3.8 n/a 4.1 2.8 59 Wine lactone 2260 1469 Dill, buttery 9.5 8.1 8.8 6.6 8.5 60 Unknown 2301 Pepper n/a n/a n/a 4.5 5.2 61 Unknown 2365 Soapy, floral n/a 3.5 3.5 n/a 4.1 62 Unknown 2416 Perfume, floral n/a 4.2 n/a n/a n/a 63 Unknown 2437 Soapy, musty 5.4 5.3 3.2 4.8 4.4 64 Unknown 2544 Smoky, soapy 4.7 4.5 4.7 n/a n/a 65 Unknown 2556 Herbal, weeds n/a n/a n/a n/a 2.6 66 Ethyl vanillin 2575 Vanilla, cocoa 9.2 9.5 9.8 9.7 10.5 71

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67 Vanillin 2597 1412 Vanilla, chocolate 10.6 9.4 10.6 9.6 10.5 72

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73 Table 4-2. Comparison of total overall arom a intensity under various package, time and temperature conditions. Packaging Total Normalized Aroma Intensity Total Number of Aroma Active Compounds Number of Unique Compounds Day 0 267 37 5 PET day 112 (4C) 205 38 3 PET day 112 (35C) 248 46 1 Glass day 112 (4C) 196 40 0 Glass day 112 (35C) 192 41 3 15 10 5 0 5 10 15 LRI (DB Wax)Normalized Aroma Peak Intensity Day 0 Day 112ethyl butyrate myrcene octanal -pinene limonene/1,8-cineole 1-octen-3-one (Z)-4-octenal 4-mercapto-4methyl-2-pentanone (Z)-3-hexenol (E)-2-octenal acetic acid methional decanal (E,E)-2,4-heptadienal (Z)-4-decenal (E,Z)-2,6-nonadienal linalool undecenal (E,E)-2,4-nonadienal -undecalactone ethyl vanillin vanillin m-cresol 4-vinylguaiacol wine lactone bis-(2-methyl-3-furyl) disulfide trans-4,5-epoxy-(E)-2-decenal -ionone (E)-2-undecenal -damascenone geraniol carvone (E,Z)-2,4-decadienal (E,E)-2,4-decadienal guaiacol Figure 4-1. Aroma comparison of day 0 and 112 (35C) in glass packaging.

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15 10 5 0 5 10 15LRI (DB Wax)Normalized Aroma Peak InIntensity Day 0 Day 112ethyl butyrate -pinene myrcene limonene/1,8-cineole (E)-2-hexenal octanal 1-octen-3-one 2-methyl-3-furanthiol (Z)-4-octenal 4-mercapto-4methyl-2-pentanone (Z)-3-hexenol (E)-2-octenal acetic acid methional (E,E)-2,4-heptadienal decanal (Z)-4-decenal linalool (E,Z)-2,6-nonadienal undecenal butanoic acid carvone citronellol (E,Z)-2,4-decadienal (E,E)-2,4-decadienal -damascenone geraniol -ionone furaneol m-cresol eugenol sotolon 4-vinylguaiacol -undecalactone wine lactone ethyl vanillin vanillin guaiacol (E)-2-undecenal bis-(2-methyl-3-furyl)-disulfide trans-4,5-epoxy-(E)-2-decenal Figure 4-2. Aroma comparison of da y 0 and 112 (35C) in PET packaging. 74

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15 10 5 0 5 10 15LRI (DB-Wax)Normalized Aroma Peak Intensity 4C 35Cethyl butyrate myrcene octanal (Z)-4-octenal -pinene (Z)-3-hexenal limonene/1,8-cineole 1-octen-3-one (Z)-3-hexenol (E)-2-octenal methional decanal linalool acetic acid (Z)-4-decenal (E,E)-2,4-nonadienal butanoic acid carvone (E,Z)-2,4-decadienal (E,E)-2,4-decadienal -damascenone geraniol guaiacol -ionone bis-(2-methyl-3-furyl) disulfide trans-4,5-epoxy-(E)-2-decenal furaneol m-cresol 4-vinylguaiacol wine lactone -undecalactone ethyl vanillin vanillin eugenol Figure 4-3. Aroma comparison of orange juice stored at 4 and 35 for 112 days in glass. 75

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15.0 10.0 5.0 0.0 5.0 10.0 15.0 LRI (DB-Wax)Normalized Aroma Peak Intensity PET Day 112 4C PET Day 112 35C -pinene ethyl butyrate myrcene limonene/1,8-cineole (E)-2-hexenal octanal 1-octen-3-one 2-methyl-3-furanthiol (Z)-4-octenal 4-mercapto-4-methyl2-pentanone (Z)-3-hexenol (E)-2-octenal acetic acid methional decanal (Z)-4-decenal linalool (E,Z)-2,6-nonadienal butanoic acid (E,E)-2,4-nonadienal carvone citronellol (E,E)-2,4-decadienal -damascenone geraniol -ionone furaneol m-cresol eugenol sotolon 4-vinylguaiacol -undecalactone wine lactone ethyl vanillin vanillin bis-(2-methyl-3-furyl)-disulfide trans-4,5-epoxy(E)-2-decenal Figure 4-4. Aroma comparison of orange ju ice stored at 4 and 35 for 112 days in PET. 76

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77 15 10 5 0 5 10 15 LRI (DB-Wax)Normalized Aroma Peak Intensity Glass PET myrcene limonene/1,8-cineole -pinene ethyl butyrate (E)-2-hexenal 2-methyl-3-furanthiol (Z)-4-octenal 4-mercapto-4-methyl-2pentanone decanal acetic acid methional linalool butanoic acid (E,Z)-2,4-decadienal (E,Z)-2,4-decadienal/ citronellol -damascenone m-cresol eugenol sotolon -undecalactone 1-octen-3-one (Z)-3-hexenol (E)-2-octenal (Z)-4-decenal carvone (E,E)-2,4-decadienal guaiacol -ionone furaneol bis-(2-methyl-3-furyl) disulfide 4-vinylguaiacol wine lactone ethyl vanillin vanillin trans-4,5-epoxy-(E)2-decenal Figure 4-5. Aroma comparison of orange juice stored at 35 for 112 days in glass and PET

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CHAPTER 5 GC-OLFACTOMETRIC CHARACTERIZATION OF AROMA VOLATILES FROM THE THERMAL DEGRADATION OF THIA MIN IN MODEL ORANGE JUICE Introduction Thiamin (vitamin B1) can thermally decompose to produce highly potent aroma compounds. Previous studies have focused on identifying and characterizing decomposition products produced by thiamin under various therma l and pH conditions. The factors determining which breakdown products will be formed include temperature, pH, processing and storage time (Dwivedi and Arnold, 1973). The various products fo rmed are the result of different reactions, which are dependent upon the conditions of pH and temperature (Dwivedi and Arnold, 1973; Dwivedi and Arnold, 1972; Mulley et al., 1975). Research has show n that a greater number of degradation products are formed under basic conditions as compar ed to acidic conditions. A study by Guntert et al. (1992) examined thiamin de gradation in solutions of pH 1.5, 7.0, and 9.5. Thirty-eight, 32, and 59 compounds were formed under the respective pH conditions. Under moderately alkaline conditions, the greatest numb er of thiophenes and fewest furans would be formed. Acidic conditions showed a greater num ber of furans, furanones, and furanthiols being formed. Since orange juice is fairly acidic (typically pH 3.8) the types of compounds formed would be expected to be similar to those repor ted from acidic conditions. The primary difference is that model studies do not contain the vast a rray of reactive chemicals found in orange juice, which might produce secondary reactions. One of the most significant thiamin degradatio n products is 2-methyl-3-furanthiol. Both it, and its dimer, bis(2-methyl-3-furyl)disulfide impart a savory meaty flavor. As might be expected, it is a well documente d component of meat flavors (W erkhoff et al., 1990; Farmer and Mottram, 1990; Kerscher and Grosch, 1998). 2-Methyl-3-furanthiol and bis(2-methyl-3furyl)disulfide have also been reported in cooked brown rice (Jezussek et al., 2002), recently 78

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reported in grapefruit juice (Lin et al., 2002), and also identified as a possible off-flavor in stored orange juice (Bezman et al., 2001). Bis(2-methyl-3-furyl)disulfide is a highly potent aroma with an odor threshold as low as 2 parts in 10 14 parts water (Buttery et al., 1984). It is extremely difficult to analytically measure such potent aroma active components as they are below the detection of most instrumental techniques. Thiamin is the second most abundant water-soluble vitamin in orange juice, and is a more concentrated source for vitamin B1 than many foods that are better known sources of this vitamin, such as whole wheat bread. The thermal degradation of thiamin at high temperature for short times has been well studied as have room temperature photochemical degradations, but no prior work was found on the thermal degradation of thiamin at elevated room temperature. Because orange juice is a relatively rich source of thiamin, our goal was to determine if thiamin was the probable source of these observed off-flavors in non-refriger ated juices. To achieve this goal, the aroma active volatiles formed in thiamincontaining model orange juice solutions stored at 35 C for up to 12 weeks in the absence of light will be identified and characterized. In this study a highly sensitive pulsed flame photometric detector, PFPD, will be employed with capillary GC to quantify 2-me thyl-3-furanthiol and bis(2-methyl-3-furyl) disulfide in the model orange juices. Aroma act ive compounds in the stored model orange juice samples will be assessed using time-intensity GC-Olfactometry. Materials and Methods The following compounds were obtained co mmercially from Acros Chemical (New Jersey): glucose, sucrose, ci tric acid, 2-formyl-5-methylthi ophene, 2-methyl-3-furanthiol, dimethyl sulfide, 2-ace tylthiophene, and bis(2 methyl 3furyl) disulfide. Fructose and tripotassium citrate were obtained from Fisher (New Jersey). Thiamin hydrochloride, 2-methyl4,5-dihydro-3(2H)-thiophenone, and 2Methyl-3-(methyldithio) furan were obtained from Sigma 79

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(Steinheim, Germany). 4,5-Dimethylthiazole wa s a gift from Florida Treatt Inc. Hydrogen sulfide was obtained from Matheson Gas Products (Montgomeryville, PA). Preparation of model orange juice solutions Model orange juice (MOJ) solutions, at an adjusted pH of 3.8, were prepared according to Peleg and co-workers (1992), with modifications. A 100 g MOJ solution (% w/w) contained the following compounds: sucrose, 5.0; fructose, 2.5; glucose, 2.5; citric acid, 1.0; tripotassium citrate, 0.5, double distilled water, 88.5. Thia min hydrochloride was added at 0.024 mM. Fifty mL aliquots were transferred to 120 mL amber vials, and a nitrogen atmosphere was added by gently flowing N 2 into the vials before sealing. Sample s were then stored in the dark at 35 C for up to 8 weeks to eliminate possible photochemical reactions. A control sample was al prepared under the same conditions, ex cept without thiamin hydrochloride. so Sample preparation Thiamin-MOJ samples were taken on the following days: 0, 1, 7, 14, 28, 42, and 56. Ten mL aliquots were placed into a 30 mL vial with a septum lid and given a nitrogen headspace. Samples were placed in a 40 C water bath and equilibrated for 15 min. Samples were then exposed to SPME: 50/30 m DVB/Carboxen/PDMS StableFlex (S upelco, Bellefonte, PA) for 30 min. Gas chromatographypulse flame photometric detector (GC-PFPD) Samples were separated by SPME using an HP -5890 series II GC (Palo Alto, CA) using an O-I-Analytical 5380 PFPD with a DB-5 column (30 m 0.32 mm i.d. x 0.25 m) from J&W Scientific (Folsom, CA). In itial oven temperature was 40 C and increased to a final temperature of 290 C at 7 C/min. Injector (Gerstel, Baltimore, MD, model CIS-3) and detector temperatures were 200 and 250 C, respectively. Helium was used as the carrier gas at a flow 80

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rate of 2 mL/min. Compounds were monitored on the PFPD for sulfur in two different manners: linear and exponential responses. Chromatograms were recorded using Chromperfect (Justice Innovations, Inc., Mountain View, CA). Samples were run in triplicate. Quantitative analysis 2-Methyl-3-furanthiol and MFT MFT were quantified by means of standard calibration curves containing 0.007, 0.01, 0.05, 0.1 g/mL and 0.001, 0.01, 0.1 g/mL of MFT and MFTMFT, respectively. The standards were prepared in MOJ solutions that did not contain thiamin. The samples were extracted and analyzed in triplicate using the GC PFPD under identical conditions as the storage samples that contained thiamin. Gas chromatography An HP-5890A GC (Agilent Technologies, Pa lo Alto, CA) with a standard flame ionization detector was used to separate the model orange juice extracts using either a DB-5 (30 m 0.32 mm i.d., 0.5 m film thickness, J&W Scientific (Folsom, CA)) or DB Wax (30 m 0.25 mm i.d., 0.5 m film thickness, J&W Scientific (Fol som, CA)). Initial oven temperature was 40 C and increased to a final temperature of 265 C at 7 C/min with no hold. Injector and detector temperatures were 220 and 250 C, respectively. Data were collected and recorded using Chromperfect Software. GC olfactometry GCO equipment and conditions were identical to those described in earlier studies (Bazemore et al., 1999). The olfactometry panel c onsisted of two trained panelists, 1 male and 1 female, between 25 and 30 yrs old. Panelists were trained in a manner similar to Rouseff and coworkers (2001b), using a standard solution of 11 compounds typically found in citrus juice (ethyl butanoate, cis -3-hexenol, trans 2-hexenal, pinene, myrcene, linalool, citronellol, carvone, 81

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terpin4-ol, geranial, and neral). The standard mixture helped train panelists in a time-intensity scale, optimum positioning, and brea thing techniques. Panelists al so were trained by evaluating at least 10 commercial orange juice flavor extrac ts in order to gain experience and consistency. Panelists were not used for this study until they demonstrated the ability to replicate aroma intensity responses in th e practice juice ex tracts. Panelists ran each experimental sample in duplicate and summary reports were generated for each aromagram. Only peaks detected at least 50% of the time were included in this study. Results from each panelists aromagram were normalized with their own maximum peak inte nsity (set to 100) be fore being averaged. Gas chromatographymass spectrometry (GC MS) Sample separation was performed on a Finni gan GCQ Plus system (Finnigan Corp., San Jose, CA), using a J&W Scientific DB-5 column (60m 0.25 mm i.d. x 0.25 m film thickness (Folsom, CA). The MS was operated under posi tive ion electron impact conditions: ionization energy, 70 eV; mass range, 40 300 amu; scan rate, 2 scans/s; el ectron multiplier voltage, 1050 V. Transfer line temperature was 275 C. Initial column oven temperature was 40 C and increased at 7 C/min to a final temperature of 275 C. Injector temperature was 250 C. Helium was used as the carrier gas at a linear ve locity of 32 cm/s. When searchable spectra could not be obtained for compounds of interest because of low signal-to-noise ratio, chromatograms of selected masses were reconstructed from the MS data matrix. These selected ion chromatograms (SIC) employed at least three unique m / z values from the mass spectrum of standards were used as identification aides. Whenever possible, the molecular ion (M + ) was chosen as one of the three m / z values. 82

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Injector decomposition study A standard solution of MFT was injected onto the GC PFPD under similar chromatographic conditions outlined above with ch anges to the injector temperature. Samples were injected at three temperatures: 160, 180, and 200 C. Microbiological analysis Thiamin MOJ samples from day 0 and day 56 were plated for microbial counts using standard microbial techniques (Swanson et al., 2001). Samples were run in duplicate using orange serum agar (OSA), acidified potato dext rose agar (APDA), and plate count agar (PCA) plates. OSA and PCA plates we re incubated at 30 and 35 C, respectively, for 24 hours, while APDA plates were incubated at 25 C for 48 hours. Dehydrated media was purchased from Difco (Becton, Dickindon and Company, Sparks, MD .). Each medium was prepared according to manufacturers directions, and plates were poured using standard aseptic techniques. Results and Discussion This study differs from previous thiamin thermal degradation studies (Guntert et al., 1992; Guntert et al., 1990; van de r Linde et al., 1979; Guntert et al., 1993; Hartman et al., 1984a; Hartman et al., 1984b) in terms of time temperatures, sample matrix, detection devices, and thiamin levels employed. Whereas previous stud ies were conducted at high temperatures (110 130 C) and short times (1 6 hours), this study was conducted at relatively low temperature (35 C) and long times (8 weeks). The former conditions are typical for cooking and roasting, whereas the time temperature conditions chosen for this study represent the most extreme conditions a juice would likely encoun ter during storage. In this study, GC O is employed to identify the number, quality, and the relative aroma intensity of the thiamin degradation products. Prior studies primarily employed GC-MS to determine total volatil es without directly 83

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determining their aroma activity. Finally, thiamin concentrations chosen for this study are more typical of those found in citrus juices (0.024 mM), whereas prio r studies employed considerably higher concentrations, some as great as 296 mM or more than 12,000 times higher concentrations (Jhoo et al., 2002). Day 7 and 42 aromagrams Normalized aromagrams from thiamin mode l orange juice solutions stored at 35 C for 7 and 42 days are compared in Figure 5-1. These two dates were chosen to represent short and long-term storage conditions. Thirteen aroma volatiles were observed between the two storage times; 11 aroma active volatiles we re found after 7 day storage, but only 8 aroma volatiles were observed after 42 days storage. Six of the ei ght aroma active volatile s found in the day 42 samples were also found in the day 7 samples. Thus almost half of the aroma volatiles observed after 7 day storage were no longer observed after 42 day storage. This can be explained with sulfur compounds often being unstable. Although 5 aroma volatiles were lost between day 7 and day 42 samples (peaks 1, 2, 6, 8, and 11), two new aroma volatiles were generated (peaks 5 and 10). Total aroma intensity also decreased from day 7 to day 42. Of the aroma components detected, MFT (peak 4), roasted meaty aroma, and its dimer, MFT MFT (peak 13), roasted meat/savory aroma, were among the most inte nse. MFT is a well-established thermal degradation product of thiamin (Grosch and Zeiler-Hilg art, 1992) and has been reported in stored orange juice (Bezman et al., 2001). The intensity of MFT MFT peaks in the aromagrams in Figure 5-1 is only slightly less than that of the monomer, MFT, strongly suggesti ng that it could be a potent storage off-flavor as well. Combined, these two compounds comprise 33% of the total aroma activity after 7 day storage and 48% of the aroma peak area after 42 days storage. Because the dimer (peak 13) has 84

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only slightly less aroma intensity than MFT (peak 4) at both sampling times and there are fewer aroma volatiles at day 42, the relative impact of dimer should increase with increased storage time. Peaks 3, 9, and 12 are common to both sa mpling times and have been characterized but not identified (see Table 5-1). These peaks were characterized as havi ng tropical fruity/grape, fertilizer/ earthy, and savory/meaty/su lfury attributes, respectively. All three peaks diminish between 7 days stor age and 42 days storage. Many of the peaks that are lost after extended storage also remain to be identified. However, peak 6, with meaty, cooked attributes; peak 8, with a burnt aroma; and peak 11, with a meaty aroma, have been identified as 3-thiophenethiol, 2-acetylthiophene, and 2-methyl-3-(methyldithio) furan. The two new compounds found after 42 days storage, peak 5 with skunky/ earthy attributes and peak 10 with a meaty aroma, have been identifie d as 4,5 dimethylthiazole and 2-formyl-5methylthiophene, respectively. Their st ructures are shown in Figure 5-2. Aroma volatile identifications Table 5-1 lists the aroma active compounds observed, their linear retention index values (LRI) on DB-5 and DB Wax columns, aroma descriptors, and identification procedures employed. Linear retention index values and arom a descriptors were used to make preliminary identifications; these aroma descriptors and rete ntion values were confirmed using authentic standards. Final confirmati on was achieved by comparing GC MS data from the sample with that of standards. The PFPD is one of the most sensitive and selective detectors for studying sulfur containing volatiles. The responses from th is detector were used as further confirmation for peaks thought to be due to su lfur volatiles. The PFPD peaks in the sample that occurred at the same retention time as an authentic standard were considered additional proof of the peaks identity. Peaks 4 and 13 are the major flavor impact compounds from the thermal degradation of thiamin and have been identified as 2methyl-3-furanthiol, MFT, and bis(2 methyl 3furyl) 85

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disulfide, MFT MFT, the dimer of MFT. Identification was based on the cumulative evidence of retention matching on both DB-5, carbowax columns, aroma characteristics, PFPD data, and MS evidence. 2-Methyl-3-furanthiol was confirmed using SIC chromatograms at m / z 114(M + ), 106, and 86. In the case of MFT, all th ree SICs produced distinct peak s at the identical LRI value as the standard. The first aroma active peak shown in Figure 5-1 occurs in the region where hydrogen sulfide and dimethyl disulfide would be expected to elute. Both hydrogen sulfide (Dwivedi and Arnold, 1973; Gunter t et al., 1990) and dimethyl di sulfide (Guntert et al., 1992; Guntert et al., 1993) have been reported as thia min degradation products. Therefore, the first 6 min. of the day 7 aromagram and corresponding PFPD response is shown in Figure 5-3 to better illustrate which sulfur compound corresponds best with the first aroma peak. Hydrogen sulfide elutes before dimethyl sulfide and an unidentified su lfur peak. It is readil y apparent that the first aroma peak elutes at the same time as dimethyl sulfide. As illustrated in Figure 5-1 aroma peaks 1, 2, 6, 8, and 11 were only detected during the first few days of storage at 35 C storage. These were weak intensity aroma peaks that were completely absent after 42 days storage. Peak one has already been identified as dimethyl sulfide. The second GC O peak has been tentatively identifie d as 1-pentanol, based on its aroma description of fruity/green and its LRI values. Aroma peak 6 had a meaty, cooked aroma. It has been tentatively identified as 3-thiophenethiol on the basis of its aroma characteristics and retention characteri stics on DB-5. SIC MS chromatograms using m / z 116(M + ) and 71 (the only major peaks in the Wiley library spectra for this compound) produced peaks at the same retention time as a PDPF peak and the GC O peak in question. All of these peaks occur at the literature LRI for this compound. However, this iden tification must be considered tentative as no standard could be obtained for comparison pu rposes. Aroma peak 8 was identified as 286

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acetylthiophene on the basis of the match between its retention characteristics on DB-5 and carbowax, MS SICs of m / z of 110, 125, and 83 peaks, PFPD re sponse with identical LRI and odor match with a standard. Aroma peak 11 was identified as 2-methyl-3-(methyldithio) furan on the basis of the matching of its aroma charact eristics, retention characteristics, and MS characteristics of SICs of m / z 160, 113, and 85, compared to an authentic standard. The identities of peaks 3, 9, and 12 could not be determ ined. As seen in Figure 5-1, all three peaks were observed in samples stored for both 7 and 42 d. Peak 3 displayed a topical fruit aroma and probably does not contain sulfur, for there was no associated PFPD peak (see Figure 5-3). Its fruity aroma and early retention value suggests it might be an es ter (fruity) or a potent sulfur volatile whose concentration was above its threshol d but below the detection limits of the sulfur detector. Peaks 9 and 12 were major aroma components in the 7 day sample, but were only about half as intense after 42 days storage. Peak 12 had a DB-5 LRI value of 1403, with an aroma that was described as savory, meaty, and su lfury. It may also be due to the same aroma volatile reported by Baek and co -workers (2001) in a process flavor, because it had similar retention and aroma characteristics. It had a DB -5 LRI of 1393 and described its aroma as spicy, burnt, meaty, and roasty. They were al so unable to identify this material. Of those aroma peaks that were only seen toward the end of the storage study, peak 5 was identified as 4,5-dimethylthiazole (peak 5) and peak 10 was identified as 2-formyl-5methythiophene. SICs of m / z 114, 98, and 71 produced peaks at the identical retention values as authentic 4,5-dimethylthiazole. Aroma quality and retention values were also identical to an authentic standard. Earlier studies had found th is compound in greatest co ncentration at pH 9.5 under high-temperature short-time conditions (Gunter t et al., 1992; Guntert et al., 1990; Hartman et al., 1984a). However, at the low-temperature, acidic pH of the model orange juice in this 87

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study, it was only a minor aroma peak. Because citrus juices are highly unlikely to be stored at this temperature for this length of time, it is al so unlikely that this compound would be found in many commercial juices. The identification of peak 10 was based on its meaty aroma and the fact that it also produced a PFPD peak at the exact re tention time as 2-formyl-5-methythiophene. This peak also matched the FID LRI values on DB-5 and carbowax and the MS fragmentation data of 5-formyl-5-methylthiophene. Peak 7 ha s been identified as 2-methyl-4,5-dihydro-3(2H)thiophenone, because its sensory, chromatographic, and mass spectral properties were identical to that of an authentic standard. SICs of m / z of 116, 88, and 60 produced peaks at the identical retention value as the standard. Quantification of MFT and MFT MFT Both compounds possess a roasted meat or savory aroma, which is highly desirable in meat and savory flavors but are definite off flavors in citrus juices. MFT MFT is one of the most potent food aromas ever measured. It produces an aroma peak at levels well below that of the PFPD detector (1 pgS/s) and is thus difficult to qu antify even with the most sensitive detectors. MFTMFT has been reported in a recent GC O study of thermally concentrated grapefruit juice (Lin et al., 2002), but no qua ntitation was attempted. Thiols are known to readily oxidize into disulf ides (thiol dimers). This was demonstrated in a model study on the oxidative stability of odor-active thiols, whic h included MFT (Hofmann et al., 1996). MFT and its dimer were quantified during the course of this storage study using the PFPD. Results are shown in Figure 5-4. Even though the PFPD detector is one of the most sensitive sulfur detectors, apprecia ble aroma peaks for both MFT and MFTMFT were perceived by GCO before any PFPD peaks were observed. For example, MFT MFT was first detected on day 14 using the PFPD, whereas it produced a signi ficant aroma peak on day 7. Using a similar extraction procedure (SPME), panelists in another GC O study could detect as little as 270 ng/L 88

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MFT in stored orange juice (Bezman et al., 2001 ). As shown in Figure 5-4, MFT concentration begins to increase with increasi ng storage time up to 42 days of storage then decreases from 9.8 x 10 4 mM at day 42 to 7.0 x 10 4 mM at day 56. As expected, the dimer of MFT, MFT MFT, cannot be formed until a certain amount of the monomer has formed. Thus, its concentration will always lag behind that of the monomer. The dimer is not detected with the PFPD until day 14, with a measured concentration of 2.0 x 105 mM, which increases to 3.0 x 104 mM by 28 days and then maintains a roughly constant concentration after that The constant concentration after 28 days storage suggests that the dimer also participates in subsequent reactions and the rate of these subsequent reactions is about the same as the formation from the monomer. When comparing GCO and PFPD responses for MFT and MFT MFT as in comparing results in Figures 5-1 and 5-4, a few distincti ons must be considered. The response from the PFPD detector will be a function of the atomic sulf ur concentration irrespective of the source of the sulfur, whereas the intensity indicated by human assessors for GC O aromagrams will be a function of the human sigmoidal dose response to aroma. The aroma intensities for both MFT and MFT MFT in Figure 5-1 do not change appreciably betwee n 7 and 42 days, whereas changes in PFPD responses were observed. Human olfa ctory detection imits for some thiols are appreciably lower than that of the PFPD. Fo r example, at day 14 th e concentration of MFT MFT was 2.3 x 10 5 times greater than its aroma threshold and increased to 3.39 x 10 6 times greater than threshold at day 42. At these le vels, it should not be surprising that GC O aroma responses did not vary as they were saturated, but the PFPD response (being less sensitive) was not saturated. Thiamin as a source of MFT and MFT MFT in citrus juices It is generally accepted that both MFT and its dimer are formed during the thermal decomposition of thiamin in acid media at high temperature (van der Linde et al., 1979; 89

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Mottram, 1991). However, MFT can potentially be formed from two other pathways. It can be produced through a Maillard reacti on involving cysteine and various simple sugars (Farmer et al., 1989; Mottram and Whitfield, 1994), as well as from the reaction of nor furaneol and cysteine (Hofmann and Schieberle, 1998). Bolton et al. ( 1994) studied a thiamin/cysteine model system in order to determine the role of cysteine in th e formation of MFT. Using labeled 34S-cysteine, they determined that cysteine can contribute to MFT formation in the presence of thiamin, but that thiamin was required for the formation of MF T. Few studies have examined orange juice for the presence of cysteine. However, a recent report by Heems et al. (1998) reported no measurable amounts of cysteine in orange juice (limits of detection ) 152 g/L). Because both alternate pathways for the formation of MFT requi re the presence of cysteine and cysteine is apparently absent from orange ju ice (and probably grapefruit juice) it is therefore unlikely that MFT can be formed in any way other than the direct decomposition of thiamin. MFT can also form from the reaction of 4-hydroxy-5-methyl-3(2 H)-furanone, norfuraneol, and either cysteine or hydrogen sulfide (Hofmann and Schieberle 1998; Whitfield and Mottram, 1999). Norfuraneols presence is considered a degrad ation product of pentoses; however, a reaction pathway from hexoses was proposed by Hofmann et al. (1998). The presen ce of norfuraneol in the control model orange juice solution could point toward th e formation of MFT through the mechanism with hydrogen sulfide. To test for the presence of norfuraneol, GC O and GC-MS analyses were performed on the control model ora nge juice solution after 56 days storage. No norfuraneol was detected, thus eliminating the last alternate MFT formation pathway. Possible GC injector thermal artifacts Because thiols are unstable and readily dime rize, and because there are literature reports (Block, 1993) of sulfur artif act creation after exposure to the high temperature of the gas chromatograph injector, additional experime nts were conducted to determine if MFT MFT was 90

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formed from MFT in the GC injector. Three injector temperatures were chosen, 160, 180, and 200 C. In each case, a standard containing 0.1 g/mL MFT was injected onto the GC to determine if any dimer could be detected. In all cases, only MFT was detected by the PFPD, and its peak height did not in crease with decreasing injector temperat ure. Therefore, it appears that MFT was not degraded in the injector, and that the MFT MFT detected in this study was not an injector port artifact. Possible microbiological artifacts Microbial activity is a well -known means of producing of aroma compounds, providing they are present. However, extensive precautions were observed in this study to maintain microbial sterility in the storage samples. To confirm that none of the aroma-active compounds observed in this study were derived from microbial organisms, samples were evaluated for microbial content. Samples from day 0 and day 56 were plated using OSA for an aciduric count, APDA for a yeast/mold count, and PCA for a total plate count. Results from all plates indicated counts less than 10 cfu/mL with no visible growt h. Therefore, the aroma compounds detected in this study were not the result of microbiological contamination. Conclusions Thiamin has been shown to be the precursor to the potent aroma compounds MFT and its dimer, MFT MFT, in model orange jui ce solutions stored at 35 C. Although the study lasted for eight weeks, both compounds produced major aroma peaks after 7 days storage. Both compounds have been shown to have a profound im pact on the aroma of these stored solutions, responsible for 33 and 48% of the total aroma at day 7 and 42, respectively. The relative aroma contribution of these two compounds was shown to change with storage time. Both these meaty off flavors have been reported in prior stored and/or heated orange and grapefruit juices. 91

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Because citrus juices are rich sources of thiami n, and our model juice studies have demonstrated that, from an olfactory point of view, thes e two compounds are among the major aroma impact compounds formed, it appears that th iamin is the precursor for these off flavors in citrus juices. However, to definitively prove that thiamin is th e source of these off flavors in citrus juices, it remains for isotopically labeled thiamin to be exposed under similar conditions to see if isotopically labeled MFT or its dimer could be detected. 92

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Table 5-1. Aroma active compounds detect ed in model oran ge juice solution no. LRI a (DB5) LRI a (DB-Wax) Compound name Identification method Aroma descriptor 1 681 Dimethyl sulfide e PFPD Sulfury 2 766 1-Pentanol e LRI, odor Fruity, green 3 843 Unknown Tropical fruity, grape 4 863 1305 2-Methyl-3-furanthiol LRI, MS c odor, PFPD Roasted meat 5 928 n.d b 4,5-Dimethylthiazole LRI, MS c odor Skunky, earthy 6 967 3-Thiophenethiol e LRI, MS d odor, PFPD Meaty, cooked 7 998 1506 2-Methyl-4,5-dihydro-3(2H)-thiophenone LRI, MS c odor, PFPD Sour-fruity, musty, green 8 1085 1785 2-Acetylthiophene LRI, MS c odor, PFPD Burnt 9 1095 Unknown PFPD Fertilizer, earthy 10 1112 1785 2-Formyl-5-methylthiophene LRI, odor, PFPD Meaty 11 1178 n.d. 2-Methyl-3-(methyldithio) furan LRI, MS c odor, PFPD Meaty 12 1403 Unknown PFPD Savory, meaty, sulfury 13 1543 2150 Bis(2-methyl-3-furyl) disulfide LRI, MS c odor, PFPD Roasted meat, savory DB-5 Retention Time (min.)Normalized Peak Intensity 2 20 10Day 7 Day 42 1 2 3 4 5 6 7 8 9 10 11 12 13 Figure 5-1. SPME headspace samples of GC-O aromagrams comparing day 7 and 42, where peak intensities were inverted for da y 42 data. Peak number corresponds to compound numbers in Table 5-1. 93

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S SH 3-Thiophenethiol (peak 6) S O 2-Acetylthiophene (peak 8) O S S 2-methyl-3-(methyldithio) furan (peak 11) N S 4,5-Dimethylthiazole (peak 5) S O 2-Formyl-5-methylthiophene (peak 10) Figure 5-2. Structures of select aroma active su lfur compounds detected in the model orange juice solution. Peak numbers in parenthe ses correspond to peak numbers in Table 51. 94

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2.0 3.0 4.0 5.0 6.0 1.0 Sulfury Fruity/green Tropical fruity Roasted meat2-Methyl-3-furanthiol Hydrogen sulfide DimethylsulfideTime (min)GC-O responsePFPD response Figure 5-3. Comparison between PFPD chromat ogram and corresponding aromagram from a model orange juice solution stored for 7 days at 35C. First 6 min shown, to clearly illustrate which of the early PFPD peaks were aroma active as well as to demonstrate that there was no sulfur activ ity associated with peaks 2 and 3. SPME injection using a DB-5 column. See methods section for additional experimental details. 95

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96 0 0.0002 0.0004 0.0006 0.0008 0.001 0.0012 0.0014 0102030405060Time (days)Concentration (mM/L) 2-Methyl-3-furanthiol Bis-(2-methyl-3-furly) disulfide Figure 5-4. MFT and MFT-MFT concentrations in thiami n model orange juic e solutions stored at 35C in the absence of light as determined by PFPD.

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CONCLUSIONS The underlying objective for my study was to dete rmine what factors can affect the quality of orange juice that a consumer purchases and which of these factors can be manipulated to provide the highest quality of ora nge juice to the consumer. Fact ors that can affect the quality include determining what aroma impact compounds c ontribute to quality or ange juice as well as compounds that would negatively cont ribute towards the flavor. Othe r factors that can affect the quality of orange juice include temperature, p ackaging and flavor precursors such as thiamin. Aroma impact compounds were determined in commercially purchased orange juices that were determined organoleptica lly to be of differing qualit y. Aldehydes including hexanal, heptanal, octanal, nonanal, decanal, undecanal and ge ranial were determined to contribute to the above average quality orange juice; where as known off-flavors 4-viny lguaiacol and methional contributed to the detriment of the below average juice. A second study determined how the aroma impact compounds from the above study change over time, temperature and packaging. Aldehydes including (Z)-3-hexenal (green banana aroma), octanal (lemon aroma) and decanal (woody, green aroma) diminished and/or were lost over time and temperature. Off-flavor compounds such as carvone (licorice aroma) and m-cresol (manure aroma) were not found at day 0 and were formed over time. Polyethylene terephthalate samples had known off-flavor compounds that were not in glass samples, including 2-methyl-3furanthiol (meaty aroma), eugenol (burnt sugar aroma) and soto lon (cooked, spicy aroma). The last study determined the probable sour ce of the off-flavor compounds 2-methyl-3furanthiol and bis(2-methyl-3-f uryl) disulfide through a model orange juice study to be the second most abundant water soluble v itamin in orange juice, thiamin. Orange juice manufacturers can use the inform ation from this study to tailor add-back flavor packages with the arom a active compounds that contribute to quality orange juice. 97

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Manufacturers can also take into account the type of packaging that is used and the shelf-life of the product at higher real world temperatures and the affect it has on orange juice quality. Finally, with a recent trend toward s fortification of orange juice and beverages with vitamins and phytochemicals, for example calcium fortified orange juice; this study show s that the levels of thiamin present in orange juice can cause off-fl avor production in an orange juice matrix. Additional fortification with thiamin would cause an increase in the off-flavors 2-methyl-3furanthiol and bis(2-met hyl-3-furyl) disulfide. 98

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BIOGRAPHICAL SKETCH J. Glen Dreher grew up in West Palm Beac h, FL. He attended Purdue University and graduated in 1997 with a B.S. in Food Science. In spring 1999 he entered graduate school at the University of Florida in food science. He spent a year in Gainesville, FL taking course work and then relocated to Winter Haven, FL for his doc toral research at the Citrus Research and Education Center in Lake Alfred, FL. He t ook a job at Jim Beam Brands in Clermont, KY February, 2003 working in new product developmen t. He has since completed his Ph.D. in 2007. 111