High pressure processing of orange and grapefruit juices

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High pressure processing of orange and grapefruit juices
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Goodner, Jamie Kirkpatrick, 1971-
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Food Science and Human Nutrition thesis, Ph. D   ( lcsh )
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Thesis (Ph. D.)--University of Florida, 1998.
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Includes bibliographical references (leaves 113-118).
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Typescript.
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Vita.
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by Jamie Kirkpatrick Goodner.

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HIGH PRESSURE PROCESSING OF ORANGE AND GRAPEFRUIT JUICES


By

JAMIE KIRKPATRICK GOODNER



















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

1998















ACKNOWLEDGMENTS


I would like to thank my committee, Drs. Braddock,

Parish, Gregory, Sims and Powell, for the time and effort

each spent on my behalf. They provided guidance as well as

set an example of how to be a fine scientist. I would also

like to thank Cynthia, Lorrie, Prashanthi, Renee, Bev and

Rockey for their friendship and many helpful discussions of

all things scientific. Most of all, thanks go to my husband

Kevin, who could always see the light at the end of the

tunnel, even when I was sure it had gone out.
















TABLE OF CONTENTS




ACKNOWLEDGMENTS ........ ..................... .ii

LIST OF FIGURES ........ ...................... v

LIST OF TABLES ........ .................... vii

ABSTRACT .......... ....................... viii

INTRODUCTION ........... ...................... 1

BACKGROUND .......................................... 4
Physical Effects ......... .................. 4
Operating Principles ........ ................ 5
Contributing Factors ........ ................ 8
Enzyme Inactivation ....... ................ 9
Pectinesterase ........ ................... .11
Quality Effects ....... .................. 14
Separation Procedures ..... ............... ..17

MATERIALS AND METHODS ....... .................. .22
Pectinesterase Inactivation .... ............ .22
Juice Preparation ...... ............... ..22
Pressurization ...... ................ 22
PE Activity Determination .... .......... .. 28
Enzyme Isolation Study ...... ............... ..29
Enzyme Extraction ...... ............... ..30
Sample Preparation ..... .............. 31
Cloud Loss ......... ..................... .31
Storage Study of Valencia Orange Juice .. ....... ..32
Statistics ......... ..................... .34

RESULTS AND DISCUSSION ...... ................. ..35
Enzyme Inactivation ...... ................ 35
Kinetics ....... ................... .36
Heat Generation by Pressure .... .......... .44
Decimal Reduction Values .... ........... .50
Enzyme Sensitivity .... ............. 51
PH Effects ....... .................. 52


iii









Enzyme Isolation .................. 53
Cloud Loss ........ ..................... .66
Storage Study .................................. 78
Statistical Analysis of Flavor Volatiles . 78
Principal Component Analysis ... ......... .95
Discriminant Analysis .... ............ 103

CONCLUSIONS ........... ...................... 1i1

REFERENCES ........ ...................... 113

BIOGRAPHICAL SKETCH ....... .................. .. 119















LIST OF FIGURES


Figure page

1. Reactions of Pectin and Pectinesterase .. ....... ..13
2. Schematic Diagram of Isoelectric Focusing System 20
3. Typical Pressure Profile Obtained by Stansted
Isostatic High Pressure Unit in Rapid
Compression/Decompression Mode ................ 25
4. Stansted "Plunger Press Food Lab 9000" High Pressure
Food Processing Unit ...... ................ ..27
5. Inactivation of Orange Pectinesterase at Pressures of
600-900 MPa. Data Points Represent the Average of Two
Measurements ................................... 38
6. Inactivation of Grapefruit Pectinesterase at Pressures
of 600-900 MPa ........ ................... .40
7. High Pressure Inactivation of Pectinesterase at 500
MPa. Data Points Represent the Average of Two
Measurements ........ .................... .46
8. Log of PE Inactivation at 500 MPa ... ......... .48
9. SDS-PAGE Separation of Pectinesterase from Untreated
Valencia Juice .............................. .55
10. SDS-PAGE Separation of Pectinesterase from Valencia
Juice Pressurized at 800 MPa for 1 Minute ...... .57
11. SDS-PAGE Separation of Pectinesterase from Valencia
Juice Pasteurized at 75 0C for 1 Minute ...... .. 59
12. SDS-PAGE Separation of Pectinesterase from Valencia
Juice Pasteurized at 90 0C for 1 Minute ......... ..61
13. Cloud Stability of Orange Juice Treated at 500 MPa 68
14. Cloud Stability of Orange Juice Treated at 600 MPa 70
15. Cloud Stability of Orange Juice Treated at 700 MPa 72
16. Cloud Stability of Orange Juice Treated at 800 MPa 74
17. Cloud Stability of Orange Juice Treated at 900 MPa 76
18. z-3-hexenol Composition Over Storage Time ...... .81
19. a-pinene Composition Over Storage Time .. ....... ..83
20. Sabinene Composition Over Storage Time .. ....... ..85
21. Myrcene Composition Over Storage Time .. ....... ..87
22. Octanal Composition Over Storage Time .. ....... ..89
23. d-Limonene Composition Over Storage Time ........ ..91
24. Compilation of Monitored Peak Correlation ...... .94









25. Principal Component Analysis of 6 Volatiles During
Storage Study Utilizing Factor 1 and Factor 3.
Ovals Represent 95% Confidence Limits .. ....... ..97
26. Principal Component Analysis of 6 Volatiles During
Storage Study Utilizing Factor 1 and Factor 2.
Ovals Represent 95% Confidence Limits ...... 102
27. Discrimination Among Treatments Based on Peak Areas
of Sabinene and Myrcene. Ovals Represent 95%
Confidence Limits ...... ................ 105
28. Discriminant Analysis on Volatiles to Determine
Grouping Based on Juice Treatment .. ........ 108















LIST OF TABLES


Table rpage

1. Percent Inactivation of Pectinesterase in Orange and
Grapefruit Juice at Different Pressures ...... ..43
2. Specific Activity of Isoelectric Focussed Fractions of
Pectinesterase Isolated from Untreated Late Season
Valencia Orange Juice ..... ............... .63
3. Specific Activity of Isoelectric Focussed Fractions of
Pectinesterase Isolated from Late Season Valencia
Orange Juice Treated at 75 0C for 1 Minute ..... ...64
4. Specific Activity of Isoelectric Focussed Fractions of
Pectinesterase Isolated from Late Season Valencia
Orange Juice Treated by High Pressure (800 MPa)
for 1 Minute ........ .................... .65
5. Plate counts on OSA at days 49 and 89 of cloud loss
study. Counts are reported using standard plate
counting protocol ...... ................. ..79
6. Factor Analysis for Principal Component Analysis . 99
7. Factor Loadings Used in Discriminant Analysis . 109


vii















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy



HIGH PRESSURE PROCESSING OF ORANGE AND GRAPEFRUIT JUICES

By

Jamie Kirkpatrick Goodner

December, 1998

Chairperson: Dr. Robert J. Braddock

Major Department: Food Science and Human Nutrition

The enzyme pectinesterase (PE) reduces the quality of

citrus juices. Current commercial inactivation of the enzyme

is accomplished by heat pasteurization. Pressurized

treatments of orange and grapefruit juices to by-pass the

use of extreme heat during processing is explored. PE

inactivation using high pressure processing (HPP) in the

range of 500-900 MPa was accomplished in orange and

grapefruit juices. The higher pressures (>600 MPa) caused

instantaneous inactivation of the heat labile form of the

enzyme, but did not inactivate the heat stable form of PE.

Heat labile grapefruit PE was also more sensitive than

orange to pressure.

Isoelectric focusing and polyacrylamide gel

viii











electrophoresis were used to isolate and examine extracted

PE from Valencia juice. Untreated juice displayed a narrow,

well defined band at 36,000 molecular weight. Juice treated

at 900 C showed a complete loss of PE and no activity was

detectable. Samples treated with HPP at 800 MPa or

pasteurized at 700 C exhibited a decrease in PE activity and

an aberration of the band at 36,000 molecular weight.

HPP was also investigated as a means to preserve cloud

in fresh squeezed orange juice. Pressures from 500 MPa to

900 MPa were investigated at hold times of 1 second, 1

minute and 10 minutes. Cloud preservation was directly

proportional to increased pressure and longer processing

times. All treatments yielded a microbially stable product.

A 90 day shelf life under refrigeration conditions was

achieved using pressures of 700 MPa and higher combined with

treatment times of at least 1 minute.

A storage study ascertained the difference in volatile

profiles over time in high pressure treated juice versus

traditional pasteurization. It was found that both

statistical techniques were able to satisfactorily

distinguish the two treatments from each other and the

untreated control. Myrcene, a-pinene, sabinene, and d-

limonene were the flavor compounds most affected by

treatment.











INTRODUCTION

In order for food to have a longer shelf life, it must

be processed to dramatically increase the stability over

fresh, or unprocessed food. One of the most common and

important techniques for prolonging shelf life and safety of

food products is achieved by killing the microorganisms that

cause spoilage and food borne illness by heat preservation.

Often, enzymes that cause degradation after harvesting are

also inactivated by thermal pasteurization or retort

operations. However, heat processing can dramatically

changes the fresh flavor and quality of the food product.

As an alternative to heat pasteurization, high pressure

processing (HPP) has been shown to reduce microbial levels

(Takahashi et al., 1993; Ogawa et al., 1992), affect

properties and functionalities of proteins (Messens et al.,

1997; Masson, 1992), and influence enzyme activity

(Seyderhelm et al.1996; Basak and Ramaswamy, 1996). As such,

it is rapidly gaining interest as a tool for food processing

and an alternative to heat. Growth of pressure resistant

bacterial spores is inhibited in high acid food, and thus,

orange and grapefruit juices, which are commercially treated

using heat, are prime candidates for HPP.

Current processing of citrus juice employs a

pasteurization step, which has the purpose of reducing

1











microbial levels as well as inactivating pectinesterase

(PE), the enzyme responsible for cloud loss in citrus

juices during holding and storage. The severe levels of

heating used in commercial pasteurization treatments are

necessary to inactivate PE, and these are in excess of what

is necessary to make the product microbially safe.

Pasteurization results in a more stable product, but one

that has also lost its more delicate volatile components.

Heat treatment of orange juice can also lead to cooked off-

flavors and caramelization of sugars, neither of which

contributes positively to "good flavor." The first purpose

of this project was to determine the effectiveness of high

pressure for PE inactivation while maintaining flavor

integrity. Since it has been generally observed that

constituents of food can have a protective effect on the

enzyme against inactivation by heat or pressure (Seyderhelm

et al., 1996; Ogawa et al., 1990), PE was investigated in

two of its biochemical environments: orange and grapefruit

juice.

The second goal of this research was to determine the

cloud stability of pressure treated juice versus a non-

pressurized control. Juice cloud loss is the result of

demethylated pectin interacting with calcium ions, causing a

precipitation into a clear serum layer on top of a viscous

layer of settled pectin and insoluble solids. Cloud is











retained by protecting the natural pectin in the extracted

juice from enzymatic deesterification and degradation by

pectinesterase (Rouse and Atkins, 1952). Turbidity is a

desired characteristic of citrus juice, and cloud content is

one of the criteria of quality (Rothschild and Karsenty,

1974). Thus, cloud loss is considered a quality defect in

citrus juice, and inactivation of pectinesterase is one of

the main reasons for the level of heating in commercial

pasteurization. In this work, high pressure treatment is

shown to inactivate the heat labile form of pectinesterase

in orange and grapefruit juices. Since some PE activity

remains in juice after pressurization, it was of interest to

determine the stability of the cloud after pressure

treatment.

The final goal of this project was to ascertain if high

pressure treatment of fresh orange juice was discernable

from fresh and pasteurized juice. Volatiles were monitored

by purge-and-trap gas chromatography (GC) during storage to

determine degradation of volatiles over time.











BACKGROUND


Physical Effects



The idea of controlling microorganisms by high pressure

treatment is not novel. At the turn of the century,

scientists had discovered that pressure extended the shelf

life of milk by reducing the initial bacterial load by 5 to

6 log cycles (Hite, 1899). This author also investigated

fruit juices and vegetables (Hite et al.,1914). It is now

proposed that HPP affects the secondary bonds of large

molecules such as proteins, sugars, and cell membranes

(Mertens, 1996). Only recently has high pressure been

considered as a food processing technique. Two companies

already have pressurized products (e.g. jam, citrus juices,

and yogurt) on the market in Japan.

Much of the work in this area to date has dealt

exclusively with the control and inactivation of microbial

populations through pressure treatment (Ogawa, et al., 1990,

Shigehisa et al., 1991, Styles et al., 1991, Takahashi et

al., 1991; Butz et al., 1990; Carlez et al., 1993, Ludwig et

al., 1992; Raffalli et al., 1994; Sato et al., 1994; Smelt

and Rijke, 1992; Yasumoto et al., 1993), so there is less

known about chemical changes and enzyme inactivation due to

4









5

high pressure processing. One study has evaluated the

changes in mandarin orange juice associated with high

pressure treatment, both microbial and chemical(Takahashi et

al., 1993). They found what they termed an insignificant

change in a list of volatiles in Satsuma mandarin single

strength juice.

A thorough analysis of physical characteristics after

pressure treatment was reported, where standard methods were

used to determine microbiological activity, chemical

composition, pH, color, aroma and viscosity (Donsi et al.,

1996). These results show no substantial compositional

change in vitamins, sugars, pH, organic acids and several

aroma compounds. Microbial inactivation was achieved at 350

MPa. Only viscosity was measurably affected by pressure

treatment; a reduction in non-Newtonian viscosity (the ratio

of shear stress and the corresponding velocity gradient) was

noted.





Operating Principles



Two general scientific principles govern the action of

high pressure on food. The first is LeChatelier's

principle, which states that a reaction equilibrium will

shift to minimize the effect of an external force applied to












the system, such as heat or the addition of product or

reactant. This means a shift in the reaction resulting in a

smaller volume will be enhanced by high pressure treatment,

including chemical reactions as well as possible changes in

molecular conformation. The second theory important to

understanding the effect of high pressure is Pascal's

principle, stating that as pressure is applied it travels

instantaneously and uniformly throughout the sample.

Pascal's principle is true regardless of the sample size or

volume, which means that the whole sample (or food) will be

treated uniformly throughout. This is in direct contrast to

thermal processing, which results in hot spots and

overheating of the surface to obtain the desired temperature

in the center of the sample. The energy required to process

by pressure is also lower since no additional energy is

required to maintain the desired temperature level once it

has been reached. This makes process time irrelevant to

energy concerns, unlike heating. For reference,

approximately the same energy is necessary to reach 400 MPa

and 30 C from ambient conditions(Cheftel, 1995).

Citrus juices are mostly comprised of water and thus

will experience compression of about 15% at 600 MPa,

increasing density and viscosity and therefore decreasing

coefficients of diffusion. This directly affects reaction

rates, as oftentimes with enzymes the diffusion of the two











reactants is the rate limiting step. According to the ideal

gas law,

PV=nRT EQ. 1

where P is pressure, V is volume, n represents the number of

moles, R is the ideal gas constant and T is temperature

pressurization without a corresponding decrease in volume

will result in an increase in the temperature of the sample.

This temperature increase is not adiabatic, since there can

be heat exchanged with the vessel wall. When pressure is

released the sample loses the heat it gained through

pressurization. A sample will end up close to its starting

temperature after pressurization. Morild (1992) described

the temperature change due to pressure changes as 1.86 x 10-

3 K bar-'. After adjusting the equation to the heat capacity

of our pressure medium and converting to MPa, the conversion

factor becomes 4.8 x 10-2 K MPa-1. At the highest pressure

used in this study, 900 MPa, the maximum theoretical

temperature increase is 43.2 K (43.2 0C). This absence of

temperature abuse in treated samples is the strongest case

for using high pressure to pasteurize food.











Contributing Factors

Weak acids and bases dissociate under high pressure and

pH changes can be expected in unbuffered samples (Lddemann,

1992). Changes in pH can greatly affect the microorganisms

and enzymes present in a food system and this must be

considered when attributing any type of inactivation to high

pressure alone. Greater inactivation of pectinesterase

occurs at lower pH by pressure treatment (Ogawa et al.,

1990) and heat treatment (Rouse and Atkins, 1952).

Process temperature is a major contributor to the

efficacy of high pressure in inactivating enzymes and

microorganisms. Temperatures above 50 *C or between -30 and

+5 *C during processing enhance inactivation (Cheftel,

1995). High sugar or salt content as well as low water

activity all have protective effects on pressure targets and

it has been observed that the inactivation effect of

pressure on microorganisms and pectinesterase is decreased

by increasing juice concentration (Ogawa et al., 1990),

illustrating the importance of understanding how pressure

works in real food systems.

Finally, the method of pressurization should be

considered. Some researchers claim that repeated pressure

cycling has a more devastating effect on microorganisms and

enzymes than the same total time in just one cycle. Honma

and Haga, (1991) observed a greater reduction in









9

microorganism contaminants in egg white using cycling. Curl

and Jansen, (1950 a&b) showed higher inactivation of

trypsin, chymotrypsin and pepsin using a multi-cycle

process. However, pectinesterase inactivation has not been

shown to benefit from repeated pressure cycles (Irwe and

Olsen, 1994). A cycle is defined by having only one

pressurization and depressurization step. Commercially, a

large benefit would be necessary to offset the increased

wear on the pump and the pressure vessel, since increasing

or decreasing pressure rather than holding at high pressure

that wears out the pump and fatigues the vessel.



Enzyme Inactivation



Although the mechanism of enzyme inactivation is still

unclear, the most accepted theory at present is that

covalent bonds are not disrupted by high pressure, while

electrostatic and hydrophobic interactions are. Thus, the

primary structure of the enzyme remains unchanged, but the

secondary and tertiary structure, and hence the active site

on the enzyme, is susceptible to disorder. Even small

changes in the active site can result in loss of enzyme

activity. Four kinds of enzyme inactivation have been

detailed (Miyagawa et al.1964): 1) completely and

irreversibly inactivated, 2) completely and reversibly











inactivated, 3) incompletely and irreversibly inactivated

and 4) incompletely and reversibly inactivated. The

decrease in volume caused by enzyme denaturation is the

result of both the rearrangement of conformational volume

and of solvent molecules. If the disorder caused by high

pressure is favorable enough, the enzyme protein will be

permanently denatured and disabled, while in other cases the

damage is reversible. For instance, no evidence of recovery

of pectinesterase after pressurization of 400 MPa for 10

minutes was found(Ogawa et al., 1992). Enzymes stabilized

by high pressure can be expected to have increased activity

due to pressurization. As shown by Asaka and Hayashi, (1991)

enzymatic browning due to polyphenoloxidase seems to be

enhanced. Every enzyme will therefore have a characteristic

response to high pressure treatment, and even the same

enzyme derived from different sources can react to differing

degrees. Peroxidase was shown to have non-similar

inactivation profiles in orange juice and strawberry puree

(Cano et al., 1997). Grape, strawberry, apricot and apple

polyphenoloxidases are more pressure stable than the

polyphenoloxidase found in mushroom and potato (Hendrickx et

al., 1998). Pectinesterase, lipase, polyphenoloxidase,

lipoxygenase, peroxidase, lactoperoxidase, phosphatase and

catalase were investigated and showed a different

inactivation coefficient for each enzyme treated under the











same pressure conditions and in the same buffer solution

(Seyderhelm et al., 1997).



Pectinesterase



Pectinesterase (PE) is the enzyme in citrus juice

responsible for cloud loss, a major quality defect in the

final product. PE is found in all plant tissues and is

tightly associated with the cell wall membrane. Intact

citrus or other fruit is not rapidly degraded or softened by

PE because of this. However, upon processing the cell wall

matrix is disrupted, freeing the enzyme and allowing it to

come in free contact with its substrate, pectin. Cloud loss

occurs when the soluble pectin is deesterified and

precipitated through complex formation with calcium ions.

Figure 1 illustrates the pertinent reactions of pectin and

pectinesterase which lead to cloud loss. At present, PE is

thought to have two major forms, termed heat labile and heat

stable. These two forms, or isozymes, of pectinesterase have

different tolerances to heat treatment before inactivation

occurs. Versteeg et al. (1980) defined the levels which

could inactivate these two different forms as 70 C and 90

C. Seymour et al. (1991) purified and characterized two

forms of PE and reported a higher molecular weight and

higher percent hydrophobic amino acid content for the heat



























Figure 1. Reactions of Pectin and Pectinesterase



















OH


Pectinesterase
H20


Pectic Acid


CH3OH

Methanol


CH3


Pectin











stable isozyme of pectinesterase. The heat stable portion

is reported to represent about 10% or less of total PE

activity in oranges (Versteeg et al., 1978).



Quality Effects



As mentioned before, pressurization of food involves

some degree of heating of the sample. However, the

temperature increase due to pressurization is minimal

compared to traditional temperatures necessary to accomplish

microbial and enzyme inactivation by heat alone. Thus, the

quality of food products is more likely retained to a

greater extent. High temperature, traditional thermal

processing deteriorates color, flavor, nutrients and texture

of foods. It is well documented that orange juice subjected

to thermal pasteurization has a marked decrease in a

perception of "fresh" flavor after thermal pasteurization.

(Moshonas and Shaw, 1997; Nisperos-Carriedo and Shaw, 1990)

For example, Yen and Lin (1996) found that pressurized guava

puree retained its color, pectin, cloud and ascorbic acid

content while successfully reducing microbial count to less

than 10 colony forming units/mL. The microbial counts in

the pressurized juice remained at this level during the

storage trial of 60 days. Thermal pasteurization of the

same puree sample showed a marked deterioration in the











measured quality aspects but was more successful at

inactivating the enzymes present. Pasteurized samples were

not as microbially stable as the samples pressurized at 600

MPa.

Kimura et al., 1994 determined that strawberry jam

prepared by high pressure processing retained a higher

quality regarding volatile flavors and natural color of the

fresh fruit when compared to heat processed jam. Flavor

components important to good strawberry flavor were

determined to be in greater abundance in the pressurized

sample immediately after treatment including: trans-2-

hexenol (80 times) linalool (5 times), ethyl butyrate (6

times) 2-methylbutyric acid (7 times). In addition, a new

"sweet" aroma was created in the heated jam. Browning occurs

in heated jam during processing, but pressure treatment does

not cause browning, and vitamin C was not lost due to

pressure treatment as it was in the heated jam. The storage

study accompanying this work showed the pressurized jam

deteriorated more rapidly at room temperature. This was due

to a higher dissolved oxygen content and the presence of

active enzymes in the pressurized jam. Oxygen concentration

decreased during the storage study, indicating that it was

participating in various chemical and enzymatic reactions

which contribute to deterioration. Also, the packaging

material amenable to the two different types of processing











can cause some differences in storage stability regarding

the oxygen content. Glass was used for the heated jam, and

is a good gas barrier. Pressurization must take place in a

flexible container and thus composite films are employed.

Although these composite films were designated as gas

barriers, they are still more permeable than glass to oxygen

transfer. Pressures used in this study were 400-500 MPa,

which is capable of inactivating microorganisms, but not

particularly efficient at incapacitating enzymes. Higher

pressures are necessary to achieve inactivation of enzymes

consistent with a more shelf stable product.

Texture can be greatly affected by high pressure,

especially in protein containing systems. Juices and food

fragments do not show any documented textural changes, but

whole fruits or large fruit pieces seem to suffer softening

after pressure treatment. This is most likely due to

cellular disruption and compression of internal gas vacuoles

which lead to release of cell wall bound enzymes (Cheftel,

1992; Asaka and Hayashi, 1991). Protein solutions have been

shown to gel when pressurized, and gels formed under

pressure tend to have more tensile strength and a higher

melting temperature (Gekko and Fukamizu, 1991). Conversely,

muscle foods undergo tenderization during pressure

treatment. Again, the cause has been attributed to release

of intracellular enzymes with proteolytic activity (Ohmori











et al., 1991). It is left to the user to decide if the

potentially texture altering effects of high pressure

treatment are detrimental or beneficial in individual cases.

Clearly, not all foods are ideally suited to this

technology.



Separation Procedures





Isoelectric focusing (IEF) is a separation technique

that uses a pH gradient superimposed along an electric field

to separate components of a mixture based on their

isoelectric points. An isoelectric point, or pI, is the pH

at which the net charge on a molecule is equal to zero. A

charged molecule, such as a protein, migrates toward the

pole of opposite charge along the electrical gradient until

it comes to the point in the pH gradient where its net

charge will be zero. It comes to rest here, at its

isoelectric point, which is nearly unique to every protein.

If the molecule drifts from this zone, due possibly to

diffusion, it becomes charged again and is simply pulled

back to its pI by the electric field.

A pH gradient is established by a mixture of ampholytes

with a pH range surrounding the pI of the compounds of

interest. Each ampholyte has a different pI and migrates in











an applied electric field until it reaches the point where

it is uncharged. Ampholytes can be mixed with the sample to

be separated before applying the electric field. After

migration has stopped, usually three to five hours, the

mixture of proteins is separated with phenomenal

sensitivity, sometimes within 0.003 pH units using

immobilized gradients (Righetti et al., 1989). IEF is not

just a separation technique, it also has the purpose of

focusing the proteins into very narrow zones. This is an

especially powerful technique when combined with gel

electrophoresis to form a two dimensional separation.

Figure 2 represents the basic concept behind

isoelectric focusing. The positively charged molecule

migrates toward the cathode, while the negatively charged

molecule is attracted to the anode. The pH gradient is set

up with low pH at the anode and high pH at the cathode. As

a molecule, for example, a positively charged protein,

migrates toward the negatively charged pole, it encounters

an increasingly more basic environment which mitigates the

positive charge until it is zero. The protein will then

stop migrating at this, its isoelectric point.

Resolution of focused proteins is defined as the difference

in pI or pH between clearly distinguishable bands, or

fractions. Change in pI is proportional to the square root

of the pH gradient, and inversely proportional to the square



























Figure 2. Schematic Diagram of Iosoelectric Focusing System (Adapted from Giddings,
1991)














e- 0.--4


I I-


Low pH


High pH


Isoelectric
point









21

root of the field strength. To obtain optimal resolution,

one would use the smallest range of ampholyte pH and the

highest field strength. This must be balanced by the pI

range of proteins being separated and the heating that

results from high field strength. Heating will ultimately

decrease resolution due to diffusion so field strength and

run time must be optimized to minimize the deleterious

effects of heating while providing a good separation.
















MATERIALS AND METHODS


Pectinesterase Inactivation

Juice Preparation


Samples of orange and grapefruit juice were extracted

in the pilot plant of the Citrus Research and Education

Center in Lake Alfred, Florida using an FMC commercial

extractor (FMC, Lakeland, FL). The juice was not subjected

to a finishing step. Juice not immediately used for PE

inactivation studies was stored frozen at -23 'C and thawed

before use. Additional fresh frozen finisher pulp from

previous juice runs was added after thawing on a weight

basis at 10.7% for orange and 8.7% for grapefruit, for added

PE activity. The juices were then homogenized with a

blender for two minutes to insure small, relatively uniform

particle size and distribution. The resulting pulpy juice

was stirred before packaging samples (30 mL) into sterile

polyethylene bags (Fisher Scientific, Pittsburgh, PA) and

impulse sealed, retaining as little headspace as possible.

Samples were double bagged before being placed in the

pressure vessel.











Pressurization

Juice for enzyme analysis was pressurized using a

Stansted isostatic high pressure unit (Stansted Fluid Power,

Stansted, England) at 600, 700, 800, or 900 MPa for 1, 15

or 30 second dwell time. Runs at 500 MPa were 15 seconds and

1, 5, 15 and 60 minutes. Dwell time is defined as the time

spent at the set point pressure.

The packaged 30 mL samples were kept in an ice bath

until they were pressurized. The pressure unit was at 5-10

0C before pressurization began. A mixture of ethanol and

castor oil (85/15 v/v) constituted the pressure medium.

Time to reach the desired pressure was 10-12 seconds while

decompression time was approximately 10 seconds. Figure 3

represents a typical pressure profile obtained by the

isostatic high pressure unit with a 30 second hold time at

700 MPa. As can be seen from this graphical representation,

there were no abnormal spikes or drops in the pressure

level. The use of a chiller to cool the pressure vessel

jacket and the pressure medium ensured that samples remained

in the temperature range of 20-50 0C during processing. All

runs were done in duplicate. After pressurization, samples

were kept at 00C until PE activity could be determined.

Figure 4 is a schematic diagram of the Stansted high

pressure vessel. The Stansted unit represents the method of

direct pressure generation. In direct, or piston type


























WpON uoTssaadwom/uo~ssaaduwoo PTdpj UT
qTufl aanfsseJd Q{TH O~qqs~I paqsueqg /q Pau~pqqo aTT;OaJ anssaad TPOA, -C aab











700


600


500


400
a)

300
U)
U)
G) 200


100


0
0 5 10 15 20 25 30 35
Time (sea)



























Figure 4. Stansted "Plunger Press Food Lab 9000" High Pressure Food Processing Unit








Seal Plug

HP Fluid

Product

Mantle

Gland Seal
HP Plunger


Low Pressure
Drive Piston


Hydraulic Oil


~il


Breech Nut


Precharge Valve


Super Grade Stainless
Steel Liner


NICr HP Barrel



-------- Support Block





Base Plug











Figure 4 is a schematic diagram of the Stansted high

pressure vessel. The Stansted unit represents the method of

direct pressure generation. In direct, or piston type

compression the pressure medium is pressurized directly by

the piston, which is driven by a low pressure pump. The

hydraulic principle states that the pressure at the large

end of the piston is multiplied by the ratio of the two

piston sections. This yields the desired high pressure at

the small end of the piston. Direct pressure generation is

necessary to obtain rapid compression, but is often limited

to small diameter pressure vessels because of the

limitations of the gland seal (shown in Figure 4).

PE Activity Determination


PE was assayed using the titration method of Rouse and

Atkins (1955) using 100 mL of a 1% pectin solution in 1M

NaCl. This method is derived from the validated method of

Lineweaver and Ballou (1945), and later validated by Seymour

et al. (1991) and Warrilow and Jones (1995) The

juice/pectin mixture was brought to a pH of 7.5 and titrated

with NaOH for at least 10 minutes or 5 mL. Activity is

calculated from the equation

mL titrant x N titrant x 10'/ time x weight of sample EQ. 2

and results were reported as equivalents of enzyme

hydrolyzed per minute per gram sample, or pectinesterase











units x 10' min-' g' juice (PEu x 10') Pectin from citrus

fruits was obtained from Sigma (St. Louis, MO) and had an 8%

methoxy content. Pectin solutions were kept at a constant

temperature of 28 0C. All samples were titrated in

duplicate. Average %RSD of titrations was 8.0 for orange

juice and 7.4 for grapefruit.





Enzyme Isolation Study



PE for the detailed inactivation study was obtained

from late season Valencia juice extracted with an FMC

commercial extractor. Four groups of juice were considered:

untreated control, pressurized at 800 MPa for one minute,

heated to 70 0C for 1 minute, and heated to 90 'C for one

minute. These heating levels were chosen to represent a

light pasteurization and a commercial level of

pasteurization. After treatment, the juice was centrifuged

at 10,000 g for 30 minutes to obtain the pulp used for

extraction of the enzyme. Pulp spun out from juice was not

washed prior to extraction to prevent resuspension.

Enzyme Extraction


PE was extracted from fresh Valencia pulp via the

following method:











1. Weighed sample of pulp was washed with two volumes of

deionized water to remove soluble solids.

2. Sample homogenized in blender with 1 L 0.25 M Tris-Cl + 1

M NaCl at pH 8.0 and then stirred at room temperature for 1

hour.

3. Centrifuge at 10,000 x g for 30 minutes, retain

supernatant and filter through miracloth (Calbiochem, La

Jolla, CA)

4. Slowly add ammonium sulfate to 80% saturation, stirring

the whole time. System was kept on ice to retain PE

activity. Saturation point was calculated by mL supernatant

x 0.7g/mL for 100% saturation. Solution was refrigerated at

4 'C for 12-48 hours to allow protein to precipitate.

5. Solution was centrifuged at 10,000 x g for 30 min under

refrigeration temperatures (0'- 4 'C). Pellet was retained

and resuspended in a minimum volume of 10 mM sodium

phosphate buffer, pH 7.0 adjusted with solid sodium

hydroxide.

6. Solubilized pellet was centrifuged for 10 minutes at

10,000 x g to remove insoluble solids. Supernatant was

retained, as it contains the desired PE activity.

7. Supernatantwas dialyzed in Spectra-por dialysis membranes

against 10mM sodium phosphate buffer with pH 7 from 18 24

hours with three buffer changes. System was stirred and kept

in a cold room on ice.











The dialyzed supernatant then underwent isoelectric

focusing using a Rotofor (Bio-Rad) to separate it into

fractions along a pH gradient using ampholytes in the range

of pH 8-10.5. Since heat labile PE has an isoelectric point

of 9.5 and heat stable PE is suspected to be higher, all

fractions with a pH between 9.0 and 11.5 were assayed for PE

activity after separation. Fractions were then further

separated using SDS-PAGE followed by silver staining.

Sample Preparation


Fractions from isoelectric focusing were dialyzed to

remove ampholytes before sample prep for SDS-PAGE. A 7.5 mL

volume of sample was combined with 2.5 mL sample buffer.

Sample buffer consists of Tris-Cl buffer pH 6.8 with 2% SDS,

10% glycerol and 0.025% Bromphenol Blue. The glycerol

provides the density necessary to make the samples sink to

the bottom of the wells in the stacking gel. The Bromphenol

Blue is a tracking dye to monitor sample progress through

the gel during the run.



Cloud Loss



Cloud loss of treated samples was monitored as an

indicator of residual PE activity after treatment. Fresh

squeezed, mid-season Valencia orange juice from the FMC









32

extractor in the CREC pilot plant was subjected to pressures

from 400 to 900 MPa for 1 second, 1 minute or 10 minutes.

The juice was not subjected to a finishing step, and no

additional pulp was added. The juice was strained through a

U.S. standard #20 mesh screen and then homogenized in the

blender on low for 30 seconds. The method for determining

cloud loss described by Cameron et al. (1997) involves

centrifuging a 50 mL juice sample at 15,000 x g for ten

minutes and then measuring the absorbance of the supernatant

at 660 nm. Bottles were inverted five times to facilitate

mixing before samples (50 mL) were periodically drawn for

analysis. Juice bottles were stored at 4 'C between

analysis times.

Samples were monitored for microbial growth or

contamination by spread plating 0.1 mL juice sample on

duplicate orange serum agar (OSA)plates. Dehydrated OSA

(Difco)was mixed with water and plates were poured and left

24 hours to set. The plates were incubated at 30 'C for 48

hours before counting colony forming units.



Storage Study of Valencia Oranae Juice



Juice was extracted from sound, washed, refrigerated

Valencia oranges using an FMC model 291 extractor in the

CREC pilot plant. It was finished using an FMC model 35









33

finisher with a 20 mesh screen. A portion of the juice was

immediately canned and frozen for use as the study control.

Another portion was packaged in 250 mL portions in a low

permeability bag and sealed in a polyethylene bag before

being pressurized at 500 MPa for 2 minutes. Samples were

stored in an ice bath both prior to and post pressurization

until all samples were treated. A third portion of the

juice was pasteurized at 95 'C with a 15 second hold time

using a Microthermics UHT/HTST Lab, Model 25 (Raleigh, NC)

and heat sealed into the above-mentioned bags.

Bags stored at 1.5 0C were monitored for volatile

composition at approximately 4 week intervals for a total of

36 weeks. Volatiles were monitored using a Hewlett Packard

gas chromatograph, model 5980 series II with flame

ionization detector. Sample introduction was accomplished

through an 01 Analytical (College Station, TX) model 4560

purge-and-trap sample concentrator. An undiluted juice

sample (1 mL) was purged for 2 minutes and analyzed on a DB-

5 column under the following GC conditions: injector: 225

0C, detector: 250 'C, initial time: 5 minutes, ramp rate

5*/minute from 45 0to 110 0C Sabinene, myrcene, z-3-

hexenol, a-pinene, octanal and d-limonene were monitored

for the three different treatments over the course of the

study. Tentative identification of peaks was determined by











Kovats indices and comparison to known standards. These

compounds were chosen because of their contribution to

"fresh orange flavor" (Redd et al, 1996).



Statistics

All statistics presented in this study were computed

using Statistica (Statsoft, Tulsa, OK). Principal component

analysis and discriminant analysis were performed and all

results are reported at the 95% confidence level.

Samples were monitored at approximately 4 week

intervals, and duplicates of each sample were run in random

order. The values presented represent an average of the

peak areas of each separate compound at each sampling time.
















RESULTS AND DISCUSSION


Enzyme Inactivation



Fresh Valencia juice has PE activity in the range of 2-

6 PEu x 103 (Snir et al., 1996). Blending fresh frozen pulp

into the sample juices increased juice PE activity to 10-12

PEu x 101, the point that at least a log cycle reduction

resulting from pressure treatment could be measured by the

traditional wet chemical assay. Juice pulp was chosen over

prepared enzyme to approximate the natural food system,

since Pollard and Kieser (1951) found that enzyme

inactivation in a raw juice was distinctly different than

the results of a purified enzyme preparation. Also,

commercial citrus PE is prepared from citrus peel and may

not have the same ratio of isozymes found in the internal

parts of the fruit. Since the activity in juice is due

mainly to the pulp it was important to use a pectinesterase

source that would most closely mirror the typical food

system.














Figures 5 and 6 show remaining PE activity versus the

dwell time at four different pressures. Inactivation of PE

with higher isostatic pressure was bi-phasic, in accordance

with the different forms of the enzyme. Prior to this

study, two separate slopes of inactivation have been

reported for thermal inactivation (Versteeg et al., 1980;

Wicker and Temelli, 1988). The first drop in activity after

pressurization has been described as an "instantaneous

pressure kill" by Basak and Ramaswamy (1996). These

researchers investigated pressure effects on PE in the range

of 100 to 400 MPa, and observed a much less pronounced

initial drop than is seen in Figures 5 and 6 which

illustrate higher pressures and shorter compression time.

The time to reach the set point (come-up time) was longer on

their pressure equipment, taking up to 3 minutes. They

pointed out the come-up time at the lower pressures should

not have much effect. Dwell times in their study were as

long as 720 minutes. Since a hold time of this duration is

commercially impractical, the need for information at higher

pressures is clearly indicated.

Seyderhelm et al. (1996) reported the effect of higher

pressures on PE, but the data given was for commercial PE in

pH 7 tris buffer at 45 'C. The shortest processing time





























Figure 5. Inactivation of Orange Pectinesterase at Pressures of 600-900 MPa.
Data Points Represent the Average of Two Measurements











6 600 > 700
5


x
O3

w
a. 2


0

0 5 10 15
Dwell Time (sec)


-800 -z 900


20


25


30





























Figure 6. Inactivation of Grapefruit Pectinesterase at Pressures of 600-900 MPa.
Data Points Represent the Average of Two Measurements











10 -E 600 -0- 700 800 YI 900
8



a-
2
0
0 5 10 15 20 25 30
Dwell Time (sec)

CD









41

shown, 2 minutes, was sufficient to completely inactivate PE

at 900 MPa. An approximately 45 'C increase in temperature

can be expected at 900 MPa (Morild, 1992), so it is possible

that the complete inactivation was augmented by heat. At

600, 700 and 800 MPa, less inactivation of PE was

experienced in buffer (Seyderhelm et al. 1996) compared to

the 15 second results in orange juice (Figure 5). Although

data on enzymes isolated from their native environment are

important, this discrepancy stresses the need for empirical

data using the natural enzyme in the appropriate biochemical

model to assess applicability to real food systems.

It was suspected that the initial drop in activity was

due to inactivation of heat labile PE, while the remaining

activity illustrated the effect of pressure on the heat

stable PE (Figs 5 & 6) (Irwe and Olsson, 1994). The heat

labile PE comprises from 86 94% of the total enzyme in

Valencia juice (Snir et al., 1996), and at higher pressures

the rapid inactivation is very close to this percentage.

Sun and Wicker (1996) confirmed that exposing juice to

pH extremes (pH 2 for 5 minutes) also can inactivate the

heat labile form, but this treatment was ineffective against

the heat stable form. Subjecting the orange and grapefruit

juice to a pH of 2 for five minutes caused 91% inactivation

of total PE activity in orange juice. The residual PE

activity following higher pressure treatments of juice was











similar to the activities reported after low pH treatment,

which suggests that the remaining activity represented heat

stable PE.

Table 1 is a summary of the inactivation percentages

for orange and grapefruit juice at varying pressures and

acid treatments. Subsequent heating of a pressurized (1

minute at 700 MPa) orange juice sample for 2 minutes at 70

'C did not reduce PE activity, while heating for 2 minutes

at 90 0C resulted in a marked decline in PE activity, from

0.2 to 0.1 PEu x 10', substantiating that only the heat

stable form remained after pressurization. These

temperatures were chosen because they represent two levels

of heating that can distinguish the two isozymes (Versteeg

et al. 1980).

Heat Generation by Pressure

The question of whether or not the heat generated by

pressurization was sufficient to inactivate PE was

considered. Samples were placed in the unit at 5- 10 C and

reached temperatures between 20 0and 50 C (measured by a

thermocouple) depending on set point pressure. Immediate

cooling occurred upon decompression. Morild (1992) described

the temperature change due to pressure changes as

aTV/Cp = 1.86 x 10-3 K bar-3. The pressure medium used in

this study was 15% castor oil, which has a heat capacity































Table 1 Percent Inactivation of Pectinesterase in Orange
and Grapefruit Juice at Different Pressures

Treatment (1 Orange Grapefruit

second)

600 MPa 10 50

700 MPa 61 82

800 MPa 82 87

900 MPa 93 85











(CP) of 2.1 J/g 0C and 85% ethanol. The heat capacity of

pure ethanol is 1.4 J/g 0C. Ethanol was 5% water,, so the

heat capacity of the ethanol component was 1.5 J/g C. A

combination of these values at the appropriate ratio results

in a heat capacity of the pressure medium equal to 1.6 J/g
'C. Substituting this value of Cp for that of water in the

equation results in a conversion of 4.8 x 10-3 K/bar. One

bar is equal to 0.1 MPa, so after adjusting the equation to

the heat capacity of the pressure medium and converting to

MPa, the conversion factor becomes 4.8 x 10-2 K MPa-'. At

the highest pressure used in this study, 900 MPa, the

maximum theoretical temperature increase is 43.2 0C. Again,

the heating will not be adiabatic because of heat exchange

with the chilled vessel wall. This confirms that

temperatures generated by pressures used in this study were

not sufficient to thermally inactivate PE.

Figure 7 shows pressure treatment at 500 MPa,

illustrating the difference in curve shape between lower

(<600 MPa) and higher (600-900 MPa) pressures. At <600 MPa,

it is possible to observe the first order inactivation of

heat labile PE. A plot of the log of the data from 500 MPa

(Figure 8) gives a regression equation of

y = (-1.9 x 104)x + 1.08 EQ. 3

The slope can be used to determine the decimal reduction

value, DP and the inactivation coefficient, k. DP and k





























Figure 7. High Pressure Inactivation of Pectinesterase at 500 MPa. Data Points
Represent the Average of Two Measurements


















2000


3000


4000


Dwell Time (seconds)


14
12


08
x6
w


1000





























Figure 8. Log of PE Inactivation at 500 MPa










1.1
y=(-1.9 x 10A4)x +1.08
<0.9
0
"" 0.8
0.7

0.4

0.3
0 1000 2000 3000 4000
Dwell Time (sec)











values will be discussed later. At 700 MPa and above,

pressure application inactivates this fraction more rapidly

than the 1 second minimum dwell limitation of the equipment,

leaving the heat stable form active (see Figures 5 and 6).

The time required to reach the set point pressure was

approximately 10 seconds, so the enzyme spent some time at

the lower pressures before starting the dwell time counter,

contributing to the inactivation of the enzyme. Higher

pressures inactivate the heat labile form too quickly to

measure this decline.

Longer processing times at >600 MPa did not inactivate

the remaining heat stable form. This result, coupled with

the observation that pectinesterase does not recover from

high pressure treatment (Ogawa et al., 1992) puts this

enzyme in the group that is incompletely and irreversibly

inactivated (Miyagawa et al., 1964). The existence of a

maximum pressure above which no extra inactivation is

apparent has also been observed in trypsin, chymotrypsin and

chymotrypsinogen (Curl and Jansen, 1950a &b). Samples held

at 700 and 800 MPa for as long as 1 minute had little

decrease in activity over samples held for a 15 second dwell

time. These results showed that dwell times of 15 seconds or

less were sufficient to reduce PE activity in orange juice,

with inactivation increasing significantly with increasing

pressures in both juices.









50

Analysis of variance (ANOVA) designated the probability

of difference between PE inactivation and pressure levels as

100% for both juices. Varying dwell times at the higher

pressures did not cause significantly different PE

inactivation in grapefruit juice (80% probability of

difference, p=.193) but caused significant differences in

orange juice PE at a probability level of 100% (p=0.000).

Decimal Reduction Values


The time necessary to reduce activity one log cycle, or

90% at a given pressure, is defined as the DP value. The DP

value of PE in orange juice for 600 MPa was 143 seconds (2.4

min), while the Dp value for 500 MPa was 5000 seconds (83.3

minutes). The slope of the regression line of 500 MPa

adjusted by 2.303 for the natural log yields a k of 4.4 x

10-1 s-1, while the k value at 600 MPa is 7.5 x 10-3 s-'. Not

surprisingly, the enzyme is more rapidly inactivated at 600

MPa than at 500 MPa. This represents inactivation of the

heat (and pressure) labile forms of PE only. Since the

enzyme has been shown to be biphasic, reporting one decimal

reduction value for both forms is inaccurate. No

inactivation of the heat stable form was noted, thus no

inactivation kinetics are reported.

The time or pressure necessary to cause a 90% reduction

in the D value is termed the z value. The zp value









51

corresponding to the 500-600 MPa range was 65 MPa. Basak and

Ramaswamy (1996) report a D. value of 260 minutes at pH 3.7

and 14 minutes at pH 3.2 at 400 MPa. The juice used in this

study was in the middle of this pH range at 3.45. For

comparison, the temperature necessary to accomplish 90% PE

inactivation in orange juice in less than a minute was

reported as 850C by Rouse and Atkins (1952).

Enzyme Sensitivity


Comparison of Figures 5 and 6 shows that grapefruit PE

was initially more sensitive to pressure treatment than

orange. The same observation was made for the sensitivity of

grapefruit PE to thermal inactivation (Rouse and Atkins,

1952). Due to the high percentage of grapefruit PE rapidly

inactivated at 600 MPa, no Dp value was calculable from the

pressure data of this study. Table 1 shows that grapefruit

PE inactivation is not greater than 85% even at the

highest pressures used. Comparing this to the values

presented for orange, one may initially form a contrary

conclusion about the sensitivity of grapefruit PE, but it is

hypothesized that since grapefruit PE has a percentage of

heat stable enzyme as high as 33% (Rombouts et al., 1982),

it will not experience as much total inactivation as orange

PE by high pressure.











It is proposed that the heat labile form of PE in

grapefruit was more sensitive to pressure treatment. To

substantiate the assertion that only the heat stable form

remained after high pressure treatment, pressurized

grapefruit juice samples (700 MPa for 1 minute) were heated

to either 70 CC or 90 C and then assayed for PE activity

after the sample was cooled to 4 'C. The 70 C treated

sample showed no change in activity, while the 90 'C sample

decreased in PE activity from 1.5 PEu to 0, showing complete

inactivation of both forms.



A major contributor to this sensitivity is the fact

that grapefruit juice is of a lower pH than orange,

sometimes as much as 0.5 pH units. It has already been

mentioned that enzyme inactivation takes place more readily

in a lower pH solution when either heat or high pressure is

the method of inactivation. This fact is thoroughly

established in citrus juices by the work of Rouse and Atkins

(1952, 1953). Their results showed that PE was inactivated

at lower temperatures if the starting juice was at a lower

pH.











Enzyme Isolation



Enzyme isolation and separation was performed to

compare the effects of heat and pressure treatment on PE

isolated from pulp. Figures 9-12 show the results of the

SDS-PAGE separation of the IEF fractions of PE isolated from

fresh and treated Valencia orange juice. Figure 9

represents the control, which has a tight band at 36,000

Dalton, typical of PE (Seymour et al.,1991). Figure 12

represents the juice treated at 90 'C, approximating

commercial pasteurization. The absence of a protein band at

36,000 D in the gel in Figure 12 is an indication that the

pectinesterase enzyme protein has been denatured and

precipitated before analysis by electrophoresis. This is

confirmed by the absence of any measurable PE activity in

the 90 'C heated sample.

The figures (9 & 10) depicting the sample pressurized

at 800 MPa for 1 minute and the juice heated to 75 0C show

very similar results by gel electrophoresis. There is an

ill resolved band around 36,000 molecular weight indicating

that although pectinesterase is present, it is different

from the control. The possibility of sample overloading was

considered but ruled out, as protein concentration was

similar in all samples. Both the pressurized and the 75 OC

samples show a decrease in total activity over the control.




























Figure 9. SDS-PAGE Separation of Pectinesterase from Untreated Valencia Juice
Lane 1= Mid Range Ladder, 2=pH 9.1, 3=pH 9.35, 4=pH 9.62, 5=pH 9.95, 6=pH 10.81, 7=pH
11.59, 8-10= raw extract





















36,000MW -0


1 2 3 4 5 6 7 8


9 10




























Figure 10. SDS-PAGE Separation of Pectinesterase from Valencia Juice Pressurized at
800 MPa for 1 Minute. Lane 1=pH 9.16, 2=Mid Range Ladder, 3=pH 9.3, 4=9.41, 5=pH
9.55, 6=pH 9.72, 7=pH 10.01, 8=pH 10.32 9=pH 11.06, 10=pH 11.96
























36,000 MW -0


1 2 4 5 6 7 8 9 10




























Figure 11. SDS-PAGE Separation of Pectinesterase from Valencia Juice Pasteurized at
75 'C for 1 Minute. Lane 1=Mid Range Ladder 2=pH 9.15, 3=pH 9.37, 4=pH 9.52, 5=pH
9.67, 6=pH 9.89, 7=pH 10.89, 8=pH 10.33 9=pH 11.15 10=pH 12.52













..... ., .,. , qk.' Mr r r ,


36,000MW


2 3 4 5 6 7 8


9 10




























Figure 12. SDS-PAGE Separation of Pectinesterase from Valencia Juice Pasteurized at
90 0C for 1 Minute. Lane 1=pH 9.07, 2=pH 9.30 3=pH 9.52, 4=pH 9.50, 5=pH 9.84, 6=pH
11.09, 7=pH 11.71, 8=Mid Range Ladder, 9-10= Raw Extract



























36,000MW 0.


1 2 3 4 5 6 7 8


9 10









62

Tables 2-4 list activities of the control and the differing

treatments as well as the pH of the fractions from

isoelectric focusing. It is important to compare fractions

with similar pH, because the protein will be found in the pH

range that brackets its pI. For PE this is 9.5, and

consideration of Tables 2-4 illustrates that these pH values

are indeed those with the highest specific activity. As

expected, the control had the highest pectinesterase

activity.

The 75 'C and pressure treatments were expected to

inactivate approximately 90% of the total PE activity as was

seen with extracted pressurized pulp, but this was not the

case. Since the specific activity is reported per mg of

total protein in the IEF fraction it is possible that some

other contaminating protein is present in the control, which

would make the reported specific activity lower.

Several theories exist regarding the existence of heat

labile and heat stable forms of PE. Some believe the heat

stable form to be at a higher molecular weight than the

36,OOOD of the heat labile form. Other evidence points to

the possibility that both forms have the same molecular

weight, but differ in their ability to tolerate heat. The

heat stable form of PE has a higher percentage of

hydrophobic amino acids than the heat labile fraction

(Seymour et al., 1991), which could account for its greater




























Table 2. Specific Activity of Isoelectric Focussed Fractions
of Pectinesterase Isolated from Untreated Late Season
Valencia Orange Juice

Fraction Specific Activity pH
(PEu/mg protein)

15 0.024 9.1

16 0.068 9.35

17 0.071 9.62

18 0.036 9.95

19 0.008 10.81




























Table 3. Specific Activity of Isoelectric Focussed Fractions
of Pectinesterase Isolated from Late Season Valencia Orange
Juice Treated at 75 'C for 1 Minute

Fraction Specific Activity pH
(PEu/mg)

13 0.059 9.52

14 0.047 9.67

15 0.045 9.89

16 0.033 10.89

18 0.038 11.15



























Table 4. Specific Activity of Isoelectric Focussed Fractions
of Pectinesterase Isolated from Late Season Valencia Orange
Juice Treated by High Pressure (800 MPa) for 1 Minute

Fraction Specific Activity pH
(PEu/mg)

14 0.048 9.41

15 0.046 9.55

16 0.039 9.72

17 0.037 10.01

18 0.033 10.32











resistance to heat and pressure denaturation. Higher

hydrophobic amino acid content in an aqueous ionic system

(citrus juice) will result in unfavorable solvent

interactions causing the sphere of hydration of the

denatured enzyme to be greater than the native form. Thus,

denaturation will be more thermodynamically unfavorable in

the heat stable form because of solvent interaction and an

increase in hydration volume. In addition, the increase in

hydration volume will, according to LeChatelier's principle,

be restricted by the application of high pressure.



Cloud Loss



Figures 13-17 illustrate the effectiveness of HPP in

preserving cloud in orange juice. Five different pressure

levels were employed and the cloud loss over time was

compared to cloud loss in an untreated control. Treatment

at 500 MPa (Fig 13) did not yield any significantly greater

stability over the untreated control, although a 10 minute

hold time was able to increase shelf life by two weeks.

Treatment at 600 MPa (Fig 14) had better results, with 10

minute treatment yielding a cloud stable product for the

duration of the 90 day study. As might be expected, shorter

treatment times were less effective, but a 1 minute

processing time afforded much greater stability over the



























Figure 13. Cloud Stability of Orange Juice Treated at 500 MPa (1 sec,E; 1 min,
0; 10 min, A; control,V)






70
60
50
40-/
X03030

20
10
0
0 10 20 30 40
days


50





























Figure 14.


Cloud Stability of Orange Juice Treated at 600 MPa (1 sec,M; 1 min,
0; 10 min, A; control,V)







70
60
50
40
%30-
20
10


0 20 40 60 80 100
days


























Figure 15.


Cloud Stability of Orange Juice Treated at 700 MPa (1 sec,M; 1 min,
0; 10 min, A; control,V)







70
60
50
.40
NR30

20
10


0 20 40 60 80 100
Days





























Figure 16.


Cloud Stability of Orange Juice Treated at 800 MPa (1 sec,M; 1 min,
0; 10 min, A; control,V)







70


60
50
.40

%30-
20
10
0
0 20 40 60 80 100
Days



























Figure 17. Cloud Stability of Orange Juice Treated at 900 MPa (1 sec,M; 1 min,
0; 10 min, A; control,V)







70


60
50
i.40
%30
20
10


0 20 40 60 80 100
Days









77

control and extended cloud stability to 49 days. At 700 MPa,

treatment by high pressure is much more effective, as a 1

minute treatment at 700 MPa stabilized cloud for the full 90

day duration of the study. Samples treated for 10 minutes

were stable until the last week of the study, when they

completely lost all cloud. These samples were found to be

microbially stable and no explanation was discovered. Only

molds are able to clarify juice through the production of

extracellular enzymes such as pectinesterase (Nussinovitch

and Rosen, 1989), so absence of contamination by a yeast or

bacteria would not be unusual, as was the case in this

sample. It is possible that mold growth was present but not

detected by plating. Visible mycelia in the sample was

absent and is not necessary to accomplish cloud loss or

clarification of the sample. Cloud was monitored for 90 days

because this point signifies flavor deterioration in

commercial, packaged, not-from-concentrate refrigerated

orange juice.

Pressures of 800-900 MPa (Figs 16-17) were much more

successful in preserving cloud at shorter processing times.

One second treatment at these higher pressures was effective

at preserving cloud for up to 80 days. Longer processing

times of 1 or 10 minutes maintained a cloud level that was

unchanged from the initial cloud content of the juice at

time zero. Thus, the samples at 800 and 900 MPa at











processing times of 1 minute or above are considered to

suffer no appreciable cloud loss at refrigerated storage (4
0C) over a period of 90 days.

Table 5 summarizes plate counts taken during the cloud

loss study. A dashed line in the table indicates that a

particular sample was not cloud stable at the time of

microbiological sampling, so that particular juice sample

was eliminated from microbial analysis. The two samples in

Table 5 listed as "TNTC" were most likely contaminated post-

processing by Rhodotorula yeast, which has not been shown to

affect cloud in citrus juice. Rhodotorula was identified by

its characteristic pink color and examination under a

microscope to determine cell size was that of yeast and not

bacteria.

Storage Study

Statistical Analysis of Flavor Volatiles


Figures 18 to 23 are the peak areas of the six

monitored compounds used in this study versus the storage

time in weeks. Figure 18 (z-3-hexenol) has little

interpretive merit as there are no obvious trends or

patterns and the treatments are not well separated from each

other. Figure 19 (a-pinene) shows a definite difference

among the three treatments. There are three distinct levels

of x-pinene corresponding to the three treatments. The



























Table 5. Plate counts on OSA at days 49 and 89 of cloud loss
study. Counts are reported using standard plate counting
protocol

Treatment Time 600 MPa 700 MPa 800 MPa 900 MPa

1 second(49) 40 est. TNTC

1 minute(49) 145 est. <10 cfu/mL 10 est. 15 est.

10 minutes(49) 50 est. <10 cfu/mL 75 est. 5 est.

1 second(89) 5 est.

1 minute(89) 15 est. 5 est. 145 est.

10 minutes(89) <10 cfu/mL 5 est. 5 est. TNTC

























Figure 18. z-3-hexenol Composition Over Storage Time
Treatments: c=Control, p=Pressurized, h= Heat Pasteurized












38000


34000 -


30000


26000-


22000 ____


18000


14000


10000


10 15 20


30 35 40


o TREAT: c
E3 TREAT: p
* TREAT: h


WEEK


























Figure 19. a-pinene Composition Over Storage Time
Treatments: c=Control, p=Pressurized, h= Heat Pasteurized












2.4e6 .... .... .... ..- - -


2e6


1.6e6


1.2e6 .


8e5


4e5


0
-5 0 5 10 15 20


25 30 35 40


o TREAT: c
o TREAT: p
o TREAT: h


WEEK




























Figure 20. Sabinene Composition Over Storage Time
Treatments: c=Control, p=Pressurized, h= Heat Pasteurized










1.1 e6 -- -


9e5


7e5


5e5


3e5


le5


-15 0 5
-5 0 5 10 15 20 25 30


35 40


o TREAT: c
1 TREAT: p
* TREAT: h


WEEK


























Figure 21. Myrcene Composition Over Storage Time
Treatments: c=Control, p=Pressurized, h= Heat Pasteurized












4e6


3.4e6


2.8e6 -


2.2e6


1.6e6


le6 _
0 TREAT: c
4e5 13 TREAT: p
-5 0 5 10 15 20 25 30 35 40 0 TREAT:h
WEEK

o


























Figure 22. Octanal Composition Over Storage Time
Treatments: c=Control, p=Pressurized, h= Heat Pasteurized












5.4e5

5e5

4.6e5 A il

4.2e5

: 3.8e5


3.4e5

3e5

2.6e5 TREAT: c
o. TREAT: p
2.2e5O TREAT: p

-5 0 5 10 15 20 25 30 35 40 0 TREAT:h
WEEK

co




























Figure 23. d-Limonene Composition Over Storage Time
Treatments: c=Control, p=Pressurized, h= Heat Pasteurized









3.8e7


3.6e7

3.4e7

3.2e7


3e7

2.8e7

2.6e7

2.4e7

2.2e7
-5 0 5 10 15 20 25 30


35 40


o TREAT: c
O TREAT: p
o TREAT: h


WEEK