Pressure Assisted Thermal Processing: Tomato Carotenoid ...

Pressure Assisted Thermal Processing:

Tomato Carotenoid Stability during Processing and Storage and Feasibility of Using

Chemical Markers for Evaluating Process Uniformity

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of

Philosophy in the Graduate School of The Ohio State University

By

Rockendra Gupta, M.Tech.

Graduate Program in Food Science and Technology

The Ohio State University

2011

Dissertation committee:

V.M. Balasubramaniam, Advisor

Steven J. Schwartz

Sudhir K. Sastry

John Litchfield

Copyright by

Rockendra Gupta

2011

ii

Abstract

Lycopene is a major carotenoid in tomatoes and epidemiological studies suggest

that consumption of food rich in carotenoids lowers the risk of developing certain types

of cancer and chronic diseases. Combined pressure temperature processing has the

potential to reduce the product thermal exposure and helpful in preserving tomato

products with better quality and improved functional properties. However, very little is

known about its fate in tomato products subjected to a range of combined pressure-

temperature (P-T) treatments This study investigated the effect of pressure-thermal

treatments on post processing extractability, isomerization, bioaccessibility and storage

stability of lycopene in tomato juice. In a separate study, the feasibility of using an

intrinsically formed chemical M-2 (4-Hydroxy-5-methyl-3(2H)-furanone) as a potential

marker for understanding combined P-T process non-uniformity was investigated.

Raw and hot break (~93oC, 60 sec) tomato juice from Roma tomatoes was

subjected to combined pressure-temperature (P-T) treatments (high pressure processing

(HPP; 500-700 MPa, 30oC), pressure assisted thermal processing (PATP; 500-700 MPa,

100oC), & thermal processing (TP; 0.1 MPa, 100

oC)) for up to 10 min. Furthermore, hot

break juice from two different tomato cultivars (high and normal lycopene content) was

subjected to HPP (700 MPa, 45 °C, 10 min), PATP (600 MPa, 100 °C, 10 min) & TP

iii

(0.1 MPa, 100°C, 35 min) and its storage stability was evaluated over a 52 week period

at 3 storage temperatures viz. 4, 25 and 37oC.

Combined pressure-thermal (PATP, HPP) treatments resulted in up to 12%

increase in lycopene extractability over TP and unprocessed control juice. In addition, all-

trans lycopene showed stability to isomerization in juice samples subjected to HPP,

PATP and TP. The post processing retention of β-carotene was a function of processing

time, temperature, pressure, cultivar used and type of juice (raw vs. hot break).

During storage, lycopene degradation varied as a function of cultivar, processing

method, storage temperature, and time. Increase in storage temperatures also increased

degradation. Among the stored juices, HPP processed juice showed the least lycopene

degradation. Also, HPP and PATP juice samples better retained lycopene cis isomers and

color during storage. β-carotene showed good stability in the processed samples during

storage. A two-step first order equation was used to predict the changes in lycopene

concentration over the course of storage. The processed juice samples also showed

microbial stability over the course of 52 week storage at 4, 25 and 37oC.

The in-vitro bioaccessibility studies showed that regardless of the processing

conditions, less than 0.5% of the lycopene originally present in the processed juice got

micellarized. When raw juice was processed, the amount of lycopene present in micelles

of raw juice (control), HPP, PATP and TP samples was 25.4±1.8, 27.3±2.2, 27.2±1.6 and

26.7±1.3µg/100g juice, respectively. No significant difference was found between the

amount micellarized between various treatments (p>0.05). Likewise, when hot break

juice was processed, the amount of lycopene in the micelles of hot break juice (control),

iv

HPP, PATP and TP samples was 21.4±0.16, 24.3±0.36, 23.8±0.2 and 26.2±0.22,

respectively. A statistically significant difference was observed in the amount of lycopene

micellarized in control, HPP, PATP and TP samples (p<0.05). All-trans β-carotene

micellarization in the processed juice (HPP, PATP, TP) was significantly higher (p<0.05)

as compared to the raw unprocessed juice (control). The amount of β-carotene present in

micelles of raw juice (control), HPP, PATP and TP samples was 25.2±1.33, 30.0±2.1,

31.0±2.9 and 33.9±1.4µg/100g juice, respectively. Interestingly, hot break juice subjected

to P-T treatments showed 15-30% more all-trans β-carotene micellarization than the raw

juice subjected to similar P-T treatments. The amount of all-trans β-carotene in the

micelles of hot break juice (control), HPP, PATP and TP samples was 35.9±0.92,

37.5±0.41, 36.1±0.33 and 37.9±0.63, respectively.

The formation and stability of chemical marker M-2 was influenced by heat

(which favored the marker formation), pressure (which hindered marker formation) and

pH (higher pH hindered marker formation). The initial concentrations of M-2 in the gels

were 9.17 and 6.10 mg/100g at pH 6.10 and 8.25, respectively. The marker yield during

thermal treatment (at 0.1 MPa, 105oC) increased with increase in holding time (following

a first order kinetics) and decreased with increasing pH. Pressure treatments from 350 to

700 MPa at 30oC reduced the chemical marker formation for both pH values investigated.

The net final concentration of the marker formed during PATP was higher than HPP, but

lower than thermal treatments.

In summary, the present research shows that combined pressure−temperature

treatments could be an attractive approach for preserving tomato juice quality.

v

Dedication

Dedicated to the very few who walk the noble path

vi

Acknowledgements

I wish to express a deep sense of gratitude towards my advisor Dr. V.M.

Balasubramaniam and dissertation committee members Dr. Steven J. Schwartz,

Dr. Sudhir K. Sastry and Dr. John Litchfield. Their incessant support and guidance

has indeed churned the scientific professional in me and enriched my scientific

insight and worldly wisdom. I would also like to thank Dr. David Francis for

supplying the raw material needed for research. Dr. Juming Tang, Galina

Mikhaylenko and Dr. Ram Pandit at the Washington State University deserve a

note of thanks for hosting me and sharing their knowledge. Special thanks to

Rachel E. Kopec who stood by me with her knowledge and insight. I can only

appreciate the efforts of all members of the HPP lab, food industries center (Mike,

Gary, Paul, Jayne), food science faculty, staff (Julie, Tina, April, Kelly, Tony) and

students who directly or indirectly helped in improving my professional and

personal skills. The awesome Ohio State University deserves a round of applause

for its excellent library, scenic surroundings and state-of-the-art infrastructure.

Last but not the least I would like to acknowledge the sacrifices of my parents and

family, without whom I wouldn’t have seen this light.

vii

Vita

2003……………………… B.Tech. Chemical Technology/Food Technology,

Laxminarayan Institute of Technology, Nagpur

University, India

2006…………………....... M.Tech. Food Engineering and Technology,

University Institute of Chemical Technology,

Mumbai University, India

2006 to present………….. Graduate Research Associate, Department of Food

Science and Technology, The Ohio State University

Publications

Gupta, Rockendra, Balasubramaniam, V.M., Schwartz, S.J., and Francis, D.M.

(2010) Storage Stability of Lycopene in Tomato Juice Subjected to Combined

Pressure−Heat Treatments. Journal of Agricultural and Food Chemistry, 58, 8305-

8313.

Nguyen, L.T., Tay, A., Balasubramaniam, V.M., Legan, J.D., Turek, E.J., and

Gupta, Rockendra (2010) Evaluating the impact of thermal and pressure treatment

viii

in preserving textural quality of selected foods. LWT - Food Science and

Technology. 43, 525-534.

Fields of Study

Major Field: Food Science and Technology

ix

Table of Contents

Abstract........................................................................................................... ii

Dedication....................................................................................................... v

Acknowledgements........................................................................................ vi

Vita................................................................................................................. vii

List of Tables.................................................................................................. xi

List of Figures................................................................................................. xii

Chapter 1: Introduction.................................................................................

Chapter 2: Pressure Assisted Thermal Processing of Foods

1

2.1 Introduction............................................................................

2.2 In-Situ Property Measurement Under Pressure......................

2.3 Uniformity of Pressure-Heat Treatment.................................

2.4 Spore Inactivation Kinetics Under Pressure...........................

2.5 Impact of Pressure Assisted Thermal Processing on Quality

2.6 Conclusions............................................................................

References....................................................................................

5

12

21

26

33

39

40

Chapter 3: Combined Pressure-Temperature Effects on Lycopene Stability,

Isomerization and Bio-Accessibility in Tomato Juice

Abstract........................................................................................

3.1 Introduction............................................................................

3.2 Objectives...............................................................................

3.3 Materials and Methods...........................................................

3.4 Results and Discussion...........................................................

3.5 Conclusions............................................................................

References....................................................................................

63

64

66

66

74

85

87

x

Chapter 4: Storage Stability of Lycopene in Tomato Juice Subjected to

Combined Pressure-Heat Treatments

Abstract........................................................................................

4.1 Introduction............................................................................

4.2 Objectives...............................................................................



4.5 Conclusions............................................................................

References....................................................................................

104

105

107

107

113

121

123

Chapter 5: Combined Pressure-Temperature Effects on the Chemical

Marker (4-Hydroxy, 5-Methyl, 3(2H)-Furanone) Formation in

Whey Protein Gels

Abstract........................................................................................

5.1 Introduction............................................................................

5.2 Objectives...............................................................................



5.5 Conclusions............................................................................

References....................................................................................

141

142

144

144

150

155

156

Chapter 6: Conclusions................................................................................... 168

Bibliography................................................................................................... 172

Appendix A: Nutrient Content of Tomatoes………………………………...

188

Appendix B: Pressure-Temperature-Time History of Tomato Juice Samples

Processed Using Pressure Assisted Thermal Processing (PATP) and

Thermal Processing (TP)……………………………………….

190

xi

List of Tables

Table

Page

2.1 Properties of water (25oC) at different pressures.........................................

54

2.2 Heat of compression values of selected foods determined at initial sample

temperature of 25oC.....................................................................................

55

3.1 Selected attributes of fresh raw juice and hot break juice obtained from

Roma

tomatoes........................................................................................................

93

3.2 Temperature histories at different stages of high pressure processing

(HPP; 500, 600 and 700 MPa at 30oC) and pressure-assisted thermal

processing (500, 600 and 700 MPa at 100oC) of tomato juice

samples.........................................................................................................

94

3.3 Percent lycopene and β-carotene from tomato juice transferred to the

digesta and micelles during in-vitro bio-accessibility studies......................

95

4.1 Selected attributes of raw tomato flesh and the hot break tomato juice

used in the study...........................................................................................

128

4.2 Reaction rate constants and correlation coefficients of lycopene

degradation in high lycopene (FG99-218) and OX325 tomato juice

treated using HPP, PATP, and thermal sterilization.....................................

129

5.1 Temperature histories at different stages of processing during high

pressure processing (HPP; 350 and 700 MPa at 30oC) and pressure-

assisted thermal processing (PATP; 350 and 700 MPa at 105oC) of whey

protein gel samples.......................................................................................

161

5.2 Rate constants of marker M-2 formation/inhibition/degradation under

different processing conditions at gel pH 6.10 and pH 8.25........................

162

5.3 Influence of various independent variables (pressure, temperature, time,

and pH) and their interactions on chemical marker M-2 formation in

whey protein gels containing 1% ribose.......................................................

163

xii

List of figures

Figure

Page

2.1 A typical pressure-temperature profile of a food sample during

preheating, compression and holding time for combined pressure-

temperature processing (PATP). t1, t3, t4, and t5 are the preheating,

compression, come-up time and holding time, respectively.......................

57

2.2 Heat of compression values of water (experimental - dashed lines) vs.

predicted (continuous lines using NIST/ASME software) at different

initial temperatures......................................................................................

58

2.3 Change in thermal conductivity of selected foods as a function of

pressure........................................................................................................

59

2.4 Temperature and velocity distributions at 136, 182, 500 and 820 s (left to

right) for process with 500 MPa final pressure. At 136 s, forced

convection dominates, at 182 s, the holding phase begins, the flow field

reorganizes and temperature has reached its maximum. Later,

temperature goes down and fluid motion is attenuated...............................

60

2.5 Temperature distribution generated from CFD model (100 MPa) at the

end of each stage: (a) pre-loading (stage 1), (b) pressure build-up or

compression (stage 2), and (c) pressure holding (stage 3)..........................

61

2.6 Microstructures of (A) control, (B) pressure treated (700 MPa, 25oC, 5

min.), (C) pressure assisted thermal processed (700 MPa, 105oC, 5 min.)

and (D) thermally processed (105 0C, 0.1 MPa, 30 min) carrot samples....

62

3.1 Flowchart outlining the steps involved in the experiment..........................

96

3.2 Lycopene retention (▒) all-trans lycopene, (▓) cis lycopene) in fresh raw

tomato juice subjected to (a) high pressure processing (500-700 MPa,

30oC for 0, 3, 5 and 10 min) (b) pressure assisted thermal processing

(500-700 MPa, 100oC for 0, 3, 5 and 10 min) and thermal processing (0.1

MPa, 100oC for 0, 3, 5 and 10 min). Values are mean ± SD of 3

replicates......................................................................................................

97

xiii

3.3 Lycopene retention (▒) all-trans lycopene, (▓) cis lycopene) in hot break

tomato juice subjected to combined pressure-temperature processing;

HPP (500-700 MPa, 30oC for 0, 3, 5 and 10 min), PATP (500-700 MPa,

100oC for 0, 3, 5 and 10 min) and TP (0.1 MPa, 100

oC for 0, 3, 5 and 10

min). Values are mean ± SD of 3 replicates...............................................

99

3.4 Percent all-trans β-carotene retention in raw (a) and hot break (b) tomato

juice after combined pressure-heat processing; HPP (500-700 MPa, 30oC

for 0, 3, 5 and 10 min), PATP (500-700 MPa, 100oC for 0, 3, 5 and 10

min) and TP (0.1 MPa, 100oC for 0, 3, 5 and 10 min). Values are

represented as mean ± SD of 3 replicates....................................................

100

3.5 Representative light microscopic images (using 100 X oil immersion

objectives) of raw tomato juice samples processed using combined

pressure-temperature treatments; HPP (500-700 MPa, 30oC for 0, 3, 5

and 10 min), PATP (500-700 MPa, 100oC for 0, 3, 5 and 10 min) and TP

(0.1 MPa, 100oC for 0, 3, 5 and 10 min).....................................................

102

3.6 Representative electron microscopic images of hot break tomato juice

samples processed using combined P-T treatments; HPP (500-700 MPa,

30oC for 0, 3, 5 and 10 min), PATP (500-700 MPa, 100

oC for 0, 3, 5 and

10 min) & TP (0.1 MPa, 100oC for 0, 3, 5 and 10 min)..............................

103

4.1 HPLC chromatogram of lycopene isomers obtained by adding iodine

catalyst in hexane at a concentration of about 1% (w/w) of the lycopene

weight and allowing the mixture to sit for 15 min in fluorescent light of

luminance 320 lux (lumens/m2)..................................................................

130

4.2 All-trans (a) and cis (b) lycopene concentrations in high lycopene

(FG99-218) tomato juice processed using HPP (700 MPa, 45oC, 10 min),

thermal sterilization (0.1 MPa, 100oC, 35 min) PATP (600 MPa, 100

oC,

10 min) and stored in dark at different storage temperatures for

0,2,5,15,35 and 52 weeks. Untreated control had 14.65±0.66 mg all-trans

lycopene/100 g juice and 1.4±0.01 mg cis lycopene/100 g juice................

131

4.3 All-trans (a) and cis (b) lycopene concentrations in OX325 tomato juice

processed using HPP (700 MPa, 45oC, 10 min), thermal sterilization (0.1

MPa, 100oC, 35 min) PATP (600 MPa, 100

oC, 10 min) and stored in

dark at different storage temperatures for 0,2,5,15,35 and 52 weeks.

Untreated control had 8.84±0.22 mg all-trans lycopene/100 g tomato

juice and 1.38±0.01 mg cis lycopene/100 g juice........................................

133

xiv

4.4 Differential Color values (ΔE) of high lycopene (FG99-218) tomato juice

processed using HPP (700 MPa, 45 °C, 10 min), TP (0.1 MPa, 100 °C,

35 min), PATP (600 MPa, 100 °C, 10 min) and stored in dark at 4,25 and

37 °C for 0, 2, 5, 15, 35, and 52 weeks........................................................

135

4.5 Differential Color values (ΔE) of OX325 tomato juice processed using

HPP (700 MPa, 45 °C, 10 min), TP (0.1 MPa, 100 °C, 35 min), PATP

(600 MPa, 100 °C, 10 min) and stored in dark at 4,25 and 37 °C for 0, 2,

5, 15, 35, and 52 weeks...............................................................................

137

4.6 Representative experimental vs. predicted values of lycopene

concentration in tomato juice (FG99-218) processed using combined

pressure-temperature treatments and stored at various temperatures..........

▔ 700 MPa, 45 °C, 10 min. treatment followed by 4 °C storage

(---) predicted

▲ 0.1 MPa, 100 °C, 35 min. treatment followed by storage at 25 °C

(---) predicted

♦ 600 MPa, 100 °C, 10 min. treatment followed by storage at 37 °C

(---) predicted

139

4.7 Flowchart outlining the steps involved in the experiment.......................... 140

5.1 HPLC Chromatogram of chemical marker M-2 (4-Hydroxy-5-methyl-

3(2H)-furanone) extracted at 285 nm..........................................................

164

5.2 Formation and/or inhibition of M-2 marker in whey protein gel samples

(pH = 6.10) containing 1g ribose/100g gel mix subjected to various

temperatures (30 and 105oC), pressures (350 and 700 MPa) and

treatment times (up to 20 min)....................................................................

165

5.3 Formation and/or inhibition of M-2 marker in whey protein gel samples

(pH = 8.25) containing 1g ribose/100g gel mix subjected to various

temperatures (30 and 105oC), pressures (350 and 700 MPa) and

treatment times (up to 20 min)....................................................................

166

5.4 Flowchart outlining the experimental design.............................................. 167

1

CHAPTER 1: INTRODUCTION

Tomato is one of the most widely consumed food commodity across the world.

Due to its short shelf life, it is processed into several different end products which include

tomato juice, tomato paste, tomato ketchup, diced tomatoes in brine, etc. In the past

decade research on tomato products has attracted a lot of attention due to the presence of

lycopene in tomatoes. Dietary intake of lycopene (80% of which is contributed by tomato

and tomato products) has been proposed to be inversely related to the risk of certain types

of cancer or chronic diseases. However, lycopene (C40H56) with 11 conjugated double

bonds is particularly susceptible to oxidative degradation and/or isomerization upon

exposure to light, oxygen, elevated temperatures, extremes in pH and active surfaces.

Thermal processing is primarily used in the processing of tomatoes, which usually

involves canning the tomatoes and holding them in boiling water for an extended period

of time to achieve shelf stability. Low bioaccessibility of lycopene, phase separation and

watery consistency of the products such as tomato juice are the major challenges faced by

the tomato processing industry. Thus, the quest for improved quality and functionality

has led to the emergence of new technologies such as combined pressure-thermal

application for food preservation. Depending upon the treatment intensity combined

pressure-thermal treatment can provide pasteurization (high pressure processing) or

sterilization (pressure-assisted thermal sterilization) effects. Combined pressure

temperature processing minimizes the application of heat energy for food preservation

2

and attempts are being made to understand its effects on tomatoes and produce better

quality products with improved functional properties.

Although a lot of research has been done on the behavior of lycopene in thermally

processed tomato products, very limited knowledge exists on the effects of combined

pressure-temperature processing on the retention and stability of lycopene. Also,

combined P-T effects on lycopene bioaccessibility and its long term storage stability are

unknown. Thus, a comprehensive study to understand these important factors shall help

in assessing the benefits of HPP and PATP in producing high quality tomato products for

the consumer.

PATP involves the combined application of pressure (500-700 MPa) and heat

(90-121oC) over a specified holding time. Unlike high pressure processing (HPP) (<600

MPa at ambient or chilled temperature), which aims at destroying vegetative pathogenic

and spoilage microorganisms, PATP targets bacterial spores. During PATP and HPP,

rapid and uniform compression heating during pressurization and rapid cooling during

depressurization help in minimizing the thermal effects on the quality (color, texture,

flavor) of foods. It is now well established fact this heat of compression is an important

thermo-physical effect in foods exposed to high pressures. However, differences in the

thermo-physical properties between components of non-homogenous foods and between

the food and pressure transmitting fluid (PTF) give rise to temperature-pressure (T-P)

non-uniformities. Due to the lack of effective means to monitor and record real time T-P

changes under high pressure, there is a need to develop suitable method and/or

instrumentation that can map such non-uniformities and locate the cold spot.

3

Chemical markers such as furanone (that are intrinsically formed in foods at

elevated process temperatures) have been successfully used as indirect indicators of

heating patterns in advanced thermal processes such as aseptic processing, microwave

sterilization and ohmic heating. However, very limited information is available on

stability of these chemical markers during combined pressure-heat treatment. Hence, a

study on the effect of combined P-T on such intrinsically formed markers shall help in

evaluating the feasibility of their use to study pressure-temperature non-uniformities

during PATP.

Thus, the primary objective of this dissertation research was to determine the

effect of combined pressure-temperature processing on carotenoids in tomato juice and to

assess the feasibility of using a novel chemical marker M-2 as a tool to evaluate pressure-

temperature uniformity. The central hypothesis was that elevated pressures in

combination with thermal effects shall change the retention and stability of carotenoids.

Also, intrinsically formed chemical markers such as M-2, which have been successfully

employed to map temperature uniformities in thermal processes, might show similar

feasibility of use under combined P-T conditions.

The specific objectives of this dissertation were:

1. To investigate the pressure-thermal effects on post processing extractability,

isomerization and bioaccessibility of lycopene in tomato juice and to microscopically

study the changes caused by such treatments.

4

2. To study the storage stability of lycopene in tomato juice (from high and normal

lycopene tomato cultivars) processed using TP, PATP and HPP and stored up to 52

weeks at 4, 25 and 37 °C.

3. To evaluate the feasibility of applying novel chemical marker M-2 (4-Hydroxy-5-

methyl-3(2H)-furanone) for studying temperature non uniformities during PATP.

5

CHAPTER 2: PRESSURE ASSISTED THERMAL PROCESSING OF

FOODS

2.1 Introduction

Pressure assisted thermal processing (PATP) of foods involves the combined

application of pressure (500-700 MPa) and heat (90-121oC) over a specified holding

time. While high pressure processing (< 600 MPa at or near ambient temperature) aims at

destroying vegetative pathogenic and spoilage microorganisms, PATP targets bacterial

spores (Balasubramaniam and Farkas, 2008; de Heij et al., 2003). Interest in the

production of PATP processed low acid products such as soups, tea, egg products,

mashed potatoes, selected vegetables and fruits is a topic of current research and the

results have been promising (Balasubramaniam and Farkas, 2008; Nguyen et al., 2007;

Juliano et al., 2006)

Rapid and uniform compression heating during pressurization and rapid cooling

during depressurization helps in minimizing the detrimental thermal effects on the quality

(color, texture, flavor) of foods as experienced in the case of conventional thermal

sterilization. PATP also reportedly reduces the thermal impact on food quality (Rastogi et

al., 2008; Nguyen et al., 2007; Hoogland et al., 2001; Juliano et al., 2006). With increased

interest in PATP for preservation of high-quality, shelf-stable, low-acid foods (Nguyen et

6

al., 2007; Balasubramaniam and Farkas, 2008), the technology poses an attractive

opportunity to the food processors for processing heat sensitive, value-added foods with

fewer additives and cleaner ingredient labels. In addition, the possibilities of combining

PATP with other emerging technologies such as ohmic heating, pulsed electric field

processing (PEF), etc. also exists and could further the development of minimally

processed low-acid foods.

2.1.1 Basic PATP principles

During PATP, both the process pressure and temperature can influence various

reactions. Depending upon the type of reaction, the combined pressure-heat application

may be synergistic, additive or antagonistic. Application of pressure is governed by the

two basic principles viz. the Le Chatelier’s principle and the iso-static principle. The Le

Chatelier’s principle states that any phenomenon such as phase change, change in

molecular configuration, chemical reaction, etc. that is accompanied by a decrease in

volume is enhanced by pressure and vice versa (Farkas and Hoover, 2000). The iso-static

principle states that pressure is transmitted in a uniform and quasi instantaneous manner

throughout the whole sample, thus making the process independent of volume and

geometry of the product. It has been generally accepted that although iso-static principle

is assumed to be true in many high pressure food processing applications, deviations are

possible for heterogeneous large samples systems. Once the desired pressure is reached, it

can be maintained for an extended period of time without any further energy input

(Balasubramaniam, 2003). Unlike high pressure homogenization where the food is

exposed to high velocity, turbulence and shear forces, during PATP, the food is subjected

to isostatic pressure treatment. However, foods such as marshmallows, strawberry, that

7

contain large air packets, due to difference in compressibility of air and rest of the food,

pressure treatment deform them.

2.1.2 Typical processing steps involved in PATP of foods

During PATP, food is preferably vacuum packaged in a flexible, high barrier

package. At least one interface of the package should be flexible enough to transfer

pressure to the packaged food (Rastogi et al., 2007). Then the prepackaged food material

along with the pressure transmitting fluid is preheated to certain initial temperature (refer

section 2.1.1 on choosing initial temperature) in a sample basket. Subsequently the

sample basket is loaded inside a pressure vessel. The pressure vessel was also preheated

to certain temperature to minimize heat loss from the product to the surrounding through

the walls of the pressure chamber. The remainder volume of the pressure chamber is also

filled with pre-heated pressure transmitting fluid. Samples are processed at specified

pressure-heat combinations for certain holding time. Once processed, the food is

removed from the pressure chamber, and cooled immediately to minimize over

processing (Balasubramaniam and Farkas, 2008).

2.1.3 Pressure-temperature response during processing

A typical pressure-temperature response of the sample during PATP is shown in

Figure 2.1. During PATP, the food material is pressurized from atmospheric pressure

(P1) to a target pressure (P2). The time interval between P1and P2 represents the pressure

come up time. Typical commercial equipment may have pressure come-up time of

approximately 2 min to reach 600 MPa. Once the target pressure is reached, the samples

are processed for desired holding time. For economical justification and to minimize

8

thermal effects on product quality, pressure holding times of less than 10 minutes may be

desired. After processing, the samples are depressurized back to atmospheric pressure

(P4). Most of the commercial scale high pressure equipment have short (<30 sec)

decompression times.

Due to compression under pressure, the temperature of a food material increases

(Ting et al., 2002). The magnitude of this temperature change (Figure 2.1, T3-T4’)

depends on the compressibility of the substance, its thermal properties, initial

temperature, and target pressure. The maximum product temperature at the target process

pressure is independent of the rate of compression as long as heat transfer to the

surroundings is negligible. During depressurization (T6) the temperature may drop below

the initial temperature if heat is lost from the sample to the environment during pressure

holding.

2.1.4 Packaging

A variety of flexible, high barrier packaging materials may be used to contain

samples of high-pressure processing. The volume, geometry and composition (polymer

type and composition, film thickness, sealing properties and barrier properties) of the

packaging material are important considerations for selection as packaging material in

high pressure processing (Balasubramaniam et al., 2004). At least one interface of the

package should be flexible enough to transmit pressure. Thus, rigid metal containers may

not survive the pressure treatment (Rastogi et al., 2007). Presence of head space air,

particularly oxygen, can adversely affect product quality at elevated pressure-temperature

conditions. Further, air has different compressibility than water and more effort needs to

9

be made to compress the air. Thus, it would be desirable to minimize the presence of

headspace air in the packages or preferably vacuum package the product.

Various authors studied the influence of pressure treatment on packaging material

(Yoo et al., 2009; Schauwecker et al., 2002; Caner et al., 2004; Halim et al., 2009;

Fairclough and Conti, 2009; Galotto et al., 2009; Galotto et al., 2008, Koutchma et al.,

2009).

X- ray diffraction of low density polyethylene (LDPE) films exposed to pressures

from 200-800 MPa (10 min at 25 and 75oC) showed significant increases in crystallinity

with increases in pressure. While post treatment oxygen transmission rates were found to

be progressively lower with increasing pressure treatments, the melting temperature of

LDPE was found to increase with increasing pressure. However, no significant effect of

temperature and holding time was observed on either crystallinity or the melting

temperature (Yoo et al., 2009). In another study, Caner et al. (2004) used D-limonene as

the sorbate in ethanol and acetic acid solutions to test the sorption behavior of monolayer

polypropylene, multilayer polymer film and a metalized polymeric film under high

pressure conditions. Treatments at 800 MPa, 60oC for 10 min showed that type of

material, acidity and temperature had a significant effect on the sorption behavior of

packaging materials.

Schauwecker et al. (2002) studied propylene glycol (PG) migration in different

pouches under various pressure temperature conditions and observed that

polyester/nylon/aluminium/polypropylene meal ready-to-eat (MRE) type pouches treated

at 400, 600 and 827 MPa at 30, 50 and 75oC did not show detectable PG migration.

10

However, PG migration in EVOH/PE-EVOH pouches was found to decrease

significantly in pressure treated pouches (400, 600 and 827 MPa at 30, 50 and 75oC for

10 min) as compared to those treated at different temperatures (30, 50 and 75oC) for 10

min under atmospheric pressure (0.1 MPa). Pouch structures having plastic-metallic

interface as used in meal-ready-to-eat (MRE) pouches processed at elevated pressures

(≥200 MPa) and 90oC for 10 min showed signs of delamination between PP and Al

layers.

High pressure treatments (400, 800 MPa and 40, 75oC for 5 and 10 min) and

thermal sterilization at 120oC on EVOH-based packaging materials showed that high

pressure treatments do not cause crystallinity disruption of EVOH copolymers otherwise

observed in sterilization treatment at 120oC (Lopez-Rubio et al., 2005). The oxygen

barrier properties of pressure treated pouches were reported as comparable to the

untreated pouches, whereas thermally sterilized pouches showed increased oxygen

permeability. This research, however, did not investigate the combined pressure and

temperature (>100 0C) effect on the barrier properties of EVOH based packaging

materials.

Koutchma et al. (2009) studied the impact of using different packaging materials

on the rate of heat penetration into foods during preheating. Also, the effects of

preheating and HP sterilization (PATP) (688 MPa, 121oC) on package integrity, oxygen

permeability and mechanical properties for commercially available packaging materials

were studied. Four plastic-laminated materials (nylon/coextruded ethylene-vinyl alcohol,

nylon/polypropylene [PP], polyethylene terephthalate [PET]/aluminum oxide/casted PP

[CPP] and PET/polyethylene) and two aluminum foil-laminated pouches (PET/aluminum

11

[Al]/CPP and nylon/Al/PP) were used in the study. It was found that the foil-laminated

packaging had higher rate of heat transfer during preheating as compared to the thinner

polymeric materials. PATP showed increase in seal strength of foil-laminated pouches.

However, PATP altered the oxygen barrier of the composite packaging materials and it

was reported that the increase in permeability observed during PATP was due to thermal

damage due to preheating. TR processing increased oxygen permeability to a higher

extent than HP-HT processing (Koutchma et al., 2009).

More research is needed on the effect of elevated pressure-heat treatments on

different packaging materials. The changes in barrier properties of PATP packages during

extended storage are also worth investigating.

2.1.5 Pressure transmitting fluid

Pressure transmitting fluids are used to transmit the pressure to the food sample.

Water, food-grade propylene glycol-water solutions, silicone oil, sodium benzoate

solutions, ethanol solutions and castor oil are some of the commonly used pressure

transmitting fluids (Balasubramanian and Balasubramaniam, 2003). Choice of pressure

transmitting fluids is in part dependent on their ability to seal under pressure, corrosion

prevention properties, fluid viscosity changes under pressure, and heat of compression.

The composition of the pressure transmitting fluid, its thermal characteristics and ratio of

fluid to sample play an important role in governing the thermal behavior of foods under

pressure. The importance of considering the compression heating behavior of pressure-

transmitting fluid on microbial inactivation kinetics has been discussed in some studies

(Balasubramanian and Balasubramaniam, 2003; de Heij et al., 2003; Matser et al., 2004;

12

Robertson et al., 2008; Otero and Sanz, 2003). Whereas difference in compressibility and

heat of compression of the pressure transmitting fluid might result in heat transfer

between the food sample and the fluid and influence the rate of heat loss to the

surroundings, the thermal conductivity and thermal diffusivity of pressure transmitting

fluid may affect the inactivation kinetics of bacterial spores (Balasubramanian and

Balasubramaniam, 2003).

2.2 In-situ property measurement under pressure

High pressure influences both the thermal and physico-chemical properties of

food. Water is the major constituent present in most of the foods along with a complex

mixture of proteins, carbohydrates, fats, minerals, vitamins, and salts. Properties of water

under pressure are well documented after the pioneering work of Bridgman (Bridgman,

1912). Table 2.1 summarizes the data on thermal and physical properties of water

obtained from the NIST database (Harvey et al., 1996). With the exception of water, very

limited data is available on the influence of pressure on various food constituents. There

exists a unique but highly complex interaction between food constituents and availability

of free water varies from food to food.

The thermal exchange between the food, pressure transmitting fluid and the

environment through the walls of the pressure chamber can influence the uniformity of

PATP. This heat exchange can be further governed by the thermo-physical properties of

food, packaging material and the pressure transmitting fluid. Specifically the following

properties are important

Heat of compression

Thermal conductivity

13

Specific heat

Density

Thermo-fluid dynamics during combined pressure-heat treatment are influenced by the

thermo-physical and physico-chemical properties of the pressure transmitting fluid and

the food sample (Baars et al., 2007). Combined pressure-heat influence on selected

properties is discussed in the following sections.

2.2.1 Heat of compression

Although, it is assumed that pressurization would create uniform pressure and

temperature distribution within the pressure vessel, it is difficult to achieve in practice

(Denys et al., 2000; Hartmann and Delgado, 2005). Also, modeling and evaluating the

process uniformity during PATP requires reliable data for pressure-temperature

dependence on density, specific heat capacity, thermal conductivity, thermal expansion

coefficient and viscosity (Barbosa-Cánovas and Rodriguez, 2005; Min et al., 2010).

During PATP, the food material undergoes physical compression. For example,

volume of one-liter water may be reduced to 0.85 liter while under 600 MPa pressure

(~15% reduction). This physical compression also increases the temperature of the

product and this is an unavoidable thermodynamic effect (Ting, et al., 2002). The

reversible adiabatic temperature change of compression can be expressed using the

following equation proposed by Zemansky (1957):

……………………………………………………………………………………………….(1)

From the above equation it follows that the change in temperature as a result of physical

compression depends on the thermal expansivity of the substance (α), temperature (T),

14

specific volume (V), and specific heat capacity (Cp).Experimentally determined heat of

compression data of selected foods is given in Table 2.2. Water and high moisture

content foods were found to exhibit the lowest heat of compression value i.e. 3oC/100

MPa whereas fats and oils show the highest heat of compression values of 6-9oC/100

MPa (Rasanayagam et al., 2003).

From Table 2.2, it can be inferred that heat of compression of high moisture

content foods can be substantially different from non-polar compounds such as fats and

oil. While the temperature of the polar compounds such as water readily follow the

pressure curve, the temperature response of non-polar substances such as fats and oil

exhibits a time lag of up to 30 seconds to reach the maximum temperature during the

pressure holding time (Rasanayagam et al., 2003). This could be attributed to the phase

change of sample under pressure (Otero et al., 2000). For polar compounds, the

temperature increase of the sample also depends on its initial temperature, while fats and

oil, the heat of compression is fairly constant and do not depend on the initial

temperature.

2.2.1.1 Heat of compression as influenced by chemical composition

The compressibility of a substance depends on its phase and gases show highest

compression values which are drastically reduced for liquids and are further lower for

solids (Ramaswamy et al., 2005). Compression of a material decreases the average

intermolecular distance between adjacent molecules. Compressibility, thus, is an intrinsic

property of a material. A liquid contains molecules that occupy space in excess of that

needed for closed packing. It is this free volume that is decreased during compression.

15

Due to decrease in compressible volume with increase in pressure, the compressibility of

a liquid significantly decreases at elevated pressures. Material compressibility can be

estimated at constant temperature (isothermal compressibility) or constant entropy

(adiabatic compressibility) (Rasanayagam et al., 2003). Heat is generated within the

material due to work of compression against intermolecular forces. Since food is a

complex mixture of water, proteins, carbohydrates and fats, each of which has different

compressibility and specific heat capacity and thus different heat of compression values;

high pressure treatment of a food can induce non-uniform temperature distribution across

the food. This non-uniformity of temperature distribution in the food might impact

microbial and enzyme inactivation. Difference in the heat of compression values of the

pressure transmitting fluid would also contribute to this time-temperature- pressure non-

uniformity across the high pressure vessel. An interesting example of differences in

temperature profile during pressurization due to differences in chemical nature is high

pressure treatment of water and oil. Whereas water exhibits a square wave type

temperature profile by reaching its final process temperature as soon as it is pressurized,

fats and oils exhibit longer time of the order of 30 seconds to reach the maximum

temperature at the same value of applied pressure (Rasanayagam et al., 2003; Patazca et

al., 2007). Temperature delays during expansion have also been reported (Otero et al.,

2000). These phenomena may be attributed to differences in respective molecular

structure and phase transition characteristics (Otero et al., 2000; Rasanayagam et al.,

2003). Accordingly, unsaturated fatty acids show higher compression heating than

saturated fatty acids and water. Since the water molecules are small and polar and more

closely packed than the fat molecules by virtue of hydrogen bonds, they exhibit lower

16

compressibility values. Similarly, saturated long chain fats are more closely packed than

unsaturated long chain fats and less closely packed than water. Hence, they exhibit

compressibility values that are more than that of water but less than the unsaturated long

chain fats which exhibit the highest compressibility values (Rasanayagam et al., 2003).

Organic compounds such as propylene glycol and ethanol contain hydroxyl functional

groups and can form hydrogen bonds between molecules. This might influence their

thermal behavior under pressure. Propylene glycol shows heat of compression values

between water and oil, whereas ethanol shows high heat of compression values with

strong dependency on pressure (Rasanayagam et al., 2003). It should also be noted that

heat of compression values might increase with increase in the initial temperature values

as is discussed in the later sections. Since food is a complex system with numerous

interactions amongst proteins, carbohydrates, fats, vitamins, minerals and water, the

combined effect of initial sample temperature and process pressure will have significant

influence on the heat of compression of the food (Patazca et al., 2007).

While increasing the initial temperature of the high moisture foods increases the heat

of compression values (Figure 2.2), the initial temperature does not influence the heat of

compression values for fats and oils (Rasanayagam et al., 2003; Ramaswamy et al., 2005;

Patazca et al., 2007). The heat of compression values for water show strong dependence

on initial temperature (Figure 2.2) and increase with increase in the initial temperature of

water. On the contrary, heat of compression values of fats and oils showed only slight

dependency with increases in initial temperature. However, foods such as cream cheese

and honey show a complex interdependence of initial temperature and pressure.

17

With the knowledge of compression heating factors (CHm) of various food

constituents of mass fraction (Mf), the apparent temperature of the sample of mass M at

the end of the come up time can be estimated using the mixture rule given below

(Ramaswamy et al., 2005; Nguyen et al., 2007):

𝑇𝑓 = 𝑇𝑖 +( (𝐶𝐻𝑚 ∗𝑀𝑓 )𝑖

𝑀 ∆𝑃

100 + ∆𝑇H …………………………………………………….(2)

Here, Ti – is the initial sample temperature and

ΔTH – is the change in temperature of the sample due to heat gained or lost between the

test sample and the surroundings during the entire processing operation.

2.2.2 Thermal conductivity

Thermal conductivity of food materials under pressure increases as a function of

pressure, food composition and process temperature (Ramaswamy et al., 2007; Denys

and Hendrickx, 1999; Shariaty-Niassar et al., 2000). Denys and Hendrickx (1999)

determined the thermal conductivity of tomato paste and apple pulp up to 400 MPa

pressures using unsteady state method employing line heat source probe containing a K

type thermocouple. They found that the values of thermal conductivity at the studied

temperatures (30 and 65oC) for tomato paste and apple pulp were independent of the

applied pressure and were higher at higher temperature. Shariaty-Niassar et al. (2000)

reported an increase in the thermal conductivity of foods with increase in pressure and

found that thermal conductivity is also influenced by the amount of moisture present in

the food. The pressure dependency of thermal conductivity was higher at lower moisture

18

contents than at higher moisture contents possibly due to less amount of incompressible

water present in the food (Ramaswamy et al., 2005).

Ramaswamy et al. (2007) studied the thermal conductivity of apple juice, canola

oil, clarified butter, honey and high fructose corn syrup at pressures ranging from 0.1

MPa to 700 MPa at 25oC and found that that k of materials tested increases linearly with

increasing pressures up to 700 MPa. Whereas water and water like substances (apple

juice) were found to have the highest value of k (up to 0.82 W/mK at 700 MPa), fatty

foods such as canola oil and clarified butter had the lowest (0.29 to 0.4 W/m˚C

respectively at 700 MPa) values. Honey and high fructose corn syrup (HFCS) had

intermediate values (Figure 2.3).

This increase of thermal conductivity of foods under pressure could be explained

on the basis of their polarity and compressibility. Among the products tested, non-polar

clarified butter showed the highest increase (106%) in k under pressure and water showed

the lowest increase (35%). Polar materials have lower compressibility than non-polar

materials and their k values under pressure changed to a lesser extent (Bridgman, 1923).

Werner et al. (2008) studied plant oils under high pressure (400 MPa, 10-60oC) and found

an increase in thermal conductivity under pressure. The pressure and temperature

dependency of thermal conductivity was found to be linked with the coefficient of

isothermal compressibility and isobaric thermal expansion coefficient, respectively.

2.2.3 Specific heat

Since thermal effects in PATP are significantly larger than HPP, knowledge of

specific heat values under combined pressure-temperature conditions will help in

19

evaluating the thermal effects and model the heat transfer under pressure. In the absence

of experimental data on specific heat of food materials under pressure many earlier

authors assumed that specific heat of food materials remains unchanged (Hartmann and

Delgado, 2005). Hartmann and Delgado (2005) investigated in the heat and mass transfer

effects on the uniformity of high pressure induced inactivation of E. coli suspended in

UHT-milk. For this purpose the specific heat of milk under pressure was assumed to be

constant (and used specific values of the food materials estimated at atmospheric

pressure). Barbosa (2003) measured in-situ sound velocity under high pressure and

correlated the ultrasonic data with thermophysical properties at atmospheric pressure to

compute specific heat capacity as a function of pressure and temperature. 2.5. 10 and

50% (w/v) sugar solutions, 1, 5 and 10% (w/v) organic acid solutions, combination of

simple carbohydrates and organic acid solutions and pulp free orange juice were selected

for the study. Zhu et al. (2007) applied the dual-needle line-heat-source method to

estimate the thermal conductivity, thermal diffusivity and volumetric heat capacity of

fresh potato and cheddar cheese from 0.1 to 350 MPa at 5oC. Changes in volumetric heat

capacity under pressure did not show a clear pressure dependency (Zhu et al., 2007). It is

further interesting to note that high pressure decreases the specific heat capacity of water

by about 10% than those estimated at ambient pressures (Ramaswamy et al., 2005). For

example, the specific heat of water decreases from 4.18 at 0.1 MPa to 3.79 kJ/kg K at 500

MPa (Table 2.1). More research is needed to estimate the specific heat of various food

materials as a function of pressure and heat.

20

2.2.4 Density

As a product is compressed under high pressure, its volume decreases and density

increases. Estimation of density under pressure involves the estimation of sample volume

under process pressure. The change in volume is measured by converting it into linear

expansion through the use of a Bellows, which is subsequently measured using an LVDT

(Linear Variable Differential Transformer) (Bridgman, 1931; Chang and Moldover,

1996). Frictional losses during displacement need to be considered when estimating

volume change using LVDT. Pressure dependent acoustic methods have also been used

to calculate density changes (Kovarskii, 1994). Estimation of the density of apple sauce

and tomato paste using bulk volume displacement method has been reported by Denys et

al. (2000). Min et al. (2010) employed a piezometer with movable copper piston at one

end to determine density changes in various foods. The movement of piston under

pressure caused impedance change in the coil wound around the piezometer. This change

in impedance was calibrated against known volume changes in water exposed to high

pressures. It was found that the density of sucrose solutions (2.5-50%), soy protein

solutions (2.5-10%), soybean oil, chicken fat, clarified butter, chicken breast, ham,

cheddar cheese, carrot, guacamole, apple juice and honey increased as the process

pressure was increased. However, with increasing pressure, the rate of increase in density

of these samples was found to decrease.

2.2.5 pH

High pressure is believed to cause ionic dissociation of water molecules with a

corresponding decrease in pH (Cheftel, 1995; Hoover et al., 1989). Studies conducted by

21

Heremans (1995) indicate a lowering of pH in apple juice by 0.2 units per 100 MPa

increase in pressure. pH plays an important role in phenomenon such as gelation, enzyme

activity, protein denaturation and microbial inactivation kinetics and most

microorganisms show increased susceptibility and inability to recover from sub-lethal

injuries at low pH values (Hoover et al., 1989). At this time reliable instruments for

measurement of pH under pressure are not readily available. More research is needed to

characterize transient pH shift under combined pressure-heat treatment as a function of

various food composition.

2.3 Uniformity of pressure-heat treatment

Variations in both temperature and pressure can contribute to the development of

non-uniformity within a processed volume during PATP experiment. Though, for

practical purpose, pressure treatment assumed to be transmitted uniformly and quasi

instantaneously throughout the sample volume, pressure non-uniformity may exists in

heterogeneous large samples (Minerich and Labuza, 2003). Authors reported that

pressure at the geometric center of a large food such as ham is approximately 9 MPa less

than 600 MPa delivered by the process system.

While many food materials have heat of compression value about 4-6oC/100 MPa

at target pressure-temperature conditions, walls of the pressure vessel have negligible

(~0oC / 100 MPa) heat of compression. As a result, product fraction near the vessel wall

cools down and does not reach the same temperature as that at its center (de Heij et al.,

2002; Ting et al., 2002). Transient temperature exchange occurs between the food,

packaging material, pressure transmitting fluid and the walls of the pressure chamber

likely occur.Also, if the pressure vessel is not properly insulated, the vessel would tend to

22

lose heat to the surroundings thereby resulting in loss of product temperature. Hence, it is

necessary to keep the vessel temperature at or near the desired final product temperature

under process pressure and provide suitable lagging to prevent loss of heat to the

surroundings.

Hartmaan and Delgado (2005) numerically simulated the non-uniformities arising

due to the effect of convective transport and heterogeneous heat transfer during high

pressure (400-500 MPa) induced inactivation of E. coli and B. subtilis alpha amylase.

They proposed that under high pressure processing conditions a fluid flow develops

which interacts with temperature changes effecting inactivation under transient

conditions. Differences in the temperature distribution in the vessel under high pressure

are also a function of the applied pressure and the rate of compression and could be of the

order of 6K at 500 MPa and rapid compression rates (Hartmann et al., 2004).

Mathematical modeling of combined pressure-heat treatment indicates that both the

forced and free convection arise due to pressure-temperature interactions during PATP.

Similarly, changes in rheological characteristics of the food and pressure transmitting

fluid can also influence the process non-uniformity (Hartmann et al., 2004; Baars et al.,

2007). Other factors influencing process nonuniformity during PATP may include

position of the sample within the high pressure chamber (Denys et al., 2000) and sample

phase transition characteristics (Siegoczyski et al., 2007).

2.3.1 Mathematical modeling of process non-uniformity

Basic equations of thermo-fluid dynamics viz. three dimensional heat transfer

equation, equation of continuity, Navier-Strokes equation, expressed appropriately with

23

dimensionless numbers can be used to numerically simulate the non-uniformity effects in

a pressure vessel (Baars et al., 2007; Hartman and Delgado, 2005).

Hartmann and Delgado (2005) employed the finite volume method to solve the

equations of fluid dynamics. The numerical method also considered effects due to

viscosity and heat transfer properties of packaging. Similarly, Carroll et al. (2003) used

the conductive heat transfer equation (neglecting convective effects) to model heat

transfer/ temperature distribution within the pressurizing fluid under high pressure

processing conditions (equation 3):

∇2𝑇 −𝜌𝑐𝑃

𝐾

𝜕𝑇

𝜕𝑡=

𝑇∝

𝐾

𝜕𝑃

𝜕𝑡……………………………………(3)

Assuming that the fluid is being heated at a constant rate (which would occur at

constant pressurization and over pressure range across which the physical properties of

the fluid are constant), the temperature profile during pressurization is obtained by

analytically solving the above equation (Carroll et al., 2003). Similarly progressively

solving heat transfer equations, Carroll et al. (2003) obtained heat transfer equations for

holding phase and cooling phase. Also, heat transfer between the compressed fluid and

the vessel walls could be modeled using the basic heat transfer equation in radial

direction and for top and bottom surfaces (Hartman et al., 2004). In general, the thermal

effects under high pressure in a pressure vessel have been modeled by modifying the

basic heat transfer equation proposed by Fourier (Caroll et al., 2003; Denys et al., 2000;

Chen et al., 2007). Khurana and Karwe (2009) used the basic equations of mass

momentum and energy to simulate fluid flow and temperature distribution in a high

24

pressure vessel. Boussinesq approximation was used to model natural convection in the

high pressure vessel assuming very small changes in density. After considering time

constant of thermocouples used they reported a good agreement between numerically

predicted and experimentally obtained temperature data at lower initial temperatures. At

higher starting temperatures, enthalpy balance to correct numerically predicted data

showed better agreement between the predicted and experimental values. Attempts have

also been made to use neural networks for predicting the process parameters (Torrecilla

et al., 2004).

Hartmann et al. (2004) employed mass, momentum and energy conservation

equations to model thermo-fluid dynamics under high pressure conditions (up to 500

MPa). Temperature distribution simulated using computational fluid dynamics is shown

in Figure 2.4. It was found that temperature increases from the bottom to the top of the

vessel. Also it has been reported that temperature maxima exists at the top corner and the

top central zones (Hartmann et al., 2004; Hartman and Delgado, 2005).

In another study, Chen et al. (2007) used computational fluid dynamics to predict

transient temperature, pressure and velocity distribution vectors during different stages of

pressure cooling of pork (Figure 2.5). Three dimensional equations of energy and

momentum transfer were used in modeling and differential scanning calorimetry was

employed to determine enthalpy of pork at different temperatures. The results show that

temperature within the sample decreases radially from the center of the vessel towards

the boundaries. However, in the pressure vessel itself, the cold region exists towards the

bottom of the vessel (Chen et al., 2007).

25

2.3.2 Use of enzyme/protein based biochemical markers as indicators of process

non-uniformity

Few researchers have proposed the use of enzyme/protein based markers for

estimating thermal non-uniformities under combined pressure-temperature processing

conditions (Grauwet et al., 2010; Gogou et al., 2010).Gogou et al. (2010) studied

Thermomyces lanuginosus xylanase enzyme up to 600 MPa and 50-70oC and found a

synergistic effect of pressure with temperature on the enzyme inactivation. First order

inactivation kinetics was reported. Likewise, Grauwet et al. (2010) evaluated the

potential of using Bacillus amyloliquefaciens α-amylase based indicator for mild

pressure-temperature conditions. After exposing the above enzyme to isobaric-isothermal

treatments up to 680 MPa and 10-45oC and dynamic pressure-temperature treatments

(350-600 MPa and initial temperature 10-25oC), the enzyme activity was measured. Due

to our limited understanding, further research is needed to develop real-time, reliable,

sensitive and versatile industrially relevant sensors that can be used for mapping

pressure-heat related non-uniformities with precision, accuracy and reproducibility

especially during high pressure sterilization or pressure assisted thermal sterilization

(PATS) conditions.

2.3.3 Reporting PATP data

For successful development of PATP, it is important that researchers are able to

compare and validate results obtained from various laboratories. However, a number of

factors can make this difficult.

1. Differences in the equipment design, material of construction, and insulation

characteristics.

26

2. Differences in the pressurization and depressurization rate

3. Neglecting to consider thermal effects under pressure and control initial sample

temperature

4. Inadequate description of the experimental procedures (example – some research

papers do not clarify whether or not pressurization and depressurization times are

included in the stated pressure holding times, whether or not stated temperature is

initial sample temperature or sample temperature under pressures)

5. Type of pressure transmitting fluid used and the ratio of pressure transmitting

fluid: to sample

The recommended experimental practices in high pressure research have been suggested

by Balasubramaniam et al. (2004).

2.4 Spore inactivation kinetics under pressure

Whereas high pressure has been shown to effectively inactivate vegetative cells

(Dogan and Erkmen, 2004), pressure alone has little effect on the destruction of spores.

At ambient temperatures, bacterial spores can survive pressures above 1000 MPa (Farkas

and Hoover, 2000). It has been found that combined pressure-temperature treatment has

synergistic effect on the spore inactivation (Cheftel, 1995; Hoover et al., 1989; Rovere et

al., 1998, Ananta et al., 2001). However, few studies have addressed PATP spore

inactivation kinetics over a range of pressure and temperature conditions (Rovere et al.,

1998; Miglioli et al., 1997; Ananta et al., 2001; Margosch et al., 2004a,b, Margosch et al.,

2006, Rajan et al., 2006, Rodriguez et al., 2004; Reddy et al., 2006; Ahn et al., 2007; Bull

et al., 2009; Zhu et al., 2008).

27

Spore formation is a unique survival strategy of some bacteria and among them

spores of Clostridium botulinum are the most important in food safety considerations

(Heinz and Knorr, 2001). Possibly due to differences in mechanism of inactivation

between thermal and combined pressure-heat treatment, some of the traditional surrogate

organisms such as Bacillus sterothermophilus and Clostridium sporogenes appear to be

sensitive to PATP treatments. Bacillus amyloliquefaciens, a mesophilic organism that

forms highly pressure resistant spores, has been proposed as a nonpathogenic target

microorganism for assessment of sterility achieved using combined pressure-temperature

treatments (Margosch et al., 2004b; Ahn et al., 2007).

Unlike the typical linear profile on a semi-log plot of spore inactivation behavior

during thermal processing, a characteristic tailing bending the inactivation curve concave

upward is observed during PATP of spores (Okazaki et al., 2000; Ananta et al., 2001;

Rajan et al., 2006; Reddy et al., 2003). Hence, the use of non-linear kinetic models to

describe PATP spore inactivation has been suggested to be more appropriate (Peleg and

Cole, 1998; Rajan et al., 2006; Ahn et al., 2007). Some empirical non-linear models

reported in the literature include the nth

order kinetics, the log-logistic, the Weibull

distribution and the biphasic model (Ananta et al., 2001; Ardia et al., 2003; Koutchma et

al., 2005; Rajan et al., 2006; Ahn et al., 2007). The non-linear survivor curves discussed

above are thought to be due to multiple effects of high pressure and temperature on both

spores and cells resulting in shoulders and tails. Pressure affects multiple sites and

mechanisms of spore survival and not just one specific site or mechanism. Similar

concept has been proposed for microorganism cells (Hoover et al. 1989; Tay et al., 2003).

28

Sub-lethal injury to cells, presence of cell clumping and activation of dormant spores

have been proposed as possible reasons for shouldering. A variety of hypotheses explain

the tailing phenomenon during cell inactivation (Tay et al., 2003) and a similar

hypothesis needs to be developed for tailing behavior during spore inactivation.

1. Spores in suspensions may form clumps that create an artifact in estimating CFU

by plate counting techniques which results in the tailing of survivor plots.

2. Increased resistance to lethal process subsequent to lethal stresses (stress

adaptation).

3. Genetic heterogeneity in treated spore population (Heldman and Newsome, 2003;

Tay et al., 2003; Mañas and Pagán, 2005).

Studies to demonstrate that survivors of a sub-lethal pressure treatment display

increased pressure resistance during subsequent pressure treatments after being given

sufficient time to recover are in progress (Tay et al., 2003).

2.4.1 Factors affecting spore inactivation

2.4.1.1 Process pressure and temperature

Both temperature and pressure are critical process parameters influencing

microbial lethality during PATP. Rovere et al. (1998) studied the inactivation of spores

of genus Bacillus at 50, 60 and 70oC and 700, 800 and 900 MPa pressure and found that

Bacillus stearothermophillusspores showed one log reduction at 50oC, three log reduction

at 60oC and five log reduction at 70

oC. An increase in pressure from 700 to 900 MPa at a

particular temperature showed little effect on spore inactivation. Similar effect of

temperature was observed on spores of B. coagulans, B. cereus and B. licheniformis.

29

Seyderhelm and Knorr (1992) reported a two log decrease in Bacillus

stearothermophillus spore population from an initial count of 106 upon subjecting them to

200 MPa at 90oC for 30 min. The same reduction required 350 MPa at 80

oC for 30 min.

and at 70oC, 400 MPa and 45 min. little inactivation was achieved. Reddy et al. (1999)

noted greater log reduction of Clostridium botulinum spores (type E) in phosphate buffer

when the temperature was increased from 35 to 55oC at approximately 827 MPa for 5

min. A 5-log reduction of type E (Alaska) spores in phosphate buffer at 827 MPa and

40oC for 10 min holding time was observed by them. In another study, Rajan et al. (2006)

studied the effect of pressure and temperature (500-700 MPa at 95, 105, 110 and 121oC)

on inactivation of Bacillus amyloliquefaciens spores in egg patty mince. Pressure

coefficients (zp and ΔV) and temperature coefficients (zt and Ea) were used to characterize

the effects of pressure and temperature on spore inactivation. It was observed that at

elevated pressures, the spores became less sensitive to temperature changes. Similarly, as

temperature was increased the spores showed less sensitivity to changes in pressure.

2.4.1.2 Pressure come-up-time

Unlike thermal processing, during PATP, the bacterial spores are subjected to

harsher pressure-heat combinations. Pressure-come up is primarily determined as a

function of horse power of the equipment. Temperature-come-up will further influenced

by the heat transfer characteristics of the insulation used, heat of compression of the test

material, initial temperature of the product. Ahn et al. (2007) studied reported that

combined pressure-temperature treatment significantly increased the number of log

reduction of bacterial spores during the pressure come up time. Rajan et al. (2006) also

30

reported the reduction in number of spores during the pressure come up time. The

reduction varied between 0.1 and 1.2 log reduction per g egg product, depending on the

treatment conditions. Lower pressure temperature combinations resulted in lesser

reduction of spore population (500 MPa at 95 to 105oC) whereas high pressure and

temperature (700 MPa, 121oC) inactivated upto 1.2 log spores per g egg product during

the come up time period. Margosch et al. (2004a) also reported a reduction in the

population (< 0.5-log reduction) of B. amyloliquefaciensspores during a 5 min pressure

come-up time for PATP at 600 MPa and 80oC. These authors also reported reduction in

numbers of other spores tested, such as C. botulinum TMW 2.357 (1.5-log reduction), C.

thermosaccharolyticum (3-log reduction), and Bacillus subtilis (>5-log reduction) during

similar come-up times. These observations suggest that different spores are likely to have

different resistances during the pressure come-up time and highlight the importance of

documenting the PATP come-up time and the corresponding spore inactivation.

However, at least one conflicting evidence of the combined effect of pressure and

temperature on the inactivation of bacterial spores was found in the study conducted by

Ahn et al. (2007). Treatment of B. amyloliquefaciens ATCC 49763 spores with 121°C at

0.1 MPa and 121°C at 700 MPa inactivated 7.1 and 4.4 log spore/ml, respectively, during

process come-up time. This demonstrates the complex mechanism of spore inactivation

during PATP. In other words combination of pressure with temperature has some

protective effect at certain combination of pressure and temperature and this might or

might not exist for spores of other bacteria. Further studies are required to understand the

combined effect of pressure and temperature on the inactivation of bacterial spores.

31

2.4.1.3 Pressure hold time and rate of decompression

The type of spores, pressure temperature conditions and the pressure come-up-

time influence the effect of holding time on the inactivation of spores. Most the

researchers reporting combined effect of pressure and temperature on spore inactivation

kinetics, observed increasing spore inactivation with increase in pressure holding time

(Ahn et al., 2007; Rajan et al. 2006; Reddy et al., 2006; Margosch et al., 2004a,b).

Hayakawa et al. (1998) in their studies on rate of decompression on pressure inactivation

of B. stearothermophilus spores in a cyclic treatment found that rapid decompression

improved their inactivation. It was suggested that combined pressure temperature effect

induced water penetration through the spore coat and subsequent adiabatic expansion of

water caused its physical breakdown.

2.4.1.4 Type of pressure transmitting fluid

Balasubramanian and Balasubramaniam (2003) studied the inactivation of

Bacillus subtilis spores using water-glycol mix of different concentrations (water/glycol

75/25, 50/50, 25/75) and 2% sodium benzoate solution as pressure transmitting fluids. Of

the four different pressure transmitting fluids selected for the study viz. 3 water-glycol

combinations (25:75, 50:50 and 75:25) and one water-sodium benzoate (98:2)

combination, water-sodium benzoate combination showed 2 log greater inactivation

values as compared to the combination with highest compression heating values (Water:

glycol :: 25: 75). This effect was attributed to the difference in thermo-physical properties

of various pressure transmitting fluids used (Balasubramanian and Balasubramaniam,

2003). In another study, Robertson et al. (2008) reported the effect of two different

32

pressurizing fluids viz. 1:9 emulsion of food grade canola oil in water and silicon oil on

the inactivation of spores of Bacillus spp. isolated from New Zealand milk and milk

products. Spores of six species of Bacillus isolates were added to sterile reconstituted

skim milk and processed using HPP at 600 MPa, 75oC for 60 sec. The log inactivation of

all strains was reported to be higher in silicone oil than in water-based pressure

transmitting fluid. This effect was explained by pointing out the differences in

temperature increase due to compression heating of pressure transmitting fluids during

pressurization phase of combined pressure-temperature treatment (Robertson et al.,

2008). Often times, the lab scale equipment and commercial high pressure equipment

may utilize different pressure transmitting fluids. Appropriate care must be taken to

translate the microbial inactivation studies from laboratory scale equipment to

commercial scale equipment (Balasubramaniam et al., 2004).

2.4.1.5 Sporulation conditions and mineral composition of the spores

Studies on the effect of sporulation conditions (pH, temperature, composition of

the sporulation media, etc.) on the resistance of spores to inactivation during PATP

suggest that unlike thermal inactivation, decreasing the sporulation temperature increases

the pressure resistance of spores (Ratphitagsanti et al., 2008; Black et al., 2007).

Similarly, the type of mineral present in the spores can influence pressure resistance of

spores (Rathpitagsanti et al., 2008; Black et al., 2007). Also, there is no specific

correlation between the resistance of bacterial endorspores to heat and that to pressure.

Thus, two bacterial endospores showing similar resistance to pressure might show

different resistance to inactivation by heat only or inactivation by a combined pressure-

33

temperature treatment (Black et al., 2007). The unpredictable nature of spore inactivation

using PATP and enigmatic observations in spores such as superdormancy continues to

intrigue the researchers (Black et al., 2007). More insight is needed to establish the inter-

relationships between spore composition, composition of the spore environment

including process conditions and the mechanisms underlying inactivation of a variety of

spores so that a reliable spore inactivation model under PATP conditions could be

developed.

Further research in PATP induced spore inactivation is needed to evaluate the effect

of physic-chemical properties such as pH, water activity, food matrix and food

component interactions on spore inactivation. It is also desirable to develop suitable

chemical or enzymatic markers as indicators of spore inactivation. Minimizing tailing

during inactivation of spores in PATP is also needed. Data on molecular mechanisms of

spore inactivation under PATP would only help improve the process design to effect

sterility of food products (Balasubramaniam and Farkas, 2008). Kinetic database on the

inactivation kinetics of different spores in various food products would help establish

pressure-temperature conditions for PATP and help the regulatory authorities in

approving the process for commercialization.

2.5 Impact of pressure assisted thermal processing on quality attributes of foods

Pressure assisted thermal processing of foods to achieve sterility has not been studied

much for quality changes. Conventionally high pressures in combination with lower

temperatures have been employed for processing number of foods such as milk, jams,

jellies, guacamole, inactivation of enzymes in fruit and vegetable products, etc. Several

34

research articles reporting such studies are available in the literature. However, the

present discussion aims to highlight some studies on changes in the quality attributes of

selected foods during PATP.

2.5.1 Quality changes during pressure assisted thermal processing (PATP)

Nguyen et al. (2007) studied the effect of pressure assisted thermal processing on

texture, color, carotenoid content and microstructure of carrots. Pressure treatments

between 500-700 MPa employing temperatures between 95 and 121oC showed that

pressure assisted thermal processing resulted in better product quality as compared to

thermal treatments using corresponding product heating profile and temperatures as that

in PATP. The holding time was however higher in case of thermal processing in order to

achieve the required sterility. Textural loss in PATP showed a typical dependence on

pressure at lower temperatures (95 and 105oC) and this dependence on pressure decreased

at higher temperatures (121oC). At 121

oC and lower pressures (500 MPa), textural loss

was higher as compared to that at 121oC and 700 MPa due to higher preheating

temperature and time required before pressurization. Color and carotene retention was

much better in PATP processed samples as compared to the thermally processed samples.

Also, the microstructures of PATP processed carrots (700 MPa, 105oC, 5 min) were very

similar to the fresh control sample (Figure 2.6). Thermal processing at 105 0C and 30

min showed a collapsed structure (Nguyen et al., 2007). Similar findings of PATP

treatments on carrot texture were reported by de Roeck et al. (2008). They found that

PATP resulted in minimal softening and very little changes in intercellular adhesion. A

35

clear difference was noticed between PATP and thermally processed carrot pectin. Unlike

thermal sterilization, texture was preserved after PATP treatments.

In continuation of their previous work, Rastogi et al. (2008) reported the positive

influence of pressure (100-400 MPa), temperature (50-70oC) and calcium (0.5-1.5% w/v)

pretreatments and their combinations on the hardness of PATP treated carrots. They

found a distinct increase in hardness with each of the pretreatments and with combination

treatments a far greater increase in hardness as compared to individual pretreatments. In

each case calcium had positive influence on the hardness. It was found that combined

pressure, temperature and calcium pretreatment helps in preserving the cell

microstructure.

PATP also affects the retention of lycopene and rheology of tomato puree

(Krebbers et al., 2003). Pressure assisted thermal sterilization at 700 MPa and 121oC

results in color improvement and better retention of lycopene compared to 40% loss after

thermal sterilization at 118oC for 20 min. The viscosity of PATP treated tomato puree is

lower as compared to the thermally sterilized sample. PATP also results in 99%

inactivation of PG and PME (Krebbers et al., 2003). Juliano et al. (2006) reported a 30%

reduction in textural properties such as hardness, cohesiveness and water loss after PATP

of egg patties formulated with Xanthan gum and cheese. The pressure-temperature

combinations employed were 700 MPa for 105 or 121oC and 3 or 5 min holding time.

Also, low vacuum packaging in egg patties modified using Xanthan gum and PATP

processed at 700 MPa, 105oC, 5 min showed texture comparable to original patties.

Textural attributes and syneresis were significantly influenced by preheating rates. Also,

36

water addition to the defrosted patties increases the water holding capacity and helps

maintain hardness within acceptable values after PATP at 105 and 121 0C and 700 MPa.

Pressure assisted thermal processing has also been shown to retain textural

properties and nutrients and significantly inactivate enzymes. High pressure sterilization

(PATP) using pulses (1000 MPa, 105oC, 80 sec process time, pulse gap 30 sec) results in

better retention of firmness and ascorbic acid in green beans as against thermal

sterilization (118oC, 30 min). Pulsed PATP also results in 99% inactivation of

peroxidase (POD) as against high pressure processing (HPP) at 500 MPa, 45oC, where

76% initial POD activity is retained (Krebbers et al., 2002).

Butz et al. (2002) studied the effect of combined pressure and temperature on

broccoli, carrots, tomatoes and tomato pulp and found very less effect of high pressure on

chlorophyll a and b in broccoli, and carotenoids and lycopene in tomatoes. Antioxidant

capacity of the water soluble fraction of tomatoes and carrots and antimutagenicity of

high pressure treated tomato pulp was almost the same as that of control. High pressure

treated tomato pulp showed higher water binding ability and increased glucose

retardation index. Extractability of carotenoids from coarse carrot homogenate was found

to decrease.

PATP between 500-700 MPa and 90-115oC was shown to affect the chemical

pectin conversions (de Roeck et al., 2009). Apple pectin solutions at pH 6.5 were

subjected to increasing temperature treatments at atmospheric pressure (0.1 MPa) and

PATP (combined pressure temperature) treatments. Whereas, a temperature-only effect

resulted in higher rate constants for demethoxylation of pectin than beta elimination,

increase in temperature showed higher rate of increase of beta elimination than

37

demethoxylation. However, under PATP conditions, demethoxylation was found to be

stimulated and beta elimination was retarded.

Leadley et al. (2008) compared the effects of PATP and thermal sterilization on

color, texture and microbial quality of green beans and found that PATP treated samples

were darker in appearance and had a significantly firmer texture as compared to the

thermal sterilized samples.

Compared to the studies on how HPP influences interactions between the

components of various food products, effects of PATP on the quality attributes of foods

are relatively less studied. There is a need to study the interactions of food components

and changes in the physico-chemical properties of foods under PATP conditions. Also,

further research is needed to quantify the effect of PATP on the bioavailability of

nutrients after processing and storage. Data on consumer perception and acceptability of

PATP processed products is also required before commercializing products processed

using this technology (Balasubramaniam and Farkas, 2008).

2.5.2 Effect of PATP on nutrient content of foods

Several studies have reported the positive effects of pressure treatment at

temperatures ≤ 60oC on maintaining the fresh like nutrient content of foods (Sanchez-

Moreno et al., 2009). However, very few studies have investigated the effect of pressure

at elevated process temperatures (> 90oC) during PATP on preserving the nutrients in

foods.

Van den Broeck et al. (1998) studied the degradation kinetics of Vitamin C in an

isobaric-isothermal treatment of orange and tomato juice at 850 MPa, 65-80oC up to 400

38

min holding time. Significant degradation of Vitamin C was observed and the

degradation followed first order kinetics. However, at 850 MPa and 50oC, no degradation

of Vitamin C was observed during 60 min holding time. In general, HPPwas found to

increase the antioxidant capacity of fruit and vegetables and was influenced by food

composition, processing pressure, pressure holding time and target temperature (Sanchez-

Moreno et al., 2009).Studies on isothiocyanates present in vegetables such as broccoli

show that increase in temperature during combined pressure-temperature treatments

increase the degradation of isothiocyanates and the degradation follows first order

kinetics (van Eylen et al., 2007).

While heat, light, and oxygen can decrease lycopene during food processing,

combined pressure-temperature shows capability of preserving tomato lycopene content.

A number of studies have reported increased extractability of lycopene from tomato

products exposed to specific pressure-temperature treatments (Gupta et al., 2010;

Krebbers et al., 2003; Sanchez-Moreno et al., 2004; Sanchez-Moreno et al., 2006). In

addition, Gupta et al. (2010) reported lower rate of degradation and thus better retention

of lycopene and color in tomato juice processed using PATP (600 MPa, 100oC) and

stored for a period of 1 year at 4, 25 and 37oC. Since preserving nutritional attributes is a

major objective of any process, more studies are needed to characterize the effect of

PATP on various nutritional principles in food products.

Current regulatory status

Unlike the US Food and Drug administration’s minimum time-temperature

specifications for achieving commercial sterility in foods (21 CFR 108,113 and 114), the

39

regulatory approval for PATP process is evolving. During 2009 Feb, FDA issued no

objection to an industrial petition for processing a shelf stable food by PATP. The

petition considered the process as a thermal process (at 121oC) ignoring the pressure

lethal effects. At this time, no PATP sterilized products are commercially available.

2.6 Conclusions

Pressure assisted thermal processing (PATP) holds promise in significantly

improving the quality and stability of foods without affecting the safety. Research results

from PATP studies have demonstrated its effectiveness in lowering the sterilization time

of foods below 10 min at temperatures much lower than those used in thermal

sterilization. With the added effect of pressure, PATP expands the limits of thermal

processing in developing new products and extending their health related functionality in

the human diet.

40

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54

Table 2.1 Properties of water (25oC) at different pressures

1

Properties/pressure 0.1 MPa 100 MPa 200 MPa 500 MPa

Thermal Conductivity

(W/mK)

0.61 0.65 0.72 0.76

Specific heat (kJ/kg K) 4.18 3.99 3.83 3.79

Density (kg/m3) 997 1038 1101 1150

1Predicted values using NIST/ASME software (Adapted from Ramaswamy et al., 2005)

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Table 2.2 Heat of compression values of selected foods determined at initial sample

temperature of 25oC

Food sample Temperature increase (oC) per 100 MPa

Water 3.0

Orange juice, tomato salsa, 2% fat milk

and other water like substances

2.6-3.0

Carbohydrates 3.6-2.6a

Proteins 3.3-2.7a

Linolenic acid 9.0 - 5.9a

Soybean oil 9.1-6.2a

Olive oil 8.7-6.3a

Crude beef fat 4.4

Extracted beef fat 8.3-6.3a

Beef ground 3.2

Gravy Beef 3.0

Chicken fat 4.5

Chicken breast 3.1

Salmon fish 3.2

Egg albumin 3.0

Egg Yolk 4.5-4.3a

Egg whole 3.3

Mayonnaise 7.2-5.0a

Whole milk 3.2

Tofu 3.1

Mashed Potato 3.0

Yoghurt 3.1

Cream cheese 4.9-4.7a

Continued

56

Hass avocado 4.1-3.7a

Honey 3.2

Water/Glycol (50/50) 4.8-3.7a

Propylene glycol 5.8-5.1a

Ethanol 10.6-6.8a

a Substances exhibited decreasing temperature rise with increase in pressure

(Adapted from Rasanayagam et al., 2003; Kesavan et al., 2002; Ramaswamy et al., 2005)

Table 2.2 continued

57

Figure 2.1 A typical pressure-temperature profile of a food sample during preheating,

compression and holding time for combined pressure-temperature processing (PATP). t1,

t3, t4, and t5 are the preheating, compression, holding, and decompression times,

respectively.

58

Figure 2.2 Heat of compression values of water (experimental - dashed lines) vs.

predicted (continuous lines using NIST/ASME software) at different initial temperatures

(Adapted from Ramaswamy et al., 2005)

0

1

2

3

4

5

6

100 150 200 250 300 350 400 450 500 550 600 650

(DT

/DP

) x 1

00

(°C

/MP

a)

Pressure (MPa)

0°C 25°C 50°C 70°C

0°C 25°C 50°C 70°C

59

Figure 2.3 Change in thermal conductivity of selected foods as a function of pressure

(Ramaswamy et al., 2007)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 100 200 300 400 500 600 700

Pressure (MPa)

Th

erm

al

co

nd

ucti

vit

y (

W/m

ºC)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Water Canola oil

Honey High fructose corn syrup (HFCS)

Apple juice Clarified butter

Canola oil (linear regression) Water (linear regression)

Apple juice (linear regression) HFCS (linear regression)

Honey (linear regression) Clarified butter (linear regression)

Bridgman's formula prediction

60

Figure 2.4 Temperature and velocity distributions at 136, 182, 500 and 820 s (left to

right) for process with 500 MPa final pressure. At 136 s, forced convection dominates, at

182 s, the holding phase begins, the flow field reorganizes and temperature has reached

its maximum. Later, temperature goes down and fluid motion is attenuated. Adapted from

Hartmann et al. (2004)

61

Figure 2.5 Temperature distribution generated from CFD model (100 MPa) at the end of

each stage: (a) pre-loading (stage 1), (b) pressure build-up or compression (stage 2), and

(c) pressure holding (stage 3). Adapted from Chen et al. (2007)

62

Figure 2.6 Microstructures of (A) control, (B) pressure treated (700 MPa, 25oC, 5 min.),

(C) pressure assisted thermal processed (700 MPa, 105oC, 5 min.) and (D) thermally

processed (105oC, 0.1 MPa, 30 min) carrot samples. Adapted from Nguyen et al. (2007)

63

CHAPTER 3: COMBINED PRESSURE-TEMPERATURE EFFECTS ON

CAROTENOID RETENTION AND BIOACCESSIBILITY IN TOMATO JUICE

Abstract

This study investigated the changes in lycopene and β-carotene retention in

tomato juice subjected to combined pressure-temperature treatments ((high pressure

processing (HPP; 500-700 MPa, 30oC), pressure assisted thermal processing (PATP; 500-

700 MPa, 100oC)) and thermal processing (TP; 0.1 MPa, 100

oC)) for up to 10 min.

Processing treatments utilized raw (untreated) and hot break (~93oC, 60 sec) tomato juice

as controls. Changes in bioaccessibility of these carotenoids as a result of processing

were also studied. Microscopy was applied to visualize processing induced

microstructure changes.

TP did not alter the lycopene content of the tomato juice. HPP and PATP

treatments resulted in up to 12% increase in lycopene extractability. All-trans β-carotene

showed significant degradation (p<0.05) as a function of pressure, temperature and time.

Its retention in processed samples varied between 60-95 % of that originally present in

the control. Regardless of the processing conditions used, less than 0.5% lycopene

appeared in the form of micelles. When raw juice was processed, the amount of lycopene

present in micelles of raw juice (control), HPP, PATP and TP samples was 25.4±1.8,

27.3±2.2, 27.2±1.6 and 26.7±1.3µg/100g juice, respectively. Likewise, when hot break

juice was processed, the amount of lycopene in the micelles of hot break juice (control),

HPP, PATP and TP samples was 21.4±0.16, 24.3±0.36, 23.8±0.2 and 26.2±0.22,

64

respectively. Electron microscopic images showed denser and more prominent lycopene

crystals in HPP and PATP processed juice than in thermally processed juice. However,

lycopene crystals did appear to be enveloped regardless of the processing conditions

used.

The processed juice (HPP, PATP, TP) showed significantly higher (p<0.05) all-

trans β-carotene micellarization as compared to the raw unprocessed juice (control). The

amount of β-carotene present in micelles of raw juice (control), HPP, PATP and TP

samples was 25.2±1.33, 30.0±2.1, 31.0±2.9 and 33.9±1.4µg/100g juice, respectively.

Interestingly, hot break juice subjected to combined P-T treatments showed 15-30% more

all-trans β-carotene micellarization than the raw juice subjected to combined P-T

treatments. The amount of all-trans β-carotene in the micelles of hot break juice

(control), HPP, PATP and TP samples was 35.9±0.92, 37.5±0.41, 36.1±0.33 and

37.9±0.63, respectively.

This study demonstrated that combined pressure-heat treatments increases

lycopene extractability. However, the in-vitro bioaccessibility of carotenoids (more

specifically, lycopene) was not significantly different among the treatments investigated.

3.1 Introduction

Dietary intake of lycopene (80% of which is contributed by tomato and tomato

products) has been proposed to be inversely related to the risk of certain types of cancer

or chronic diseases (Giovannucci et al., 1995; Hadley et al., 2002). However, lycopene

(C40H56) with 11 conjugated double bonds is particularly susceptible to oxidative

65

degradation and isomerization upon exposure to light, oxygen, elevated temperatures,

extremes in pH and active surfaces (Nguyen and Schwartz, 1999).

Combined pressure-heat treatments can be utilized for food pasteurization (High

pressure processing (HPP); 400-600 MPa treatment at chilled or mild process

temperatures) and sterilization (Pressure-assisted thermal processing (PATP); 500-700

MPa, 90-120oC) (Balasubramaniam and Farkas, 2008; Rastogi et al., 2007). They help

the food processors in producing quality foods with minimal effects on taste, texture,

appearance, or nutritional value.

Although lycopene is fairly stable to degradation and isomerization during

conventionally practiced thermal treatments (Nguyen and Schwartz, 1999; Nguyen et al.,

2001), high pressure processing has shown an increase in the extractable lycopene from

tomato products (Qiu et al., 2006; Krebbers et al., 2003, Hsu et al., 2007). Krebbers et al.

(2003) studied the fate of lycopene in tomato puree subjected to HPP (300, 500 and 700

MPa at 20oC for 2 min) and PATP (700 MPa, 90

oC for 30 sec.). Authors observed an

increase in the amount of extractable lycopene from the tomato puree. Similar results

were reported for pressure treated tomato puree (100-600 MPa, 20oC, 12 min) (Qiu et al.,

2006) and tomato juice (300-500 MPa, 25oC, 10 min) (Hsu et al., 2007). However, none

of the prior studies investigated the effects of different juice extraction methods (raw

juice vs. hot break juice) on carotenoids. In addition, the impact of combined pressure-

heat treatments (at elevated process temperatures >60oC) on carotenoids is not well

understood. Hence, a comprehensive study is needed to understand the changes in

carotenoids occurring during combined P-T processing.

66

Bioavailability of active phytochemicals such as lycopene is an important

consideration for any novel processing technology. Garrett et al. (1999) developed an in-

vitro digestion method to assess carotenoid bioavailability from meals and reported that

micellarization of lycopene from the meal was less than 0.5%. The meal contained 7.5%

tomato paste, 3% chicken fat. It was microwaved for 50 sec on a high energy setting.

Since lipophilic food components are absorbed from the intestine in the form of micelles,

the very low micellarization of lycopene from the (<0.5% lycopene micellarized) food

meals limits the bioavailability of lycopene. Several other studies have reported the

limited micellarization of lycopene from tomato based test meals after thermal processing

(Reboul et al., 2006; Goni et al., 2006; Huo et al., 2007; Failla et al., 2008; Aherne et al.,

2009; Colle et al., 2010). However, the impact of combined pressure-temperature

treatments on micellarization of lycopene has not been reported.

3.2 Objective

Thus, the overall objective of this study was to investigate the pressure-thermal

effects on post processing extractability, isomerization and bioaccessibility of lycopene

and β-carotene in tomato juice and to microscopically study the changes caused by such

treatments.

3.3 Materials and methods

Fully ripe red Roma tomatoes were purchased from a local store and processed

within 48 hours of procurement. Figure 3.1 presents the overall experimental approach

used during the study. The study utilized raw (untreated) tomato juice and hot break juice

as controls. Samples were subjected to various thermal, combined pressure-heat

67

treatments. Impact of these processes on tomato carotenoids was evaluated. Further

microscopic analysis was carried out to evaluate microstructure changes.

3.3.1 Juice preparation

Tomato juice was extracted at ambient conditions (22oC) on the day of purchase

using a lab scale juicer (Juiceman Jr.), immediately filled in polypropylene pouches (5cm

X 3 cm) (3 mil Deli, Thomson Equipment and Supply, Cincinnati, OH) and carefully heat

sealed using a hand impulse heat sealer (American International Electric, Whittier, CA)

after manually removing any trapped air bubbles. In order to minimize undesirable

effects of active enzymes, the pouch containing juice were immediately immersed in an

ice bath, stored in a refrigerator (4oC) and processed within 6 hours.

To prepare hot break juice, the raw juice was rapidly heated to 93.3oC in a hot pan

with constant stirring, held for 60 sec to inactivate the enzymes, and rapidly cooled to

21±0.6oC. Subsequently about 4 g of hot break juice was packaged in polypropylene

pouches. The packaged juice samples were stored in a refrigerator at 4oC and subjected to

various pressure-heat treatments within one day of extraction. The initial pH and %TSS

of the fresh tomato juice were 4.45 and 5.1, respectively. Hot break juice also had pH and

%TSS values of 4.44 and 5.3, respectively (Table 3.1).

3.3.2 High-pressure kinetic tester

Packaged tomato juice was treated in high-pressure kinetic tester (pressure test

unit PT-1, Avure Technology Inc., Kent, WA) (Ratphitagsanti et al., 2009). A 54-ml

stainless steel (SS-316) pressure chamber was immersed in a temperature-controlled bath

to maintain the desired process conditions (30oC for HPP and 100.5

oC for PATP).

68

Propylene glycol (57-55-6, Avatar Corporation, University Park, IL) was used as the

pressure transmitting medium as well as heating medium in the temperature controlled

bath. The desired pressure was generated at the rate of 18.42 MPa/s using an intensifier

(M-340 A, Flow International, Kent, WA) connected to a hydraulic pump (model

PO45/45-OGPM-120, Interface Devices, Milford, CT). The depressurization rate was

approximately 2 s. The unit was used for both high pressure processing and pressure-

assisted thermal processing experiments as outlined below.

3.3.3 High pressure processing

The tomato juice samples were pressure treated at 500, 600 and 700 MPa for 0, 3,

5 and 10 min at 30oC. Prior to pressure treatment, the juice samples were chilled in ice–

water mixture for 10 min. The chilled samples were placed inside a 10-ml polypropylene

syringe (model 309604, Difo, Becton Dickinson), which served as the sample holder. The

sample holder was also filled with ~6 ml of chilled water to ensure that immediate

vicinity of the sample pouch had similar temperature and heat of compression

characteristics as that of the tomato juice. To minimize heat exchange with the

surrounding glycol, the sample holder was wrapped with two layers of insulating material

(Sports Tape, CVS pharmacy Inc., Woonsocket, RI) (Nguyen et al., 2007). The initial

temperatures of the samples were determined by using the following formula (Nguyen et

al., 2007) and verified by performing preliminary experiments.

𝑇3′ = 𝑇3 +( (𝐶𝐻𝑚 ∗𝑀𝑓)𝑖

𝑀 (

∆𝑃

100) + ∆𝑇H ………………………………………… (1)

69

T3

’ is the target temperature, T3 is the initial sample temperature, CH is the heat of

compression value of the sample (defined as temperature increase per 100 MPa during

sample pressurization), and ΔP is the process pressure. Owing to its high moisture

content, CH value of tomato juice was assumed to follow that of water (Rasanayagam et

al., 2003). ΔTH is the temperature gain by the test sample from the surrounding glycol

bath during pressure process time and early stages of pressure holding time.

The pressurization started when the sample temperature reached the

predetermined value T3. Sample temperature history at various stages of combined

pressure-temperature treatment is given in Table 3.2. After processing, the samples were

immediately withdrawn and stored at 4oC until analyzed.

3.3.4 Pressure assisted thermal processing

PATP experiments were carried at 500, 600, 700 MPa at 100oC for 0, 3, 5 and 10

min. Before PATP, the samples and the sample holder 10-ml polypropylene syringe were

preheated (Table 3.2) in a hot water bath (Isotemp 128, Fisher Scientific, Pittsburgh, PA)

for a period of 5 min. The water bath was maintained at respective predetermined

temperatures for each of the pressures. The preheated syringe containing samples were

then transferred to the pressure chamber. Temperature of the pressure chamber was

maintained at the desired process temperature. The pressurization started when the

sample temperature reached the predetermined value T3 (Table 3.2). More details of the

experimental technique are described in sections above and elsewhere (Nguyen et al.,

2007). After processing, the samples were immediately cooled in ice–water mixture and

70

subsequently stored at 4oC until analyzed. Details of pressure-thermal history during

various combined pressure-heat treatment are summarized in Table 3.2.

3.3.5 Thermal processing

Boiling water in a steam jacketed kettle was used for thermal processing (100oC

for 0, 3, 5 and 10 min) of tomato juice. 4 g Juice vacuum packaged in (5cm X 3 cm)

pouches, immersed in the boiling water and held for 10 min. Subsequently the samples

were immediately cooled in ice–water mixture and refrigerated at 4oC until analyzed.

Details of temperature history during thermal process are given in Table 3.2.

3.3.6 Analysis

3.3.6.1 Carotenoid extraction and high performance liquid chromatography (HPLC)

Tomato carotenoids were extracted using a method developed by Ferruzzi et al.

(1998). Briefly, 4 g tomato juice was mixed with 4 g celite and 1 g calcium carbonate. 50

ml methanol was added and the mixture was homogenized at 10000 RPM for 1 min.

Carotenoids were extracted three times with 25 ml HPLC grade hexane/acetone (1:1 v/v)

(Fisher Scientific, USA). The combined hexane layer was collected quantitatively after

filtering through anhydrous sodium sulfate and the volume was made to 100 ml using

HPLC grade hexane. 2ml aliquots were dried under nitrogen, reconstituted in methyl tert-

butyl ether (MTBE)/ methanol (1:1 v/v) (HPLC grade, Fisher scientific, USA), filtered

through 0.2 µm 13 mm nylon syringe filter and 50 µl was injected for HPLC (Agilent

technologies, Model HP 1050) equipped with Waters 996 Photodiodearray (PDA)

detector. The mixture was separated on a Waters YMC C30 HPLC column (4.5 mm x150

mm, 5 μm particle size). Separations were achieved using gradient elution with different

71

concentrations of methanol: MTBE: 2% aq. ammonium acetate in reservoirs A (88:5:7)

and B (20:78:2). The following gradient was used: at 0 min 0% B, linear gradient to 85%

B over 20 min, followed by a linear gradient to 100% B over 10 min, returning to 0% B

and holding for 5 min.

Isomerization of the lycopene standard was performed in hexane by adding iodine

catalyst at a concentration of about 1% (w/w) of the lycopene weight and allowing the

mixture to sit for 15 min in fluorescent light of luminance 320 lux (lumens/m2). HPLC

analysis was then performed on the isomerized sample. Lycopene isomers in tomato juice

were identified by comparing chromatograms of tomato juice with the chromatograms of

isomerized lycopene standard (Gupta et al., 2010).

To quantify lycopene in tomato juice samples, a calibration curve was generated

using authentic all-trans lycopene standard. Spectra were extracted at lycopene

absorption maximum of 471nm. Levels of cis lycopene isomers are given in all-trans

lycopene equivalents.

β-carotene was identified by its characteristic spectra at an absorption maximum

of 450 nm. Levels of lycopene in processed samples are based on the initial amount

present in the control fresh juice and reported as percent retained.

3.3.6.2 Transmission electron microscopy (TEM)

Selected samples of untreated and processed tomato juice were examined by

transmission electron microscopy using the published procedure by Nguyen et al. (2001).

Briefly, tomato juice samples were lightly centrifuged in 2ml vials at 2000 RPM (24 X

3,75 g) using a eppendorf centrifuge (model 5424, Hauppauge, NY). The tomato juice

72

cells were then re-suspended for 30 min in a fixative consisting of 2.5% glutaraldehyde in

0.1 M phosphate buffer at pH 7.4 and centrifuged. After rinsing 3 times with 0.1 M

phosphate buffer containing 0.1 M sucrose buffer, the cells were re-suspended in a small

amount of warm 2% agarose, centrifuged and chilled in ice–water mixture for 10 min. to

set the agarose. The cloudy part containing cells was cut in 1 mm size blocks and fixed in

1% osmium tetroxide in 0.1 M phosphate buffer for 1 hour. The fixed samples were then

dehydrated using 10 min transfers through a graded ethanol series (50%, 70%, 80%,

95%, 100%, 100%) followed by propylene oxide. After embedding the samples in epon

resin and polymerizing overnight at 60oC, the samples were sliced and resulting sections

were transferred to carbon reinforced grids. The sections were examined on a FEI tecnai

spirit transmission electron microscope (FEI, Hillsboro, Oregon) at the campus

microscopy and imaging facility, the Ohio State University.

3.3.6.3 Light microscopy

Processed and unprocessed tomato juice samples were examined using Zeiss

Axioskop Widefield LM (Carl Zeiss Microimaging GmbH, Goettengen, Germany) at

100X with oil immersion objectives.

3.3.6.4 Bioaccessibility studies

The bioaccessibility studies were performed by adapting the method reported by

Garrett et al. (1999). Briefly, 5 grams of tomato juice was mixed with 3% olive oil and

incubated in a 37oC shaking water bath at 85 rpm for 10 min. The pH was adjusted to

2.0±0.1 using 1.0 M HCl and after adding 2 ml porcine pepsin (20 mg/ml in 0.1 M HCl),

the volume was made up to 40 ml using 120 mM NaCl. The mixture was blanketed with

73

nitrogen and sealed with paraffin. It was then incubated in a 37oC shaking water bath at

85 rpm for 60 min. After incubation the pepsin reaction was immediately stopped by

placing the reaction tubes in crushed ice. The pH was adjusted to 6.0 ±0.1 using 1 M

sodium bicarbonate followed by addition of 2ml pancreatin/lipase solution (10mg/ml

pancreatin + 5 mg/ml lipase in 100mM sodium bicarbonate) and 3ml bile solution (40

mg/ml bile extract solution in 100 mM sodium bicarbonate). The pH was then adjusted to

6.5±0.1 using 1 M NaOH and the volume was made up to 50 mL using 120 mM saline

solution. The mixture was blanketed with nitrogen, capped and sealed with paraffin. It

was then incubated in a 37oC shaking water bath at 85 rpm for 120 min. The enzymatic

reactions were ceased by placing the tubes in crushed ice. The solution was mixed well

and was centrifuged at 5000xg for 35 min at 4oC. The supernatant (aqueous phase

containing micelles) was then filtered through 0.22µm syringe filter (25mm diameter,

Fisher Scientific, Pittsburgh, PA) to remove microcrystalline non-micellarized

carotenoids that were not removed as pellets during centrifugation (Garrett et al., 2000).

Aliquots (1 ml) of the filtrate containing micelles were combined with 1 ml ethanol and

extracted three times with 2 mL of 1:1 acetone/hexane. 1ml distilled water was added to

the pooled extracts and the samples were re-extracted into 2mL hexane thrice. The pooled

hexane extract was evaporated under a stream of nitrogen. The dried samples were

reconstituted in 1 ml 1:1 MTBE/methanol and analyzed by high performance liquid

chromatography (Agilent Technologies, Model HP 1050) equipped with Waters 996

Photodiodearray (PDA) detector. The mixture was separated on a Waters 4.5mm x150

mm C-30 reverse phase HPLC column. Lycopene was quantified using a standard

lycopene curve generated using lycopene standard obtained from Chromadex inc.

74

3.3.6.5 Total soluble solids (0Brix) and pH

Total soluble solids (% TSS) were measured using Atago Digital Handheld

Pocket Refractometer (Cole-Parmer Instrument Company, Vernon Hills, Illinois). The pH

of raw tomatoes and the juice was measured using a portable handheld pH meter (Model

PHH-81A, Omega Engineering, Stamford, CT).

3.3.6.6 Statistical data analysis

Data was analyzed with Minitab software, version 14.1 (Minitab, State College,

PA). Data is expressed as mean±SD of three replicates for post processing lycopene

stability and five replicates in case of bioaccessibility studies. Pairwise comparisons for

the means of factors were evaluated with Tukey’s test at 5% significance level (P<0.05).

3.4 Results and discussion

Juice extraction and its effect on carotenoids (lycopene and β-carotene)

The total lycopene content (all-trans + cis isomer) of raw and hot break tomato

juices did not differ significantly. However, the lycopene cis isomer content of hot break

juice was significantly (approx. 25%) greater than that of the raw juice. Contrary to this,

hot break process degraded the all-trans β carotene content of the juice by 15% (of that

present in the raw juice). Selected attributes of fresh and hot break juice are given in

Table 3.1.

Impact of pressure-thermal effect on lycopene degradation

The stability of tomato lycopene as influenced by processing (HPP, PATP and

TP) and juice preparation (untreated raw and hot break) are presented in Figures 3.2 and

75

3.3. Raw juice had 6.86 mg/100g all-trans lycopene and 0.44 mg/100g cis lycopene.

Pressure treatment (500 MPa 30oC) for 0 min yielded approximately 13% lesser all-trans

lycopene isomer and approx. 29% less cis isomers as compared to the control (Figure

3.2a). However, prolonged pressure holding time at 500 MPa or elevated pressures did

not cause any additional degradation. Pressure treatment at 600 MPa, 30oC resulted in

similar all-trans lycopene values as that of control, however, the cis isomer content was

significantly lower than that present in the control (P<0.05). 700 MPa pressure treatment

at 30oC resulted in increased all-trans lycopene extractability (up to 12%) than the

control. These observations were consistent with the findings of earlier researchers

(Krebbers et al., 2003; Sanchez-Moreno et al., 2006; Hsu et al., 2008; Gupta et al., 2010).

The reason(s) for decrease in lycopene content at 500 MPa are unclear.

Studies in the literature have previously reported anomalous behavior in lycopene

extractability from tomato products exposed to certain pressure conditions (Krebbers et

al., 2003). Pressure is known to cause a decrease in activation volume and reactions that

are favored by increase in pressure and decrease in volume proceed faster under high

pressure conditions. Pectinases (Krebbers et al., 2003) and lipoxygenases (Peeters et al.,

2004) are naturally present enzymes in tomatoes that are difficult to inactivate under high

pressures and near ambient temperatures (Shook et al., 2001; Krebbers et al., 2003;

Peeters et al., 2004). Peeters et al. (2004) discovered that approximately 25%

lipoxygenase activity is retained in tomato juice treated at 500 MPa for 5 min at 20oC. On

the contrary, Krebbers et al. (2003) reported a 5.5 to 6.5 fold increase in tomato

pectinmethylesterase activity at ambient process temperatures regardless of the pressures

used (<700 MPa). Up to 36% residual polygalacturonase activity was reported under

76

same treatment conditions. The increased interaction of one or a combination of these

enzymes with their respective substrates due to decrease in activation volume (under

pressure) coupled with changes in their conformation and activity might explain some of

the degradative reactions observed at lower pressures such as 500 MPa and ambient

process temperatures. Past studies on the enzymatic and oxidative metabolites of

lycopene show that addition of lipoxygenase to lycopene solutions significantly increases

the production of lycopene metabolites (Ferreira et al., 2004). Likewise, Biacs et al.

(2000) studied the co-oxidation of carotenoids from tomato in an experimental system

containing lipoxygenase and reported approximately 25% degradation of lycopene during

a 15 min holding time. In addition, several studies have discovered the presence of

carotenoid cleavage enzymes in plants (Schwartz et al., 2001; Fleischmann et al., 2002;

Fleischmann et al., 2003) and animals (Fleischmann et al., 2002). Further studies

correlating enzyme activity to lycopene degradation and decrease in activation volume

under elevated pressure conditions are necessary to understand this phenomenon.

PATP of raw juice at 500, 600, 700 MPa and 100oC for different holding times up

to 10 min did not change the all-trans lycopene extractability from the juice (Figure

3.2b). Both PATP and thermally (0.1 MPa, 100oC for up to 10 min) processed juice

samples showed all-trans lycopene content similar to that of the raw juice and a small

increase in the cis isomers of lycopene (up to 5%) as compared to the raw juice (P<0.05).

Preheating fresh juice at 65oC for 5 min resulted in up to 8% decrease in lycopene (Figure

2b). However, this decrease did not transfer to the samples analyzed for lycopene content

after PATP, possibly due to the increased extractability of lycopene from tomato juice

after PATP.

77

Combined pressure-temperature treatment of hot break juice resulted in a minor

(up to 10%) increase in the all-trans lycopene extractability as compared to the hot break

and raw juice controls. Although small, changes in cis isomer content of processed

samples were statistically significant (P<0.05). PATP and thermally treated samples

showed marginally higher cis lycopene isomers compared to hot break juice and HPP

processed samples (Figure 3.3).

All-trans lycopene in tomato products is fairly stable during traditional thermal

processing (Nguyen and Schwartz, 1998; Lin and Chen, 2005; Nguyen and Schwartz,

1999). Furthermore, cis lycopene has been reported to be fairly stable to pressure

treatments (Qiu et al., 2006). It has been proposed that isomerization of carotenoids is a

structurally and thermodynamically specific phenomenon and thus the degradation and/or

isomerization of all-trans lycopene is governed by its structural specificity. Also, the

differences in shape of the carotenoid molecule influence its crystalline state,

hydrophobicity, solubility and other such properties which individually or in combination

might affect its stability (Nguyen et al., 2001). Lycopene, being a linear molecule, forms

multilayers or aggregates (Ray and Mishra, 1997) and the aggregated form lycopene

molecules might be able to resist further structural changes (Nguyen et al., 2001). Since

pressure is known to decrease the activation volume and compress food components, it is

possible that pressure favors the formation of compact lycopene aggregates. Cis forms of

lycopene being bent structures, formation of all-trans lycopene aggregates might prevent

its isomerization during combined pressure-heat treatments.

78

β-carotene: Raw and hot break juice samples subjected to pressure treatment at 30oC

retained 75-93% of all-trans β-carotene (Figure 3.4). PATP or TP treatment of raw juice

at 100oC better retained all-trans β-carotene than pressure treatment at 30

oC (Figure

3.4a).

PATP and thermal treatments showed similar all-trans β-carotene retention.

Under PATP and thermal conditions a typical dependency of all-trans β-carotene

retention on time was observed with higher processing times yielding lower values

(Figure 3.4b).

Among studies reported in the literature, the fate of β-carotene in processed

tomato juice is not in good agreement. Conversion of all-trans to cis form has been

reported in tomato products subjected to thermal processing at 100oC for 30 min (Nguyen

et al., 2001, Lin and Chen, 2005). However, the magnitude of these changes is quite

different between the two studies. On the contrary, no changes in the β-carotene

extractability were observed after thermal (0.1 MPa, 95oC, 60 min) and high pressure

processing (600 MPa, 20oC, 60 min) of tomato homogenate (Fernandez-Garcia et al.

2001). Based on differences in carotenoid extractability with different extraction solvents

(tetrahydrofuran vs. petroleum ether), changes in the microstructure caused by different

processing methods were suggested (Fernandez-Garcia et al. 2001). It has also been

proposed that all-trans β-carotene crystal aggregates in the Languir-Blodgett film, may

not be able to easily assemble into an ordered structure and stabilize, thus showing its

susceptibility to isomerization (Ray and Misra, 1997). In another study, a 35% increase in

all-trans β-carotene extractability was reported after 400 MPa pressure treatment at 25oC

for 15 min. and a significant increase (9%) in all-trans β-carotene extractability after

79

thermal processing at 90oC for 1 min followed by immediate freezing ( Sanchez-Moreno

et al., 2006). However, the effect of freezing on the increase in β-carotene extractability is

not clear. The results from the present study show that stability of β-carotene is

influenced by type of juice (raw vs. hot break), processing method (HPP, PATP and TP)

and the holding time under these processing conditions. Differences among different

studies could be attributed to variations in the tomato cultivars utilized, method of juice

preparation, extraction and processing (Nguyen and Schwartz, 2000)

In-vitro micellarization of carotenoids

In-vitro model to assess bioaccessibility of carotenoids involves studying the

micellarization of carotenoids from the food matrix (raw and hot break tomato juice in

this study). This approach has been shown to be a cost-effective model for screening the

bioavailability of carotenoids from foods (Garrett et al., 1999). Table 3 compares the

respective amounts of β-carotene and lycopene transferred from the tomato juice into the

digesta and subsequently into the micelles during a simulated digestion process.

Up to 16% of all-trans β-carotene got transferred from the tomato juice into the

digesta and up to 12% of that present in the juice got micellarized. In-vitro digestion of

the hot break juice showed significantly higher percentage of β-carotene in digesta and

micelles as compared to that present in the digesta and micelles of raw juice (P<0.05).

As compared to all-trans β-carotene, significantly lower (P<0.05) quantity of lycopene

got transferred into the digesta (up to 6% of that present in the tomato juice) and lesser

amount in the micelles (0.4% of that present in the juice). Also, unlike all-trans β-

carotene above, no significant difference was found between the quantity of lycopene in

80

the digesta of hot break and raw juice. Same was true for micellarized lycopene. The

results obtained are very similar to that reported by Garrett et al. (1999) who found

16±0.5% carotenes and 5.1±0.2% lycopene in the digesta respectively. Also, the amount

of lycopene in micellar form was less than 0.5% of that originally present in the meal

sample used for their study.

Effect of processing conditions on bioaccessibility of all-trans β-carotene

Significant differences were observed in the amount of all-trans β-carotene

present in the digesta prepared from raw juice processed using different techniques (TP,

PATP, and HPP). Digesta from HPP and PATP (700 MPa for 5 min at 30 and 100oC,

respectively) samples had significantly higher (P<0.05) all-trans β-carotene than the

digesta from raw juice and TP juice (0.1 MPa, 100oC for 5 min) (Table 3.3).

However, no significant difference was observed in the amount of micellarized

all-trans β-carotene in HPP, PATP and thermally processed samples. Micelles from

processed samples (HPP, PATP and thermal) had significantly higher (P<0.05) all-trans

β-carotene as compared to the raw juice (Table 3.3).

Similarly, when hot break juice was processed, no significant difference was

found in the amount of all-trans β-carotene present in the micelles obtained by in-vitro

digestion of HPP, PATP and thermally treated samples. However, the amount of all-trans

β-carotene present in micelles obtained from these samples was significantly greater than

that present in the micelles of processed raw juice (Table 3.3). Also, the %

micellarization of β-carotene ranged between 70% – 100% of that present in the digesta

81

with maximum % micellarization observed in raw juice thermally treated at 0.1 MPa,

100oC for 5 min.

Effect of processing conditions on bioaccessibility of all-trans lycopene

Lycopene content in the digesta from raw juice (control) and that processed using

HPP and thermal treatments did not show a significant difference. However, the lycopene

content in the digesta from PATP processed samples was significantly higher than the

other samples (Table 3.3). This increase however did not reflect in the amount of

micellarized lycopene, which was below 0.5% in the digesta of all samples. Likewise, the

micellarized lycopene in the digesta of hot break juice and its HPP, PATP and thermal

variants was not much different and was less than 0.5% of that present in the original

juice.

The percent micellarization of lycopene mentioned in the literature varies

depending upon the type of product, processing conditions and method of assessment.

However, one common aspect of all research on lycopene bioaccessibility is its limited

micellarization (Aherne et al., 2009; Colle et al., 2010; Reboul et al., 2006; Huo et al.,

2007; Goni et al., 2006; Garrett et al., 1999). High pressure homogenization of tomato

juice can negatively impact the bioaccessibility of lycopene from tomato juice. This

negative impact on lycopene bioaccessibility was found to increase with an increase in

the homogenization pressure (Colle et al., 2010). Also, high pressure homogenization

followed by thermal processing at 90oC for 30 min negatively affected the in-vitro

micellarization of lycopene. Based on these results the authors hypothesized that fibrous

network formed during homogenization made lycopene less accessible to digestive

82

enzymes and bile salts. Subsequent thermal processing was not sufficient to break the

network and could not make lycopene more bioaccessible (Colle et al., 2010). It was

further reported that bioaccessibility of carotenoids was dependent on three prime factors

viz. type of carotenoid, food matrix and method of processing (Reboul et al., 2006). A

good correlation was found between the in-vitro bioaccessibility data, human-derived

bioaccessibility data and mean bioavailability data reported in studies involving healthy

humans. In-vitro digestibility studies on raw and processed tomatoes showed that

micellarization of lycopene increased from 0.1% (raw tomatoes) to 1.60% (processed

tomatoes) and that of β-carotene increased from <0.1% (raw tomatoes) to 5.97%

(processed tomatoes) (Reboul et al., 2006). The chain length of the fatty acids (added to

the test meal (salad puree) during in-vitro digestion) also influenced the micellarization of

carotenoids. It was found that regardless of their degree of unsaturation, increasing the

chain length of fatty acids (added to the salad puree during in-vitro digestion) increased

the in-vitro bioaccessibility of carotenoids. Up to 5.6% micellarization of lycopene was

reported with the addition of 2.5% c18:2 triglycerides during in-vitro digestion (Huo et

al., 2007). In addition to variations during processing and assessment, the geographical

origin of a cultivar can affect the bioaccessibility of its carotenoids. Aherne et al. (2009)

recently reported a greater impact of geographical location on the carotenoid content of

tomatoes and their bioaccessibility than the differences in the variety. 4 different Irish

tomato varieties with their corresponding Spanish counterparts were studied for

micellarization of β-carotene, lutein and lycopene. The micellarization of β-carotene in

Spanish varieties was distinctly higher than the corresponding Irish varieties and up to

5% micellarization was noted. However, a mixed effect of tomato variety and

83

geographical location was reported on lycopene micellarization, which ranged between

0.2-0.8% in Irish varieties and between 0.1-1.3% in Spanish varieties. No specific

correlation was found between the amounts of lycopene and β-carotene originally present

in the tomatoes and their amounts present in the micelles. Attempts to significantly

increase the micellarization of carotenoids (than what is typically observed in processed

samples) have shown that extreme processing conditions (stir frying tomato paste with

fresh vegetables and oil at 177oC, 4 min) are capable of increasing lycopene

bioaccessibility (Garrett et al., 1999). On the contrary, milder processing conditions, like

those observed in emerging technologies such as combined pressure-temperature

processing have a distinct advantage of improving the product quality and preserving

sensitive nutrients (Verlent et al., 2006; Krebbers et al., 2003; Gupta et al., 2010, Hsu et

al., 2008; Garcia et al., 2001). It has been previously reported that combined P-T

processing of tomato products results in excellent consistency (Verlent et al., 2006),

viscosity, improved water binding capacity (Krebbers et al., 2003) and better color

retention (Gupta et al., 2010; Krebbers et al., 2003) as compared to the thermally

processed samples. Likewise, the vitamin C content and antioxidant activity of combined

P-T processed products is very well retained and is close to the fresh control (Fernandez-

Garcia et al., 2001; Sanchez-Moreno et al., 2009). Combined P-T processing has been

found to produce a shelf stable tomato juice with a holding time of 10 min at pressures

above 600 MPa and temperature ≥ 45oC (Gupta et al., 2010). However, holding times

commercially used to achieve shelf stability of tomato juice are significantly more (up to

35 min at 100oC; Barringer et al., 2004) than that used in this study (10 min at 100

oC).

Longer processing time would obviously lead to adverse effects on nutrients and quality.

84

Microscopic changes in tomato juice due to combined pressure-heat processing

Both raw and hot break tomato juice processed using combined pressure-

temperature treatments were observed microscopically for changes in the microstructure

(Figure 3.5). Freshly extracted raw juice (Figure 3.5) showed distinct cellular

components including chromoplasts dispersed in the cytoplasm. HPP (700 MPa, 30oC, 10

min) treated juice showed closer resemblance to the control untreated juice. A denser

matrix with less resolution between cell components was observed. On the other hand,

thermal processing (0.1 MPa, 100oC, 10 min) showed a continuous matrix with little or

no resemblance to the control juice and indistinguishable cellular components. PATP

(700 MPa, 100oC, 10 min) showed the characteristics of both HPP and thermally

processed juices. Differences between the juice matrix of all 4 types of juices is evident.

Unprocessed hot break juice showed a more continuous network with no

separation of phases (data not shown). HPP and PATP processed hot break juice sample

shows a matrix different than that observed in unprocessed hot break juice. However,

thermally processed sample shows little or no matrix with indistinguishable cellular

components.

Carotenoid biosynthesis and development of carotenoid bearing structures begin

during ripening of tomato tissue and maturation of chromoplasts (Ljubesic et al., 1991).

A rapid increase in lycopene and its subsequent accumulation results in crystallization

(Bathgate et al., 1985) and these crystals remain enveloped by a membrane in the

chromoplast. Likewise, β-carotene crystals too are enveloped by a membrane. However,

the membrane that envelopes β-carotene crystals has been suggested to be different than

85

the one that envelopes lycopene (Rosso et al., 1968). Although processing conditions are

sufficiently rigorous in disrupting cell walls and organelles, from the electron

micrographs (Figure 3.6) it can be seen that lycopene and β- carotene remain enveloped

even after combined pressure-temperature processing. Similar findings with thermal

processing only have been reported by Nguyen et al. (2001). Whereas β-carotene is

associated with plastoglobulin-type structures and get dissolved in the lipid material of

the globules, lycopene is present as crystal deposits and associated with the thylakaloid

membrane (Rosso et al., 1968; Ben-Shaul et al., 1969; Mohr et al., 1979).

It can be seen from Figure 3.6 that changes in the microstructure due to different

processing conditions did not necessarily affect the crystals of lycopene. The dispersion

of lycopene crystals in the matrix is evident regardless of the processing method used,

although it is worth nothing the prominently strong contrast of lycopene crystals in HPP

and PATP processed samples as against thermally processed samples. Increased lycopene

extractability experienced in case of HPP and PATP of tomato juice could be correlated

to the differences in tomato juice matrix observed in microscopic images (Figure 3.6).

However, the resistance of lycopene from processed samples to micellarization can be

attributed to the subsistence of its native enveloped crystalline form even after processing

(Figure 3.6).

3.5 Conclusions

This study suggests that the type of juice (raw juice vs. hot break juice) has a

significant impact on the stability of carotenoids in the processed juice. Combined

pressure-temperature processing (HPP, 700 MPa, 30oC; PATP, 500-700 MPa, 100

oC)

86

increased the extractability of lycopene from the tomato juice (both raw and hot break)

whereas thermal processing (TP, 0.1 MPa, 100oC) had negligible effect on its stability.

On the other hand, β- carotene degradation was dependent on the processing temperature,

processing time, processing pressure and type of juice (raw vs. hot break). In general,

increasing the holding time during PATP (500-700 MPa, 100oC) and TP (0.1 MPa,

100oC) had an adverse effect on β- carotene content of the juice. Bioaccessibility of

lycopene was limited regardless of the processing method used and apparently the

treatments were not severe enough to solubilize the lycopene crystals and facilitate its

micellarization. However, β- carotene showed better micellarization and processing

(HPP, PATP and TP) further improved its micellarization. Based on the results of this

study and studies on quality of tomato juice reported in the literature, combined pressure-

temperature processing poses a promising alternative for producing good quality tomato

products.

Acknowledgements

Support for this research was provided by USDA-CSREES-NRICGP grant 2006-

35503-17571, by the Ohio Agricultural Research and Development Center (OARDC),

and by The Ohio State University. References to commercial products or trade names are

made with the understanding that no endorsement or discrimination by The Ohio State

University is implied.

87

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Inc.

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carotenoids in different tomato varieties. J. Sci. Food Agric., 81 (9), 910-917.

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93

Table 3.1 Selected attributes of fresh raw juice and hot break juice obtained from Roma

tomatoes.

oBrix

(%TSSb)

pH Lycopene contenta

mg/100g juice

Total

lycopenea

mg/100g

juice

All-trans β

carotenea

(%of fresh

juice)

All-trans

mg/100g

Cis

mg/100g

Fresh

Juice

5.1 4.45 6.86±0.26 0.44±0.01 7.3±0.27 100±1.31

Hot break

juice

5.3 4.44 6.54±0.420 0.55±0.02 7.09±0.44 81.94±2.46

a Values are mean±SD of three replicates. b Percent total soluble solids present in the

juice.

94

Table 3.2 Temperature histories at different stages of high pressure processing (HPP; 500, 600 and 700 MPa at 30oC) pressure-

assisted thermal processing (PATP; 500, 600 and 700 MPa at 100oC) and thermal processing (TP; 0.1MPa at 100

oC) of tomato juice

samples.

Treatment

Processing

Pressure

(MPa)

Processing

time

(s)

Temperature at different stages during processing (oC)

Time required at different

stages of preprocessing (s)

Preprocess

(T1)

Immediately

before

pressurization

(T2)

Immediately

after

pressurization

(T3)

holding

(T3-T4)

Depressurization

(T5)

Preprocess

(t1)

come up

time

(t2)

HPP

30oC

500 0 2.0±1 13.9±0.5 28.9±1.2 30.0±0.6 17.1±0.8 329±5 23±1

600 2.0±1 13.9±0.5 28.9±1.2 30.0±0.6 17.1±0.8 329±5 23±1

600 0 2.0±1 10.1±0.6 28.2±1.1 30.3±0.4 15.2±0.6 329±5 30±1

600 2.0±1 10.3±0.6 28.2±1.1 30.3±0.4 15.2±0.6 329±5 30±1

700 0 2.0±1 7.3±0.5 29.3±0.9 30.6±0.6 13.9±0.5 329±5 35±2

600 2.0±1 7.3±0.5 29.3±0.9 30.6±0.6 13.9±0.5 329±5 35±2

PATP

100oC

500 0 72.0±0.4 78.0±0.5 99.6±0.7 100.4±0.7 79.6±0.9 329±5 23±1

600 72.0±0.4 78.0±0.5 99.6±0.7 100.4±0.7 79.6±0.9 329±5 23±1

600 0 68.0±0.5 72.0±0.6 100.4±0.5 100.1±0.6 74.9±0.8 329±5 30±1

600 68.0±0.5 72.0±0.6 100.4±0.5 100.1±0.6 74.9±0.8 329±5 30±1

700 0 65.0±0.4 68.8±0.8 99.2±0.8 100.3±0.4 73.1±1.5 329±5 35±2

600 65.0±0.4 68.8±0.8 99.2±0.8 100.3±0.4 73.1±1.5 329±5 35±2

TP

100oC

0.1 0 21.7±0.3 --- --- 100.0±0.1 --- --- 73±3

0.1 600 21.7±0.3 --- --- 99.9±0.2 --- --- 73±3

94

95

Table 3.3 Percent lycopene and β-carotene from tomato juice transferred to the digesta and

micelles during in-vitro bio-accessibility studies.

Treatment

Concentration µg/100 g juice

Lycopene β-carotene

Digesta

Micelles

Digesta

Micelles

Raw juice

Control1

321.5±38.1 25.4±1.8 35.0±2.6 25.2±1.3

HPP2

307.4±24.5 27.3±2.2 42.2±2.1 30.0±2.1

Preheat3

257.9±10.7 22.8±1.3 29.2±2.5 25.6±2.2

PATP4

442.6±51.9 27.2±1.6 45.4±1.6 31.0±2.9

TP5

274.1±16.3 26.7±1.2 34.3±1.1 33.9±1.4

Hot break juice

Control1

370.8±6.1 21.4±0.1 44.4±2.4 35.9±0.9

HPP2

300.9±41.9 24.3±0.3 45.4±0.3 37.5±0.4

Preheat3

348.2±17.7 24.9±0.8 42.9±0.7 33.5±0.4

PATP4

386.4±15.0 23.8±0.2 48.7±0.4 36.1±0.3

TP5

366.2±3.3 26.2±0.2 46.3±0.3 37.9±0.6

Values are represented as mean ± SD of 5 replicates.

1 Respective unprocessed raw juice and hot break (~93

oC, 60 sec) juice

2 High pressure processed (HPP) juice (700 MPa, 30

oC, 5 min)

3 Preheated for PATP processing (0.1 MPa, 65

oC, 5 min)

4Pressure assisted thermally processed (PATP) juice (700 MPa, 100

oC, 5 min)

5Thermally processed (TP) juice (0.1MPa, 100

oC, 5 min)

96

Figure 3.1 Flowchart outlining the steps involved in the experiment.

Lycopene extraction

followed by High

Performance Liquid

Chromatography

Light Microscopy

Electron microscopy

Bioaccessibility

studies

Data analysis

High pressure

processing

(500, 600 and 700

MPa), 30oC for

0,3,5 and 10 min.

Pressure assisted

thermal processing

(500, 600 and 700

MPa), 100oC for

0,3,5 and 10 min.

Thermal processing

(0.1 MPa, 100 oC for

0,3,5 and 10 min)

Fresh, ripe, Roma

tomatoes

Raw Juice Hot break juice

(93.3oC 60 sec)

97

Figure 3.2 Lycopene retention (▒) all-trans lycopene, (▓) cis lycopene) in fresh raw

tomato juice subjected to (a) high pressure processing (500-700 MPa, 30oC for 0, 3, 5 and

10 min) (b) pressure assisted thermal processing (500-700 MPa, 100oC for 0, 3, 5 and 10

min) and thermal processing (0.1 MPa, 100oC for 0, 3, 5 and 10 min). Values are mean ±

SD of 3 replicates.

a

Continued

98

Different letters above the bars indicate significant differences in the total lycopene

content (P<0.05).

b

Figure 3.2 continued

99

Figure 3.3 Lycopene retention (▒) all-trans lycopene, (▓) cis lycopene) in hot break

tomato juice subjected to combined pressure-temperature processing; HPP (500-700

MPa, 30oC for 0, 3, 5 and 10 min), PATP (500-700 MPa, 100

oC for 0, 3, 5 and 10 min)

and TP (0.1 MPa, 100oC for 0, 3, 5 and 10 min). Values are represented as mean ± SD of

3 replicates. Different letters above the bars indicate significant differences in the total

lycopene content (P<0.05).

100

Figure 3.4 Percent all-trans β-carotene retention in raw (a) and hot break (b) tomato juice

after combined pressure-heat processing; HPP (500-700 MPa, 30oC for 0, 3, 5 and 10

min), PATP (500-700 MPa, 100oC for 0, 3, 5 and 10 min) and TP (0.1 MPa, 100

oC for 0,

3, 5 and 10 min). Values are represented as mean ± SD of 3 replicates. Different letters

above the bars indicate significant differences in β-carotene levels (P<0.05).

a

Continued

101

b


g

f h

102

Figure 3.5 Representative light microscopic images (using 100 X oil immersion

objectives) of raw tomato juice samples processed using combined pressure-temperature

treatments; HPP (500-700 MPa, 30oC for 0, 3, 5 and 10 min), PATP (500-700 MPa,

100oC for 0, 3, 5 and 10 min) and TP (0.1 MPa, 100

oC for 0, 3, 5 and 10 min).

Chromoplast Distinct organelles freely

suspended in the juice matrix

Distinct organelles in matrix closely

resembling the raw juice

Disrupted dense matrix with hard to

distinguish organelles

Partially disrupted matrix with some

distinct organelles Distinct organelles in matrix closely

resembling the raw juice

103

Figure 3.6 Representative electron microscopic images of hot break tomato juice samples processed using

combined pressure-temperature treatments; HPP (500-700 MPa, 30oC for 0, 3, 5 and 10 min), PATP (500-

700 MPa, 100oC for 0, 3, 5 and 10 min) and TP (0.1 MPa, 100

oC for 0, 3, 5 and 10 min).

Dense Plastoglobulin-type

sacs with β-carotene Dense Lycopene crystals

Extremely prominent and denser

carotenoid containing structures with

distinct boundaries

Prominent and dense carotenoid structures dispersed in

the cytoplasm with signs of solubilization

Low density dispersed carotenoid containing

organelles with signs of solubilization

104

CHAPTER 4: STORAGE STABILITY OF LYCOPENE IN TOMATO JUICE

SUJBECTED TO COMBINED PRESSURE-HEAT COMBINATIONS

Abstract

A study was conducted to characterize the storage stability of lycopene in hot-

break tomato juice prepared from two different cultivars and processed by various

pressure−heat combinations. Samples were subjected to pressure assisted thermal

processing (PATP; 600 MPa, 100°C, 10 min), high pressure processing (HPP; 700 MPa,

45°C, 10 min), and thermal processing (TP; 0.1 MPa, 100°C, 35 min). Processed samples

were stored at 4, 25, and 37°C for up to 52 weeks. HPP and PATP treatments

significantly improved the extractability of lycopene over TP and control. All-trans

lycopene was found to be fairly stable to isomerization during processing, and the cis

isomer content of the control and processed juice did not differ significantly. During

storage, lycopene degradation varied as a function of the cultivar, processing method,

storage temperature, and time. This study shows that combined pressure−temperature

treatments could be an attractive alternative to thermal sterilization for preserving tomato

juice quality.

105

4.1 Introduction

A large portion of lycopene in the North American and European diet comes from

the consumption of tomato and tomato products (Clinton, 1998). Many epidemiological

studies suggest the consumption of carotenoid rich foods such as tomato products reduces

the risk of developing diseases such as cancer and cardiovascular diseases (Gann et al.,

1999; Sesso et al., 2005; Sesso et al., 2003; Bruno et al., 2007). With increased consumer

demand for healthy foods, studying the fate of lycopene subjected to various preservation

methods has gained interest (Henry et al., 2000; Oms-Oliua et al., 2009; Odriozola-

Serranoa, 2008).

Lycopene (C40H56) is a carotenoid that occurs in the form of all-trans and cis

isomers in red tomatoes. Red tomatoes normally contain 94-96% all-trans lycopene,

which is thermodynamically most stable form (Porrini et al., 1998). However, lycopene

from heat-induced cis-isomer-rich tomato sauce is reportedly more bioavailable than

from all-trans-rich tomato sauce (Unlu et al., 2007). Also, human plasma and tissues

have been shown to contain 40-80% cis isomers (all-trans, 5-cis, 9-cis, 13-cis, and 15-cis

being the most common isomers) (Clinton et al., 1996).

Lycopene in tomato and tomato products exposed to traditional thermal

treatments is fairly stable during processing (Nguyen and Schwartz, 1998). However, the

magnitude of lycopene degradation during storage reported in the literature varies greatly

and is influenced by temperature, water activity, presence or absence of light and oxygen

(Shi and Le Maguer, 2000; Xianquan et al., 2005). Inter-conversions between all-trans

106

and cis forms of lycopene in thermally treated tomato juice have also been reported (Lin

and Chen, 2005).

Due to consumer interest in health, wellness, flavor, and freshness, the food

industry is exploring alternative minimal processing approaches such as high pressure

processing (Balasubramaniam and Farkas, 2008). High pressure studies on tomato juice

and/or tomato puree show an increase in the amount of extractable lycopene over

untreated or thermally processed juices (Sanchez-Moreno et al., 2006; Krebbers et al.,

2003; Hsu et al., 2008). Pressure treated tomato puree samples better retain color over

thermally pasteurized and sterilized samples (Sanchez-Moreno et al., 2006; Krebbers et

al., 2003). Hsu et al. (2008) studied the fate of lycopene in hot break tomato juice

extracted from red daydream tomatoes. The hot break tomato juice was processed using

high pressure processing (HPP) (300-500 MPa, 25 °C, 10 min) and heat treatment (98 °C,

15 min) and stored at 4 °C for 28 days. Pressure treatment increased the lycopene

extractability from tomato juice relative to control or thermally treated juice, and

improved its storage stability. In another study, Qiu et al. (2006) pressure treated tomato

puree at 100-600 MPa, 20±1 °C for 12 min and studied the degradation and isomerization

of lycopene at 4±1 °C and 24±1° C for a period of 28 days. Pressure treatment increased

the extractable lycopene content and retained its stability during storage. However

microbiological stability of the product was not reported. Most of the current storage

studies are limited to less than 30 day duration and the stability of pressure treated tomato

juice during an extended storage period is not known. Also the storage stability of

107

lycopene in pressure assisted thermally processed (500-700 MPa, 90-120 ° C) tomato

juice has not been studied.

4.2 Objectives

The objective of this study was to investigate the storage stability of lycopene in

tomato juice stored up to 52 weeks. Juice from two tomato cultivars that differed in

lycopene content was processed using thermal processing, pressure assisted thermal

processing, and high pressure processing. Samples were subsequently stored at

temperatures of 4, 25 and 37°C. Product was evaluated on the basis of color, lycopene

isomer profile, and microbial stability.


Two fully ripe tomato (Solanum lycopersicum L.) cultivars; high lycopene FG99-

218 (Old gold crimson ogc homozygous; dark green (dg) homozygous) and commercial

variety OX325 (Old gold crimson ogc homozygous; alcabaca (alc) heterozgygous) were

freshly harvested from the North Central Agricultural Research Center, Fremont, OH and

transported to the processing pilot plant in Columbus, OH (~ 2 hour travel). The initial

quality attributes of the raw tomato product are summarized in Table 4.1. It is worth

noting that FG99-218 (high lycopene) had nearly twice the amount of total lycopene than

that of OX325 and had higher “a” values on the CIE color scale.

4.3.1 Tomato juice processing

The tomatoes were washed and juice was extracted within 48 hours of harvesting

the tomatoes. The juice was extracted using hot break process (91±2°C) employing a

108

pilot scale tomato juice extraction system consisting of a tomato crusher (W.J. Fitzpatrick

co. Chicago, IL), concentric tube heat exchanger, and a screw type tomato juice extractor

(Chisholm Ryder Co., CJE-350 D-28). The juice was collected and cooled to 20±1 °C.

The juice was immediately vacuum packaged (Spiromac vacuum sealer, model 450 T,

Québec, Canada) in polypropylene pouches (Thomson Equipment and Supply, Cincinnati,

OH). Each pouch contained 50±3 g tomato juice. The pouches were stored in a walk-in

refrigerator (4±0.5°C) under dark. The stored tomato juice samples were subjected to

various pressure-heat treatments (see following sections for details) within 2-days of

juice extraction. Unprocessed hot break juice was also used as control to compare the

effect of various processing conditions on juice quality attributes during storage.

Selected quality attributes of the hot-break juice are summarized in Table 4.1.


The tomato juice samples were high pressure processed using 5 liter sample

holding capacity, pilot scale high pressure machine (Iso-lab high pressure food processor,

Stansted Fluid Power Ltd, Essex, UK). Pressurization and depressurization rates of ~5.3

MPa/s and ~8.3 MPa/s were used. 100 % propylene glycol (Brenntag Midsouth Inc.,

Henderson, KY) was used as the pressure transmitting fluid. The samples (pre-

conditioned at an initial temperature of 20°C) were high pressure processed (700 MPa, 45

°C for 10 min). Sample temperature under high pressure conditions were monitored using

T-type thermocouples (Omega Engineering, Stamford CT). The thermocouple was fed

through the pouch using a C-5.2 stuffing box (Ecklund-Harrison Technologies, Fort

109

Myers, FL). After processing, the samples were immediately withdrawn and stored at

different temperatures.

4.3.3 Pressure-assisted thermal processing

For processing tomato juice using pressure-assisted thermal processing, the

tomato juice samples were first preheated to an initial temperature of 80°C in a water

bath (Fisher Scientific, Pittsburgh, PA). Similarly the initial temperature of glycol was

also adjusted to 80°C. The preheated samples were loaded into a stainless steel sample

holder and then loaded inside a pressure chamber preheated to 105°C. Samples were

pressure treated at 600 MPa, 100 °C for 10 min. The equipment had a pressurization time

of <140 s. More details of the experimental techniques used are provided elsewhere

(Nguyen et al., 2010). After processing, the samples were depressurized (< 30 s),

withdrawn from the pressure chamber, and immediately cooled in an ice–water mixture

and analyzed within 1 day after the treatment.

4.3.4 Thermal sterilization

Boiling water in a steam jacketed kettle was used for thermal sterilization (100°C,

35 min) of vacuum packaged tomato juice (Barringer, 2004). The temperature was

monitored using a K-type thermocouple (Omega Engineering) and recorded using a data

logger (IOtech, Cleveland, OH). The thermocouple was located at the geometric center of

the pouch containing the tomato juice. After processing, the samples were immediately

cooled in ice-water bath and transferred for storage at the 3 respective temperatures.

4.3.5 Storage of processed and control samples

110

The processed samples and control were immediately stored in the dark at three

different temperatures (4±0.5°C, 24±1°C and 36±1°C) and analyzed for lycopene

content, color, pH, total soluble solids and microbial stability after 0, 2, 5, 15, 35 and 52

weeks of storage.

4.3.6 Analysis

4.3.6.1 Lycopene extraction

Lycopene was extracted from tomato juice using a method reported by Ferruzzi et

al. (Ferruzzi et al., 1998). Briefly, tomato juice (5 g) was mixed with 4 g celite and 1 g

calcium carbonate. 50 ml methanol was added and the mixture was homogenized at

10,000 RPM for 1 min. Carotenoids were extracted three times with 25 mL HPLC grade

hexane/acetone (1:1 v/v) (Fisher Scientific, USA). The combined hexane layer was

collected quantitatively after filtering through anhydrous sodium sulfate and the volume

was made to 100 mL using HPLC grade hexane. 2ml aliquots were dried under nitrogen,

reconstituted in methyl tert-butyl ether (MTBE)/ methanol (1:1 v/v) (HPLC grade, Fisher

scientific, USA), filtered through 0.2 µm 13 mm nylon syringe filter and 50 µL was

injected in the HPLC (Agilent technologies, Model HP 1050) equipped with Waters 996

Photodiodearray (PDA) detector. The mixture was separated on a Waters YMC C30

HPLC column (4.5 mm x150 mm, 5 μm particle size). Separations were achieved using

gradient elution with different concentrations of methanol: MTBE: 2% aq. ammonium

acetate in reservoirs A (88:5:7) and B (20:78:2). The following gradient was used: at 0

min 0% B, linear gradient to 85% B over 20 min, followed by a linear gradient to 100%

B over 10 min, returning to 0% B and holding for 5 min.

111

Isomerization of the lycopene standard was performed in hexane by adding iodine

catalyst at a concentration of about 1% (w/w) of the lycopene weight and allowing the

mixture to sit for 15 min in fluorescent light of luminance 320 lux (lumens/m2). HPLC

analysis was then performed on the isomerized sample (Figure 4.1). Lycopene isomers in

tomato juice were identified by comparing chromatograms of tomato juice with the

chromatograms of isomerized lycopene standard.

To quantify lycopene in tomato juice samples, a calibration curve was generated

using authentic all-trans lycopene standard. Levels of cis lycopene isomers are given in

all-trans lycopene equivalents.

4.3.6.2 Colorimetry

Color of tomato juice samples was measured using a tristimulus colorimeter (CR-

300, Minolta, Osaka, Japan). A standard white tile (Y = 92.6, X = 0.3161, y = 0.3321)

was used to calibrate the instrument. The juice samples were placed in a glass petri dish

on top of the light source (15mm aperture) and L, a, and b values were directly obtained

from the colorimeter. Each measurement reported represents the average of 3 readings. L

represents lightness, +a represents redness, –a represents greenness, +b represents

yellowness, and –b represents blueness. The overall change in color (ΔE) was calculated

by the following equation (Avila and Silva, 1999):

ΔE = ………………………………………………………….(1)

4.3.6.3 Aerobic plate count (bacterial growth)

112

Aerobic plate counts of the processed and control tomato juice samples were

taken after 0, 2, 5, 15, 35 and 52 weeks of storage at 4, 24 and 37oC. Tomato juice (10 g)

from the vacuum sealed pouches was aseptically transferred in stomacher bags, and 90

mL sterile peptone water was aseptically added to each bag. The samples were

homogenized in a stomacher (Seward Lab Stomacher, Norfolk, UK) for 2 min. 1 mL

homogenized sample was then aseptically transferred to the tubes containing 9 mL sterile

peptone water and serial dilutions were spread plated on trypticase soy agar (TSA) plates.

After incubation at 37oC for 48 and 72 hrs, the viable count of microorganisms was

enumerated. Colonies were counted with a dark-field Quebec colony counter (Leica

Microsystems, Richmond Hill, Canada). The detection limit for the enumeration

procedure was 100 CFU per mL.

4.3.6.4 Total soluble solids (0Brix) and pH

Total soluble solids (%) and pH of the raw tomatoes, and the juice was measured

using Atago Digital Handheld Pocket Refractometer (Cole-Parmer Instrument Company,

Vernon Hills, IL) and portable handheld pH meter (Omega Engineering), respectively.



PA). The influence of storage temperature (T – 4, 25 and 37 °C), storage time (t –

0,2,5,15,35 and 52 weeks), cultivar (FG99-218 or OX325), treatment (HPP, PATP or TP)

as well as their interactions on the concentration of lycopene (Y) in tomato juice were

113

analyzed. Pairwise comparisons for the means of treatment and storage variables (factors)

were evaluated with Tukey’s test at 5% significance level (P<0.05).

4.3.6.6 Kinetic modeling

The degradation of lycopene as influenced by storage temperature, time, and

processing methods was modeled using two first order rate equations (Fish and Davis,

2003).

……………………………………………………..(2)

where Ct is the lycopene concentration in mg/100 g tomato juice at time t weeks, C0 is the

initial concentration of lycopene in the respective sample at week 0, S1 and S2 are

constants for each process proportional to the lycopene degradation under respective

processing and storage conditions, k1 and k2 are the rate constants for each of the two

first-order processes involved in lycopene degradation.


Effect of hot break juice extraction on lycopene

Hot break extraction process at 91±2°C was used to extract tomato juice in order

to minimize the undesirable effects of pectin degrading enzymes on the consistency of

tomato juice and extractability of lycopene. Hot break treatment of crushed tomatoes is

sufficient to completely inactivate pectin methyl esterase and retains approximately 4 %

polygalacturonase activity (Luh and Daoud, 1971). Extraction of juice from both FG99-

218 and OX325 did not significantly affect the total lycopene (all-trans + cis lycopene)

114

content of the juice. However, a significant increase in the cis isomer of lycopene was

observed in juices extracted from both cultivars. This was accompanied by a slight

decrease in the redness of the juice as shown by the “a” color value (Table 4.1). Cis

isomers of lycopene have been reported to have different physio-chemical characteristics

than the all-trans isomers including decrease in the color intensity of the tomato juice

(Nguyen and Schwartz, 1999). Although lycopene has been shown to be fairly stable to

isomerization reactions in tomato products exposed to conventional thermal processing

(Nguyen and Schwartz, 1998), presence of heat, light, oxygen and/or their combinations

during processing does influence the fate of lycopene isomers (Nguyen and Schwartz,

1999).

Combined pressure-heat treatment effects on lycopene stability

The initial temperature of HPP processed samples was approximately 20°C. The

maximum and average process temperatures at target pressure (700 MPa) over 10 min

pressure holding time were 47.0 and 45.7±0.56°C respectively. The maximum and

average process temperatures of the preheated juice (~80 °C) during 10 min PATP

holding time were 104.0°C and 101±0.87°C, respectively. Thermally processed samples

were maintained at 100°C, 0.1 MPa, for 35 min.

HPP (700 MPa, 45 °C, 10 min) and PATP (600 MPa, 100°C, 10 min) increased

the extractable all-trans lycopene in the high lycopene cultivar FG99-218 by 12% and

7% respectively over the unprocessed hot break juice that served as control. However, the

all-trans lycopene in thermally sterilized samples of FG99-218 was approximately the

same as that of the control (Table 4.1). The cis lycopene isomer content in HPP, PATP,

115

TP treated samples as well as the hot break juice (control) did not show significant

differences (p < 0.05) and constituted approximately 8% of the total lycopene in

processed and control samples (Table 4.1).

Similarly, the extractable all-trans lycopene in OX325 increased by

approximately 8% for HPP samples (700 MPa, 45°C, 10 min) but showed insignificant

change for PATP (600 MPa, 100oC, 10 min) samples. Thermally sterilized samples had a

19% decrease in all-trans extractable lycopene (Table 4.1). The cis isomers of HPP,

PATP, TP treated samples and the control varied between 12-16% of the total lycopene

(Table 4.1). It was also worth noting that OX325 had 70% less all-trans isomer than

FG99-218, the high lycopene variety. The differences in lycopene extractability from

tomato juice exposed to similar process treatments could be attributed to cultivar specific

differences. Earlier researchers have also reported increase in the lycopene extractability

from pressure treated tomato products (Sanchez-Moreno et al., 2006; Krebbers et al.,

2003; Hsu et al., 2008). However, the extent to which lycopene extractability increased in

various published studies differs greatly. For example, Krebbers et al. (2003) applied

various pressure-temperature-time combinations (300-500 MPa, 20°C, 2 min and 700

MPa, 80 and 90°C for 30 sec) and reported that tomato puree processed at 500 MPa at 20

°C for 2 min had a significant increase in lycopene extractability (~27%) than raw juice

samples. Similarly, Sanchez-Moreno et al. (2006) processed tomato puree at 400 MPa,

25°C for 15 min and reported ~48% increase in lycopene extractability as compared to

the raw unprocessed puree. Hsu et al. (2008) processed tomato juice at 300-500 MPa,

25°C for 10 min and reported a progressive increase in lycopene extractability with

116

increase in pressure. A maximum 60% lycopene extractability was reported in samples

processed at 500 MPa, 25 °C for 10 min. The differences in magnitude of lycopene

extractability among various studies may be due to the differences in the type of tomato

cultivars used, sample preparation method, type of sample used (juice, pulp, puree, etc.),

hot break vs. cold break juice extraction, and high pressure process parameters. Since

pressure-temperature treatments are known to affect membranes in vegetable cells (Shi

and Le Maguer, 2000)

and also macromolecular structures such as proteins and

carbohydrates (Butz and Tauscher, 2002), such treatments may increase lycopene

extractability. We hypothesize that increased extractability could lead to increased

bioavailability of lycopene. Research has shown that all-trans lycopene in tomato

products is fairly stable during traditional thermal processing (Nguyen and Schwartz,

1998; Nguyen and Schwartz, 1999; Lin and Chen, 2005). It has been further reported

that cis lycopene is fairly stable to pressure treatments (Qiu et al., 2006). Since

isomerization is a structurally and thermodynamically specific phenomenon, studies on

the thermodynamic effects of combined pressure-heat treatments on lycopene might be

able to explain its stability to isomerization under such processing conditions. Also, the

stability of all-trans form to degradation and or isomerization is governed by its structural

specificity. The differences in shape of the carotenoid molecule influence hydrophobicity,

crystalline state and ease of crystal formation, solubility and other such properties which

in turn might influence its stability (Nguyen et al., 2001). Lycopene is a linear molecule

and has been shown to form multilayers or aggregates (Ray and Misra, 1997) and it is

proposed that once in the aggregated form, lycopene molecules might be able to resist

further structural changes (Nguyen et al., 2001).

117

Microbial stability of the processed samples

All samples processed using HPP, PATP and TP showed microbial stability over

a storage period of 52 weeks at all three storage temperatures (4, 25 and 37°C). Control

samples stored at 25 and 37°C showed spoilage within 2 weeks (data not shown). Due to

microbial stability of HPP, PATP and TP samples, the pH and total soluble solids of the

stored samples did not show a significant change over the storage period (p<0.05).

Storage effects on lycopene stability

Figures 4.2-4.3 present the storage stability of lycopene in tomato juice subjected

to HPP, PATP, TP treatments and stored at three different storage temperatures (4, 25,

37oC) up to 52 weeks. In general, lycopene from both cultivars was fairly stable to

degradation and isomerization over the course of storage period. For cultivar FG99-218

(high lycopene, 14.65±0.66 mg lycopene/100 g tomato juice), both storage temperature

and storage time significantly influenced lycopene degradation (P<0.05). In cultivar

OX325 with lower lycopene content (8.84 ±0.22 mg lycopene/100 g tomato juice),

lycopene degradation was primarily influenced by storage time, but storage temperature

did show a statistically significant effect on lycopene degradation during storage (P <

0.05). Decrease in the concentration of cis isomers was more pronounced at 25 °C storage

for both cultivars. The present study did not consider the influence of barrier properties of

the packaging material on storage stability of the treated juice and further research is

needed to understand the role of packaging on the stability of processed samples.

Changes in tomato juice color during storage

118

Changes in color values (ΔE) of the processed and control samples over the

course of 52 weeks of storage at 4, 25, and 37°C storage temperatures are shown in

Figures 4.4-4.5. In comparison to the control sample at day zero, all the processed (TP,

HPP, PATP) samples changed color as a function of storage temperature and time.

Among the treatments, HPP samples had better color retention, while TP samples had

most color changes. PATP samples had intermediate color loss and the degradation was

also influenced by the cultivar type. In addition to increased lycopene extractability,

better retention of natural color may be an added benefit of PATP over conventional

thermal sterilization.

Since conversion of trans to cis involves decrease in the intensity of red color

(Zechmeister and Polgar, 1944) and lycopene degradation might influence color changes,

an attempt was made to correlate color changes to changes in lycopene. With few

exceptions, the changes in color values (ΔE) as a function of lycopene degradation were

fairly linear with regression coefficient values greater than 0.85 (R2 > 0.85). Earlier

studies (Krebbers et al., 2003) reported little dependence of changes in lycopene

concentration and L, a, b color values in samples subjected to thermal and combined

pressure-temperature treatments.

Kinetic modeling of lycopene degradation

The plots of lycopene content vs. storage time show that lycopene degraded

rapidly during the first few weeks of storage (weeks 0-5) and then approached a more

linear asymptotic form during subsequent storage period (Figures 4.2-4.3). The kinetic

data for degradation of lycopene during storage could not be fitted with zero, first or

119

second order rate equations. However, for tomato juice exposed to each of the variables,

a 2-step first order kinetic model could predict the degradation of lycopene within

experimental limits. The presence of 2 first order decay processes does suggest multiple

(2 or more) pathways of degradation caused by the effects of various treatments, storage

temperature and time and/or their interactions. Figure 4.6 provides experimental vs.

predicted lycopene concentration (using kinetic constants presented in Table 4.2) in

selected tomato juice samples from FG99-218. Each line in Figure 4.6 was generated

by its corresponding equation (equation 2) and offered a reasonable fit to the

experimental data within experimental limits.

The rate constants of lycopene degradation during storage are summarized in

Table 4.2. The initial phase of storage (0, 2, and 5 weeks) had larger reaction constant

values (k1) (ranging from 0.36/sec to 2.67/sec) than reaction constant values (k2)

estimated (ranging from 0.0009 to 0.0072/sec) for subsequent stages of storage (5, 15, 35,

and 52 weeks). This shows that rapid lycopene degradation occurred during the first few

weeks of storage and then gradually subsided. With few exceptions, samples stored at

4°C showed lowest rate of degradation during both first order degradation steps. This

biphasic degradation behavior of lycopene was also observed in watermelon samples

stored for one year at -20 and -80°C (Fish and Davis, 2003). However, the rate constants

(k1, k2) of lycopene degradation in watermelon tissue under frozen storage were

approximately one-tenth of the values reported in this study. This could be attributed to

difference in lycopene level in the studied food matrices, process treatment, and storage

temperature.

120

Processing methods and cultivars were found to influence the lycopene stability

over 52 weeks of storage. For example, with FG99-218, in comparison to pressure treated

samples at day 0, pressure treated samples stored for 52 weeks showed 11, 18, and 21%

decrease in all-trans lycopene at 4, 25, and 37°C respectively (Figure 4.2a). Similarly,

thermally processed samples after 52 weeks of storage showed 26, 27 and 28% decrease

as compared to day 0 samples analyzed immediately after thermal processing. PATP

samples showed ~16% decrease at all three storage temperatures (Figure 4.2a). HPP and

PATP treatments resulted in up to 23% and 17% degradation in cis isomers, respectively,

whereas TP resulted in up to 26% cis lycopene degradation during storage. The cis

isomers of both cultivars were better retained in HPP and PATP processed samples at the

end of 52 weeks as compared to the thermally processed samples (Figure 4.2b). In

OX325 over a 52 week period, the change in all-trans isomers of lycopene in HPP, PATP

and thermally treated samples stored at 4, 25 and 37°C was 5, 10, 8% (HPP); 12, 11, 13%

(PATP) and 14, 15, 11% (TP) respectively (Figure 4.3a). The cis isomers in this cultivar

did not show a clear dependency on storage temperature (Figure 4.3b) and samples

stored at 25 °C showed more degradation than samples stored at 4 or 37°C.

Studies that report the fate of lycopene in stored tomato juice processed using

combined pressure-temperature are scarce and conducted for less than 4 weeks on

samples processed at ambient temperatures (Hsu et al., 2008; Qiu et al., 2006). These

studies conducted over a range of pressures up to 600 MPa and 25°C show that lycopene

is fairly stable in tomato juice/puree exposed to HPP treatments degrading between 1-

10% over a storage period between 2-4 weeks stored at 25°C. However, studies on the

121

fate of lycopene in thermally processed tomato products are readily available, though

large differences in lycopene stability have been reported. One study reported that

commercially canned tomato juice stored for a period of 12 months does not show

significant lycopene degradation (Agarwal et al., 2001). Additionally, Nguyen et al.

(2001) reported that extended storage of various tomato products over an 18 month

period did not change the isomer distribution of lycopene.

4.5 Conclusions

Within the range of experimental conditions used in the study, combined

pressure-temperature treatments improved the extractability of lycopene over

conventional thermal sterilization. All-trans lycopene is fairly stable to

isomerization during processing and the cis isomer content of control and

processed juice did not differ significantly. During storage, the stability of

lycopene in tomato juice is influenced by a complex interaction of method of

processing, type of cultivar, storage temperature and storage time. HPP and PATP

processed samples better retained both total lycopene and cis isomers during 52

weeks of storage. During storage, HPP and PATP samples also showed better

color retention as compared to the thermally processed samples. Tomato juice

samples processed using various treatments (HPP, PATP and thermal) were

microbiologically stable over 52 weeks of storage. A 2-step first order equation

could be effectively used to predict the changes in lycopene concentration over the

course of storage. This study suggests that combined pressure-temperature

122

treatments pose an attractive alternative to thermal sterilization for delivering

improved quality and a more functional tomato juice to the consumer.

Acknowledgements

Support for this research was provided by USDA-CSREES-NRICGP grant 2006-

35503-17571, by the Ohio Agricultural Research and Development Center (OARDC),

and by The Ohio State University. References to commercial products or trade names are

made with the understanding that no endorsement or discrimination by The Ohio State

University is implied.

123

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128

Table 4.1 Selected attributes of raw tomato flesh and the hot break tomato juice used in

the study.

Cultivar 0Brix pH Color

1 Lycopene content

1

mg/100 g juice

Total

Lycopene1

mg/100 g

juice

%TSS2 L a B all-trans cis All-trans

+cis

FG99-

218

Raw

tomato

6.3 4.52 26.02±0.21 19.86±0.16 14.89±0.09 15.02±0.26 1.02±0.05 16.04±0.3

Hot

break

juice

6.5 4.50 26.59±0.13 18.02±0.08 14.46±0.14 14.65±0.66 1.4±0.01 16.05±0.7

HPP3

6.6 4.51 25.54±0.47 17.08±0.26 12.14±0.25 16.51±0.6 1.44±0.01 17.95±0.6

PATP4

6.5 4.50 24.99±0.51 16.75±0.17 12.77±0.20 15.74±0.37 1.38±0.02 17.12±0.4

TP5

6.5 4.50 28.92±0.91 15.41±0.38 11.89±0.41 14.1±0.51 1.4±0.03 15.50±0.5

OX 325 Raw

tomato

5.4 4.38 25.89±0.11 14.52±0.19 12.66±0.25 8.96±0.15 0.88±0.06 9.84±0.2

Hot

break

juice

5.6 4.40 25.92±0.09 13.99±0.04 12.43±0.09 8.84±0.22 1.38±0.01 10.22±0.2

HPP3

5.7 4.40 26.48±0.55 14.40±0.60 12.64±1.19 9.57±0.37 1.35±0.01 10.88±0.4

PATP4

5.7 4.41 23.75±0.22 15.3±1.22 15.08±0.59 8.93±0.30 1.36±0.03 10.29±0.3

TP5

5.6 4.40 28.52±0.55 11.18±0.14 10.39±0.27 7.13±0.32 1.36±0.01 8.49±0.3

1Values are mean ± SD of three replications

2Per cent total soluble solids present in the juice

3High Pressure Processing – 700 MPa, 45

oC, 10 min

4Pressure Assisted Thermal Processing – 600 MPa, 100

oC, 10 min

5Thermal Processing – 0.1 MPa, 100

oC, 35 min

129

Table 4.2 Reaction rate constants and correlation coefficients of lycopene degradation in

high lycopene (FG99-218) and OX325 tomato juice treated using HPP, PATP, and

thermal sterilization.

Cultivar /Treatment K1 (rate constant for lycopene

degradation during 0 to 5 week

storage)

K2 (rate constant for lycopene

degradation during 5 to 52 week

storage)

Cultivar FG99-218 K1 (1/sec) Correlation

coefficient

K2 (1/sec) Correlation

coefficient

HPP

4oC 0.5124 0.99 0.0014 0.98

25oC 0.3283 0.99 0.0036 0.98

37oC 0.8086 0.93 0.0034 0.99

Thermal

4oC 0.6322 0.96 0.0015 0.89

25oC 0.637 0.97 0.0031 0.97

37oC 1.3158 0.97 0.0072 0.99

PATP

4oC 0.3631 0.83 0.0017 0.88

25oC 0.7548 0.98 0.0011 0.97

37oC 0.7342 0.97 0.0016 0.97

Cultivar OX325

HPP

4oC 2.6714 0.99 0.0009 0.98

25oC 1.3016 0.88 0.001 0.99

37oC 0.7717 0.90 0.0014 0.99

Thermal

4oC 0.4348 0.97 0.0014 0.89

25oC 0.9019 0.91 0.0016 0.99

37oC 0.545 0.99 0.0025 0.99

PATP

4oC 0.5417 0.97 0.0012 0.95

25oC 1.3124 0.94 0.0002 0.99

37oC 0.7648 0.99 0.0011 0.97

130

Figure 4.1 HPLC chromatogram of lycopene isomers obtained by adding iodine catalyst

in hexane at a concentration of about 1% (w/w) of the lycopene weight and allowing the

mixture to sit for 15 min in fluorescent light of luminance 320 lux (lumens/m2).

AU

0.00

0.05

0.10

0.15

Minutes 20.00 22.00 24.00 26.00 28.00 30.00

Cis lycopene isomers All-trans lycopene

5-cis

131

Figure 4.2 All-trans (a) and cis (b) lycopene concentrations in high lycopene (FG99-218)

tomato juice processed using HPP (700 MPa, 45oC, 10 min), thermal sterilization (0.1

MPa, 100oC, 35 min) PATP (600 MPa, 100

oC, 10 min) and stored in dark at different

storage temperatures for 0,2,5,15,35 and 52 weeks. Untreated control had 14.65±0.66 mg

all-trans lycopene/100 g juice and 1.4±0.01 mg cis lycopene/100 g juice. Values are

mean ± SD of three replications

0

2

4

6

8

10

12

14

16

18

Control HPP 4c

HPP 25C

HPP 37C

PATP 4C

PATP 25C

PATP 37C

Thermal 4C

Thermal 25C

Thermal 37 C

mg

all-trans

lyco

pe

ne

/10

0 g

to

mat

o ju

ice

Processing method/ storage temperature

0

2

5

15

35

52

Storage

time

(weeks)

Continued

a

132

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Control HPP 4c

HPP 25C

HPP 37C

PATP 4C

PATP 25C

PATP 37C

Thermal 4C

Thermal 25C

Thermal 37 C

mg cis

lyco

pe

ne

/ 1

00

g t

om

ato

juic

e

Processing method / storage temperature

0

2

5

15

35

52

Storage

time

(weeks)


b

133

Figure 4.3 All-trans (a) and cis (b) lycopene concentrations in OX325 tomato juice

processed using HPP (700 MPa, 45oC, 10 min), thermal sterilization (0.1 MPa, 100

oC, 35

min) PATP (600 MPa, 100oC, 10 min) and stored in dark at different storage

temperatures for 0,2,5,15,35 and 52 weeks. Untreated control had 8.84±0.22 mg all-trans

lycopene/100 g tomato juice and 1.38±0.01 mg cis lycopene/100 g juice. Values are mean

± SD of three replications

0

2

4

6

8

10

12

Control HPP 4c

HPP 25C

HPP 37C

PATP 4C

PATP 25C

PATP 37C

Thermal 4C

Thermal 25C

Thermal 37 C

mg

all-

tran

s ly

cop

en

e/1

00

g t

om

ato

juic

e

Processing method/Storage temperature

0

2

5

15

35

52

Storage

time

(weeks)

Continued

a

134

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Control HPP 4C

HPP 25C

HPP 37C

PATP 4C

PATP 25C

PATP 37C

Thermal 4C

Thermal 25C

Thermal 37 C

mg

cis

iso

me

r/1

00

g t

om

ato

juic

e

Processing method / storage temperature

0

2

5

15

35

52

Storage

time

(weeks)


b

135

Continued

Figure 4.4 Differential Color values (ΔE) of high lycopene (FG99-218) tomato juice

processed using HPP (□), TP (Δ) and PATP (●) and stored in dark at (a) 4oC (b) 25

oC and

(c) 37oC for 0,2,5,15,35 weeks. Values are mean ± SD of three replications

0

1

2

3

4

5

6

7

8

9

-10 0 10 20 30 40 50 60

De

lta

E

Storage time (weeks)

a

0

1

2

3

4

5

6

7

8

9

10

-10 0 10 20 30 40 50 60

De

lta

E


b

136


0

1

2

3

4

5

6

7

8

9

10

-10 0 10 20 30 40

De

lta

E

Storage time (Weeks)

c

137

Continued

Figure 4.5 Differential Color values (ΔE) of OX325 tomato juice processed using HPP

(□), TP (Δ) and PATP (●) and stored in dark at (a) 4oC (b) 25

oC and (c) 37

oC for

0,2,5,15,35 weeks. Values are mean ± SD of three replications

0

1

2

3

4

5

6

7

8

-10 0 10 20 30 40 50 60

De

lta

E


a

0

1

2

3

4

5

6

7

8

9

10

-10 0 10 20 30 40 50 60

De

lta

E


b

138


0

1

2

3

4

5

6

7

8

9

10

-10 0 10 20 30 40

De

lta

E


c

139

Figure 4.6 Representative experimental vs. predicted values of lycopene concentration in

tomato juice (FG99-218) processed using combined pressure-temperature treatments and

stored at various temperatures.

▔ 700 MPa, 45oC, 10 min. treatment followed by 4

oC storage ---- predicted

▲ 0.1 MPa, 100oC, 35 min. treatment followed by storage at 25

oC ----- predicted

♦ 600 MPa, 100oC, 10 min. treatment followed by storage at 37

oC ----- predicted

10

11

12

13

14

15

16

17

0 10 20 30 40 50 60

mg a

ll-t

ran

s ly

cop

ene/

100g t

om

ato

ju

ice


140

Figure 4.7 Flowchart outlining the steps involved in the experiment

Freshly harvested tomatoes

Cultivar A – High Lycopene

Cultivar B – Commercial Variety

(Standard lycopene content)

Juice extraction

(Hot Break) 93oC

High pressure

processing

(700 MPa, 45oC, 10

min.)

Pressure assisted

thermal processing

600 MPa, 100oC,

10min

Thermal processing

(0.2 MPa, 100 oC, 35

min.)

Unprocessed hot

break juice (control)

Lycopene extraction

followed by High

Performance Liquid

Chromatography

Storage at 4, 25 and 37oC in dark for 52

weeks(0, 2, 5, 15, 35 and 52 weeks)

pH, %TSS

measurement

Data analysis

0, 2, 5, 15, 35

and 52 weeks

Color estimation (L, a, b

values) (Colorimetry)

141

CHAPTER 5: COMBINED PRESSURE-TEMPERATURE EFFECTS ON THE

CHEMICAL MARKER (4-HYDROXY-5-METHYL-3(2H)-FURANONE)

FORMATION IN WHEY PROTEIN GELS

Abstract

Chemical markers, such as furanone, are intrinsically formed in foods at elevated

process temperatures, and have been successfully used as indirect indicators of heating

patterns in advanced thermal processes such as aseptic processing, microwave

sterilization and ohmic heating. However, very limited information is available on

suitability of these chemical markers during combined pressure-heat treatment. A study

was conducted on the formation and stability of chemical marker M-2 (4-hydroxy-5-

methyl-3(2H) furanone, a by-product of Maillard reaction) as a function of pressure,

temperature and pH. Whey protein gels (containing 1g ribose/100g gel mix) at pH 6.10

and 8.25 were subjected to pressure assisted thermal processing (PATP; 350 and 700

MPa, 105oC), high pressure processing (HPP; 350 and 700 MPa, 30

oC) and thermal

processing (TP; 0.1MPa, 105oC) for different holding times. Unprocessed gel was used as

control. The marker yield was quantified using HPLC. The initial concentrations of M-2

in the gels were 9.17 and 6.10 mg/100g at pH 6.10 and 8.25, respectively. As expected,

heat treatment at 105oC, 0.1 MPa increased M-2 concentration. The marker yield

increased with increase in holding time, following a first order kinetics and decreased

with increasing pH. Pressure treatments from 350 to 700 MPa at 30oC reduced the

chemical marker formation for both pH values investigated. Marker formation during

combined pressure-temperature (105oC, 350 and 700 MPa) was influenced by both heat

142

(which favored the marker formation) and pressure (which hindered marker formation).

The net final concentration of the marker formed during PATP was higher than HPP, but

lower than thermal treatments. This study suggests that 4-hydroxy, 5-methyl, 3(2H)

furanone may not be a suitable marker for evaluating pressure-heat uniformity during

PATP.

5.1 Introduction

Pressure-assisted thermal processing (PATP) offers new opportunities to the food

industry for processing high quality low-acid, shelf-stable foods. Although shelf-stable

low-acid foods processed by this technology are not currently commercially available, the

technology can be used for processing heat-sensitive products such as mashed potatoes,

dinner kits, meats and sauces, soups, egg products, coffee, and tea (Balasubramaniam, &

Farkas, 2008; Juliano et al., 2006). During a typical PATP process, the food is subjected

to a combination of elevated pressures (500-900 MPa) and moderate heat (90-121oC) for

a short time. One of the unique advantages of PATP is its ability to provide a rapid

increase in the temperature of treated food samples. Rapid compression heating and

subsequent expansion cooling on decompression help to reduce the severity of thermal

effects encountered with conventional processing techniques (Rajan et al., 2006).

The increase in temperature of the sample when exposed to high pressure, also

known as heat of compression, has been well documented in the literature (Patazca, et al.,

2007; Delgado, et al., 2007; Rasanayagam et al., 2003; Torres et al., 2009). However, this

increase in temperature is further influenced by food composition, product initial

temperature and target pressure. As a result, temperature gradient may exist within

143

pressure vessel possibly due to the differences in the thermal properties such as specific

heat capacity, thermal diffusivity and physicochemical properties such as composition,

density, voidage, polarity, etc. (Ramaswamy et al., 2005). Previous attempts to study the

temperature distribution profile within the high pressure chamber involve use of thermo-

fluid dynamic based mathematical models (Carroll et al., 2003; Chen et al., 2007;

Hartmann, & Delgado, 2005; Hartmann et al., 2004) and enzyme/protein based time-

temperature-pressure indicators (Van der Plancken et al., 2008; Rauh et al., 2009;

Garuwet et al., 2010; Gogou et al., 2010). Most enzyme based time-temperature-pressure

indicators have been studied at modest high pressure pasteurization temperatures below

70oC.

Development of biochemical indicators to monitor thermal process non

uniformities within a pressure chamber during PATP could help the industry to ensure

product safety and optimize the process. Kim and Taub (1993) suggested that certain

chemical compounds, such as Maillard reaction products, formed in the food during

thermal processing could be used as indicators of heating patterns. Three biochemical

markers have been commonly identified in foods, viz. 2,3-dihydro-3,5-dihydroxy-6-

methyl-4(H)-pyran-4-one (referred to as M-1), 4-hydroxy-5-methyl-3(2H)-furanone (M-

2) and 5-hydroxymethylfurfural (M-3) (Kim and Taub, 1993). These markers are the

degradation products of Maillard reaction between carbonyl group of a reducing sugar

and amine group of a suitable reactive amino acid (Lau et al., 2003; Wang et al., 2004).

Number of earlier studies (Kim et al., 1996; Ramaswamy et al., 1996) utilized these

markers for investigating temperature distribution during ohmic and aseptic processing.

The relatively fast reaction rates for the formation of marker M-2 in protein rich

144

substrates at temperature beyond 100oC make this marker particularly useful in studying

heat distribution during short-time sterilization processes (Lau et al., 2003; Pandit et al.,

2006; Pandit et al., 2007a). Also, M-2 marker yield can be positively correlated with

thermal lethality and, thus, can be effectively used to locate colds spots in packaged foods

during microwave sterilization processes (Pandit et al., 2007b).

5.2 Objectives

The objective of this study was to evaluate the feasibility of applying these novel

chemical markers for studying temperature non uniformities during PATP.


5.3.1 Materials

Whey protein isolate (97.6±0.3%; Bipro) and whey protein concentrate (80%)

were purchased from Davisco Foods International, Eden Prairie, MN. D-ribose (≥99%)

and 4-Hydroxy-2,5-dimethyl-3(2H)-furanone (purum ≥99%) (M-2 chemical marker

standard) were purchased from Sigma-Aldrich (St. Louis, MO). Morton table salt

(Morton Salt, IL) was purchased from a local grocery store. Sodium acetate (anhydrous)

and potassium bicarbonate (99.5-101.5% USP) were purchased from Fisher Scientific,

Pittsburgh, PA.

5.3.2 Buffer solutions

10% acetate buffer (pH 7.0) and 5% bicarbonate buffer (pH 10.0) were prepared

by dissolving respective amounts of sodium acetate and potassium bicarbonate in water.

145

5.3.3 Preparation of whey protein gels

Whey protein gels were prepared by modifying the formulation suggested by Lau

et al. (2003). The formulation was modified (by optimizing the concentrations of whey

protein concentrate, isolate and water) to minimize syneresis from whey protein gels

under high pressure and thus prevent the migration of M-2 marker. 200 g batches of whey

protein gel consisted of 24 g whey protein concentrate/100g gel mix, 11g whey protein

isolate/100g gel mix, 1g D-ribose/100g gel mix, 0.8g salt/100g gel mix and 63.2g de-

ionized water/100g gel mix. Briefly, respective quantities of D-ribose and salt were

dissolved in water and mixed with weighed amounts of whey protein concentrate and

isolate in a lab blender (Oster 10 speed blender, Boca Raton, FL) for 1 min to obtain a

paste like consistency. The pH of the paste was 6.10. Appropriate amounts of sodium

acetate and potassium bicarbonate buffer solutions were added to prepare paste with a pH

of 8.25. The samples were stored overnight under refrigerated conditions to ensure

complete protein hydration. 200g hydrated paste was poured in rectangular Nalgene

bottle and the bottle were covered with aluminium foils to prevent evaporation of water

during gel formation. Gel was formed by placing the Nalgene bottles containing the

hydrated paste in a water bath (Isotemp 128, Fisher Scientific, Pittsburgh, PA)

maintained at 80oC for 40 min. The whey protein gel was immediately cooled in an ice-

water mixture and refrigerated at 4oC till further processing and analysis. Earlier studies

have shown that heating at 80oC for 40 min causes negligible browning in whey protein

gels (Lau et al., 2003), the gels containing D-ribose show prominent browning at

temperatures only above 100oC (Pandit et al., 2007a).

146

5.3.4 Processing the whey protein gels

Uniform 7 cm X 2 cm X 0.1 cm sections were cut from the whey protein gel

blocks and immediately vacuum packaged (Spiromac vacuum sealer, model 450 T,

Québec, Canada) in polypropylene pouches (76.2 µm) Deli, NS1D30-155215, Thomson

Equipment and Supply (Cincinnati, OH). Vacuum packaging did not have any observable

effect on the physical characteristics of the gel. The packaged samples were subjected

various pressure-heat combinations as outlined below.

5.3.5 High-pressure kinetic tester

A high-pressure kinetic tester (pressure test unit PT-1, Avure Technology Inc.,

Kent, WA) was used to process the gel samples. A 54-ml stainless steel (SS-316)

pressure chamber was immersed in a temperature-controlled bath to maintain the desired

process conditions (30oC for HPP and 105

oC for PATP). Propylene glycol (57-55-6,

Avatar Corporation, University Park, IL) was used as the pressure transmitting medium

as well as heating medium in the temperature controlled bath. The desired pressure was

generated at the rate of 18.42 MPa/s using an intensifier (M-340 A, Flow International,

Kent, WA) connected to a hydraulic pump (model PO45/45-OGPM-120, Interface

Devices, Milford, CT). The depressurization rate was approximately 2 s. The pressure

holding times provided in Table 5.1 do not include pressurization or depressurization

times. More details about the equipment are described elsewhere (Ratphitagsanti et al.,

2009)

147


Whey protein gel samples (~3.5 g each) vacuum sealed in pouches were

compressed to 350 or 700 MPa and held for 0, 5, 10 and 20 min. at 30oC in the high

pressure PT-1 kinetic tester. Before HPP treatment, the samples were preconditioned in

ice–water mixture for 10 min. and placed inside a 10-ml polypropylene syringe (model

309604, Difo, Becton Dickinson, Franklin Lakes, NJ), which served as the sample holder.

After loading the pouch inside the sample holder, the sample holder was filled with

approximately 6 ml of chilled water (from the ice-water mixture) to ensure that

immediate vicinity of the sample pouch had similar temperature and heat of compression

characteristics as that of the whey protein gel. To minimize heat exchange with the

surrounding glycol in bath, the sample holder was wrapped with two layers of insulating

material (Sports Tape, CVS pharmacy Inc., Woonsocket, RI) (Nguyen et al., 2007). The

initial temperatures of the samples were determined by using the following equation

(Rasanayagam et al., 2003; Nguyen et al., 2007) and by performing preliminary

experiments.

𝑇3′ = 𝑇2 + (𝐶𝐻𝑖∗𝑀𝑖)𝑖

1 (

∆𝑃

100) + ∆𝑇H ………………………………………………..(1)

where, T3’ is the target temperature, T2 is the initial sample temperature, CHi is the heat-

of-compression value of component i of the sample (defined as temperature increase per

100 MPa during sample pressurization), Mi is the mass fraction of component i in the

sample, ΔP is the process pressure and ΔTH is the temperature gain (lost) by the test

sample from (to) the surrounding glycol bath. Sample temperature history at various

148

stages of high pressure treatment is given in Table 5.1. After processing, the samples

were immediately withdrawn and stored at 4oC until analyzed.

5.3.7 Pressure assisted thermal processing

Whey protein gel samples were also pressure (350 and 700 MPa) treated under

elevated heat (105oC) conditions for 0, 5, 10 and 20 min. using high pressure PT-1 kinetic

tester described earlier. The samples and the sample holder (10-ml polypropylene

syringe) were preconditioned in a hot water bath (Isotemp 128, Fisher Scientific,

Pittsburgh, PA) for 5 min. The water bath was maintained at respective predetermined

temperatures for each of the pressures (see Table 5.1). The sample pouch was then

inserted in the syringe and remainder of the syringe was filled with warm water and

immediately inserted in the pressure chamber for pressurization. The temperature history

of samples at various stages of PATP treatments is given in Table 5.1. After processing,

the samples were immediately cooled in ice–water mixture and subsequently stored at

4oC until analyzed.

5.3.8 Thermal processing

Preheated glycol at 105oC in the temperature control bath of the high pressure

vessel was used for thermal processing (105oC for 0, 5, 10 and 20 min.) of whey protein

gel samples. Whey protein gel pouches (discussed in previous sections) were immersed

in hot propylene glycol (105oC) and held for 0, 5, 10 and 20 min after which they were

immediately cooled in ice–water mixture and refrigerated at 4oC until analyzed.

149

5.3.9 Analysis

5.3.9.1 High pressure liquid chromatography (HPLC)

Chemical marker (M-1) yields were quantified using HPLC. HP 1050 system

(Hewett Packard, Plainsboro, NJ) equipped with a Waters photodiode array detector

(Waters Corp, Milford, MA) and a solvent delivery system was used. 1.2 g sample was

ground in a mortar pestle with 10 ml 10 mM H2SO4. The homogenous paste was

centrifuged (527 X g) for 10 min. and the supernatant was filtered through 0.45 µm nylon

membrane filters in HPLC vials. The filtrate was injected into an HPLC fast acid analysis

column (Bio-RAD, Hercules, CA) using automatic injection system (HP 1050, Hewlett

Packard Co., Plainsboro, NJ). 10 mM H2SO4 at a flow rate of 1 ml/min was used as the

mobile phase. Absorbance of the eluting compounds was measured at 285 nm (Kim and

Taub, 1993).

To quantify and characterize M-2 (4-Hydroxy-5-methyl-3(2H)-furanone) yield in

control and processed whey protein gels, a standard M-2 curve was generated using 4-

Hydroxy-2,5-dimethyl-3(2H)-furanone standard obtained from Sigma Aldrich (St. Louis,

MO). Concentration of the M-2 marker in processed and control samples was expressed

as mg/100 g whey protein gel.

5.3.9.2 Kinetics of M-2 formation

Formation of M-2 in whey protein gels containing 1g ribose/100g gel mix

is given by eqn. 2 (Lau et al., 2003)

𝑑𝐶

𝑑𝑡= 𝑘(𝐶∞ − 𝐶)𝑛 ……………………………………………………………………..(2)

150

𝑤ℎ𝑒𝑟𝑒 𝐶∞ is the concentration at saturation and C is the concentration at time t. k is

the reaction rate constant and n is the reaction order.



PA). The influence of treatment type (HPP, PATP, or TP), treatment time, gel pH as well

as their interaction on the formation chemical marker M-2 in whey protein gels was

analyzed. Pairwise comparisons for the means of treatment variables (factors) were

evaluated with Tukey’s test at 5% significance level (P < 0.05).


Formation of M-2 (4-Hydroxy-5-methyl-3(2H)-furanone) under combined pressure-

heat treatment conditions

A typical chromatogram of standard 4-Hydroxy-5-methyl-3(2H)-furanone at 285

nm is shown in Figure 5.1. The marker compound eluted between 5-8 minutes.

Chemical marker M-2 (4-Hydroxy-5-methyl-3(2H)-furanone) formation in whey

protein gels (containing 1g ribose/100g gel mix) as a function of pressure (350 and 700

MPa), temperature (30 and 105oC) and holding time (0, 5, 10 and 20 min) is shown in

Figure 5.2 (at pH 6.10) and Figure 5.3 (at pH 8.25), respectively.

At pH 6.10, the formation of M-2 marker in whey protein gels during thermal

processing at 0.1 MPa and 105oC increased with holding time (Figure 5.2). However,

whey protein gels treated with combined pressure-temperature combinations showed a

distinct inhibitory/degradation effect on M-2 formation. The magnitude of this inhibitory

151

and/or degradation effect increased as the pressure was increased from 350 to 700 MPa

(see Figure 5.2). Under combined pressure-temperature treatments, increase in

temperature from 30 to 100oC increased the M-2 marker yield. Within the experimental

conditions of the study, increase in time did not show a significant increase in the marker

formation for samples processed at 700 MPa, 105oC and 350-700 MPa at 30

oC. Thus,

maximum M-2 marker yield was obtained in thermally processed gels held at 0.1MPa,

105oC for 20 min, whereas the minimum M-2 marker was formed in whey protein gel

samples processed at 700 MPa and 30oC.

Kinetics constants for marker formation during various pressure-heat treatments

The marker formation during thermal processing followed a first order kinetics.

This is in agreement with that reported by other researchers for temperature only

treatments (Lau et al., 2003). The rate constants of marker M-2 formation under different

processing conditions are summarized in Table 5.2. The rate constant in thermally

processed gel samples (0.1 MPa, 105oC) was 0.077 min

-1, whereas application of pressure

(350 and 700 MPa) at 105oC inhibited the marker formation. The rate constants for

marker formation under 350 and 700 MPa at 105oC were 0.023 and 0.0033 min

-1

respectively. Treatment of gel samples at 350 and 700 MPa at low temperature (30oC)

showed negative rate constants (Table 2 -0.0013, -0.0014 min-1

respectively) for the M-2

marker formation, thus confirming degradation/inhibition effects of pressure on the

formation of M-2.

At pH 8.25 the marker yield was significantly lower (p<0.05) than that at pH 6.10

regardless of the temperature and pressure used (Figure 5.3). Although the formation of

152

M-2 followed first order reaction kinetics at 0.1 MPa and 105oC and M-2 concentration

increased with holding time, two consecutive reactions viz. formation and decay were

seen to occur during processing under 350 and 700 MPa. Under combined pressure-

temperature conditions (350 and 700 MPa, 30 and 105oC) an increase in concentration of

M-2 was seen during the first five minutes of processing after which a decrease was

observed. This observation is similar to that reported at pH 7.0 by Bristow and Isaacs

(1999). The differences in concentration and time required to reach maximum could be

attributed to differences in pH values, differences in reaction systems and processing

conditions used. At either pH (viz. 6.10 and 8.25), the extent of browning in the gel

samples could not be directly related to the M-2 marker formation. The significance of

independent variables (time, temperature, pH and pressure) and their interactions in the

formation of M-2 marker is shown in Table 5.3. It is interesting to note that each of the

variables contributed significantly toward the marker yield (p<0.05).

The effects of pressure, temperature and pH on the formation of M-2 (a Maillard

reaction product) could be explained by understanding the fate of Maillard reaction

products under different conditions. At atmospheric pressure (0.1 MPa), the Maillard

reaction is known to occur in 3-4 stages. In the first stage carbonyl group from a reducing

sugar such as ribose reacts with amino group from the amino acid to form an imine which

rearranges to the amino form (Amadori rearrangement) and subsequently is converted to

volatailes and polymerized compounds (melanoidins) (Ames, 1992). Under normal

conditions of pressure (0.1 MPa), M-2 formation in a Maillard reaction is favored by

weak acidic (pH>5.0) or alkaline (pH>7.0) conditions which promote 2, 3 enolization

leading to the formation of furanones (M-2) (Feather, 1981; Kim et al., 1996).

153

Furthermore, in a whey protein gel system containing 1g ribose/100g gel mixture, M-2

formation is known to follow first order kinetics at atmospheric pressure (0.1 MPa) and

the reaction rate is directly proportional to the process temperature (Lau et al., 2003). The

kinetic data at 0.1 MPa and 105oC in this research is in agreement with that reported

above by Lau et al. (2003).

A comparison of M-2 yields in whey protein gels of pH=6.10 held for any given

time at 105C under 0.1, 350, and 700 MPa at pH of 6.10 (Figure 5.2) clearly suggests

that high pressure, suppressed certain steps in a Maillard reaction. Although attempts

have been made to elucidate the mechanism of inhibition and/or degradation of Maillard

products under pressure (Tamaoka et al., 1991; Isaacs and Coulson, 1996; Bristow and

Isaacs, 1999), the mechanism still stands predictive. Pressure has been proposed to

suppress the condensation and browning reactions (Tamaoka et al., 1991). Use of

hyperfine ESR spectra has shown that diffusion rate of unstable free radicals is greatly

reduced in a Maillard system exposed to high pressures. Since several advanced steps in

Maillard reaction involve the generation and participation of free radicals (Tamaoka et

al., 1991), this suppression effect could have an important bearing on the fate of Maillard

products. By determining the activation volume of the formation and inhibition of

Maillard reaction products in model systems, it has been suggested that pressure

accelerates the formation of Amadori rearrangement products and inhibits their

degradation pathway (Isaacs and Coulson, 1996). Since degradation of Amadori

compounds is responsible for formation of heterocyclic Maillard products and

melanoidins, inhibition of their degradation would decrease the formation of Maillard

reaction products. Further studies suggest that reduction in the peak value of M-2 formed

154

under pressure is a result of both, retardation in its rate of formation and increase in its

rate of decomposition (Bristow and Isaacs, 1999).

Although pH>5.0 and alkaline pH (pH>7.0) favors the formation of M-2 marker,

significant differences in the formation of M-2 exist as the pH is increased from 6.10 to

8.25. At higher alkaline pH values nitrogen heterocyclic products have been shown to

form (Bristow and Isaacs, 1999). Likewise, as observed in the present study, the decrease

in the formation of M-2 marker at pH 8.25 could be attributed to a dominating pH effect.

Due to complex pressure-heat interactions, the M-2 marker investigated in study

may not be a suitable marker for evaluating thermal non-uniformities during PATP. On a

positive note, inhibition of certain stages of Maillard reaction under high pressure

processing conditions could be attractive for food both food processors and consumers

interested in adopting this technology. Further discussion on this topic is beyond the

scope of this research. Although M-2 marker might not be effective in mapping PATP

non-uniformities, it is worth noting that other enzyme/protein based markers have been

proposed for estimating thermal non-uniformities under certain high pressure processing

conditions (Garuwet et al., 2010; Gogou et al., 2010). Gogou et al. (2010) employed

Thermomyces lanuginosus xylanase enzyme up to 600 MPa and 50-70oC and reported a

synergistic effect of pressure with temperature on the enzyme inactivation. A first order

inactivation kinetics was observed. Similarly, Garuwet et al. (2010) evaluated the

potential of using Bacillus amyloliquefaciens α-amylase based indicator for mild

pasteurization conditions. After exposing the above enzyme to isobaric-isothermal

treatments up to 680 MPa and 10-45oC and dynamic pressure-temperature treatments

(350-600 MPa and initial temperature 10-25oC), the enzyme activity was measured. More

155

research is needed to develop real-time, reliable, sensitive and versatile industrially

relevant sensors that can be used for mapping pressure-heat related non-uniformities with

precision, accuracy and reproducibility especially during high pressure sterilization or

pressure assisted thermal sterilization (PATS) conditions.

5.5 Conclusions

The formation of M-2 marker at atmospheric pressure was a function of

temperature and holding time and was found to follow first order kinetics. However,

pressure exerts a dominating effect in inhibition and/or degradation of the chemical

marker M-2, thus introducing a complex effect on the formation of M-2. Also, pH has a

significant influence (p<0.05) on the formation of M-2 and increase in pH from 6.10 to

8.25 strongly inhibits the formation of M-2. Due to the confounding interaction of

pressure with temperature in yielding M-2 marker, 4-hydroxy, 5-methyl, 3(2H) furanone

(M-2) may not be a suitable marker for evaluating pressure-heat uniformity during PATP.

Acknowledgements

We gratefully acknowledge the financial support from Ohio Agricultural

Research and Development Center (OARDC) (grant # 2008-020) and thank Dr. Steven J.

Schwartz for providing analytical resources and Rachel Kopec for her suggestions.

156

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Table 5.1 Temperature histories at different stages of processing during high pressure processing (HPP; 350 and 700 MPa at 30oC)

and pressure-assisted thermal processing (PATP; 350 and 700 MPa at 105oC) of whey protein gel samples.

Treatment

Processing

Pressure

(MPa)

Processing

time

(s)

Temperature at different stages during processing (oC) Time required at different

stages of preprocessing (s)

Preprocess

Immediately

before

pressurization

Immediately

after

pressurization

Pressure

holding

Depressurization

Preprocess

Pressure come

up time

HPP 350 0.1 14.0±1 17.3±0.6 28.2±1.1 30.0±0.4 19.1±0.8 618±5 16±1

30oC 300 14.0±1 17.3±0.6 28.2±1.1 30.3±0.2 19.1±0.8 618±5 16±1

600 14.0±1 17.3±0.6 28.2±1.1 30.1±0.4 19.1±0.8 618±5 16±1

1200 14.0±1 17.3±0.6 28.2±1.1 30.3±0.2 19.1±0.8 618±5 16±1

700 0.1 1.0±1 7.0±0.5 28.4±0.7 30.6±0.6 11.6±0.9 618±5 35±2

300 1.0±1 7.0±0.5 28.4±0.7 30.8±0.4 11.6±0.9 618±5 35±2

600 1.0±1 7.0±0.5 28.4±0.7 30.4±0.7 11.6±0.9 618±5 35±2

1200 1.0±1 7.0±0.5 28.4±0.7 30.1±0.3 11.6±0.9 618±5 35±2

PATP 350 0.1 84±1 87.8±0.9 103.4±0.5 105.1±0.5 89.1±1.1 326±7 16±1

105oC 300 84±1 87.8±0.9 103.4±0.5 104.9±0.6 89.1±1.1 326±7 16±1

600 84±1 87.8±0.9 103.4±0.5 105.2±0.5 89.1±1.1 326±7 16±1

1200 84±1 87.8±0.9 103.4±0.5 105.3±0.4 89.1±1.1 326±7 16±1

700 0.1 57±1 69.5±0.9 103.9±0.7 105.5±0.4 77.1±1.2 326±7 35±2

300 57±1 69.5±0.9 103.9±0.7 105.2±0.4 77.1±1.2 326±7 35±2

600 57±1 69.5±0.9 103.9±0.7 104.9±0.6 77.1±1.2 326±7 35±2

1200 57±1 69.5±0.9 103.9±0.7 104.7±0.3 77.1±1.2 326±7 35±2

16

1

162

Table 5.2 Rate constants of marker M-2 formation/inhibition/degradation under different

processing conditions at gel pH 6.10 and pH 8.25.

Processing conditions Rate constant (k

min-1

)*

Correlation

coefficient (R2) pH Temperature

(oC)

Pressure

(MPa)

6.10

105

350 0.0232 0.99

700 0.0033 0.69

0.1 0.0772 0.99

30

350 -0.0013* 0.87

700 -0.0014* 0.97

8.25

105

350 -0.04 0.3**

700 -0.022 0.89

0.1 0.056 0.99

30

350 -0.006 0.92

700 0.004 0.34**

* Negative sign indicates degradation/inhibition of marker M-2 under combined pressure-

temperature conditions. Whereas temperature favored the formation of M-2, pressure had

a degradation/inhibition effect on M-2.

**At pH 8.25, the formation/degradation/inhibition of M-2 marker did not follow first

order kinetics. Hence the lower values of correlation coefficients

163

Table 5.3 Influence of various independent variables (pressure, temperature, time, and

pH) and their interactions on chemical marker M-2 formation in whey protein gels

containing 1% ribose.

Variable Probability (P)

(Effect of variables on M-2

formation)

Pressure (MPa) a 0.001

Temperature (oC)

a 0.000

Time (min) a 0.040

pH a 0.000

pH*Time a 0.030

pH*Pressure a 0.028

pH*Temperature a 0.005

Time*Temperature a 0.021

Pressure*Temperature a 0.005

a Significant influence on decrease in the concentration of M-2 marker (P<0.05)

164

Figure 5.1 HPLC Chromatogram of chemical marker M-2 (4-Hydroxy-5-methyl-3(2H)-

furanone) extracted at 285 nm.

165

Figure 5.2 Formation and/or inhibition of M-2 marker in whey protein gel samples

containing 1g ribose/100g gel mix ribose subjected to various time-temperature-pressure

combinations (pH = 6.10). Values represent mean ± SD of 3 independent replicates.

0

10

20

30

40

50

60

70

80

PATP 350 Mpa/105 C

PATP 700 Mpa/105 C

HPP 350 Mpa/30 C

HPP 700 Mpa/30 C

Thermal 0.1 Mpa/105 C

M-2

mar

ker

form

atin

o m

g/1

00

g g

el

Treatment type

0 min

5 min

10 min

20 min

166

Figure 5.3 Formation and/or inhibition of M-2 marker in whey protein gel samples

containing 1g ribose/100g gel mix subjected to various time-temperature-pressure

combinations (pH = 8.25). Values represent mean ± SD of 3 independent replicates.

0

2

4

6

8

10

12

14

16

18

PATP 350 Mpa/105 C

PATP 700 Mpa/105 C

HPP 350 Mpa/30 C

HPP 700 Mpa/30 C

Thermal 0.1 Mpa/105 C

M-2

Ma

rke

r C

on

ce

ntr

ati

on

(m

g/1

00

g

gel)

Treatment type

0 min

5 min

10 min

20 min

167

Figure 5.4 Flowchart outlining the experimental design.

Whey protein

gels containing

1% ribose

pH = 6.1

Whey protein gels

containing 1% ribose

pH = 8.25

High pressure

processing

(350 and 700 MPa,

30oC,

0, 5, 10 and 20

min.)

Pressure assisted

thermal

processing

(350 and 700

MPa, 105oC,

0, 5, 10 and 20

min.)

Thermal processing

(0.3 MPa,

105 oC,

0, 5, 10 and 20

min.)

Control

(0.1 MPa

30oC,

0, 5, 10 and 20

min.)

Chemical marker (M-2) extraction

followed by High Performance

Liquid Chromatography

Data analysis

pH measurement

168

CHAPTER 6: CONCLUSIONS

Combined pressure-temperature effects on carotenoid retention and bioaccessibility

in tomato juice

1. Combined pressure-thermal treatment (HPP and PATP) resulted in increased lycopene

extractability (up to 12%) as compared to the unprocessed control. TP did not alter the

lycopene content of the tomato juice and samples processed thermally showed similar

lycopene levels as the unprocessed control.

2. All-trans β-carotene showed significant degradation (p<0.05) as a function of pressure,

temperature, and time. Its retention in processed samples varied between 60-95% of

levels originally present in the control.

3. Limited micellarization of lycopene crystals was observed regardless of the processing

method used. Of the total lycopene present in the raw juice, less than 0.5% lycopene

appeared in the form of micelles.

4. Electron microscopic images showed denser and more prominent lycopene crystals in

HPP and PATP processed juice than in thermally processed juice. Lycopene crystals

appeared to be enveloped regardless of the processing conditions used.

5. The processed juice (HPP, PATP, TP) showed significantly higher (p<0.05) all-trans

β-carotene micellarization as compared to the raw unprocessed juice (control).

169

Interestingly, hot break juice subjected to combined P-T treatments showed 15-30%

more all-trans β-carotene micellarization than the raw juice subjected to combined P-T

treatments.

6. This study demonstrated that combined pressure-heat treatments increases lycopene

extractability. However, the in-vitro bioaccessibility of carotenoids (more

specifically, lycopene) was not significantly different among the treatments (TP,

PATP, and HPP) investigated.


combinations

1. Within the range of experimental conditions used in the study, combined pressure-

temperature treatments improved the extractability of lycopene from two different

tomato cultivars with different lycopene contents.

2. All-trans lycopene was fairly stable to isomerization during processing and the cis

isomer content of control and processed juice did not differ significantly.

3. During storage, the stability of lycopene in tomato juice was influenced by a complex

interaction of method of processing, type of cultivar, storage temperature and storage

time.

4. HPP and PATP processed samples better retained both total lycopene and cis isomers

during 52 weeks of storage and also showed better color retention.

5. Tomato juice samples processed using various treatments (HPP, PATP and thermal)

were microbiologically stable over 52 weeks of storage.

170

6. A 2-step first order equation could be effectively used to predict the changes in

lycopene concentration over the course of storage.

Combined pressure-temperature effects on the chemical marker (4-hydroxy-5-

methyl-3(2h)-furanone) formation in whey protein gels

1. The formation of M-2 marker at atmospheric pressure was a function of temperature

and holding time and was found to follow first order kinetics.

2. Pressure exerted a dominating effect in inhibition and/or degradation of the chemical

marker M-2, thus introducing a complex effect on the formation of M-2.

3. pH has a significant influence on the formation of M-2 and increase in pH from 6.1 to

8.25 strongly inhibited the formation of M-2.

4. Due to the confounding interaction of pressure with temperature in yielding M-2

marker, 4-hydroxy, 5-methyl, 3(2H) furanone (M-2) may not be a suitable marker for

evaluating pressure-heat uniformity during PATP.

Future research recommendations

1. Conduct research methods to improve the bioaccessibility of carotenoids from

combined pressure-temperature processed tomato juice.

2. Compare the effects of semi-continuous vs. batch P-T processing on carotenoids in

tomato juice.

171

3. Conduct a detailed consumer sensory acceptability and perception study.

4. Feasibility study on the scale-up of P-T processing for processing tomato juice.

5. Research on developing a reliable bio-marker to study P-T non-uniformities during

combined pressure-temperature processing.

172

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188

APPENDIX 1

Nutrient content of tomatoes

Nutrient

Concentration

(per 100 grams)

Proximate components (g/100g)

Water 89.44±0.32a

Energy (KJ) 134.0

Protein 1.64±0.08a

Total lipids 0.28±0.03a

Ash 1.35±0.06a

Carbohydrates, by difference 7.29

Total dietary fiber 1.90

Minerals (mg/100g)

Calcium, Ca 34±3.90a

Iron, Fe 1.30±0.17a

Magnesium, Mg 20.0

Phosphorous, P 32.0

Potassium, K 293±21.06b

Sodium, Na 132±12.38c

Zinc, Zn 0.27

Copper, Cu 0.183

Manganese, Mn 0.183

Selenium, Se (µg/100g) 0.6

Vitamins (mg/100g)

Vitamin C (Ascorbic Acid) 9.2±0.95d

Thiamin 0.08

Riboflavin 0.05

Niacin 1.22

Pantothenic Acid 0.28

Vitamin B-6 0.15

Total folate (µg/100g) 13.0

Folate, Food (µg/100g) 13.0

Continued

189

Folate, Dietary Folate Equivalent (µg DFE/100g) 13.0

Vitamin A, Retinol Activity Equivalent (µg RAE/100g) 35±3.60d

Vitamin A, IU 699±71.92d

Lipids (g/100g)

Fatty acids, total saturated 0.040

16:00 0.027

18:00 0.010

Fatty acids, total monounsaturated 0.043

16:1 undifferentiated 0.001


Fatty acids, total polyunsaturated 0.113



Amino Acids (g/100g)

Tryptophan 0.012

Threonine 0.040

Isoleucine 0.037

Leucine 0.057

Lysine 0.057

Methionine 0.013

Cystine 0.020

Phenylalanine 0.040

Tyrosine 0.027

Valine 0.040

Arginine 0.038

Histidine 0.023

Alanine 0.045

Aspartic acid 0.215

Glutamic acid 0.570

Glycine 0.038

Proline 0.030

Serine 0.042

(Source: U.S. Department of Agriculture, Agricultural Research Service (2010). USDA

National Nutrient Database for Standard Reference, Release 23. Nutrient Data Laboratory

Home Page, http://www.ars.usda.gov/ba/bhnrc/ndl)

Where applicable, values represent mean ± SEM of replications

a mean ± SEM of 31 data points;

b mean ± SEM of 12 data points;

c mean ± SEM of 27

data points; d mean ± SEM of 33 data points

Table 2.2 continued

http://www.ars.usda.gov/ba/bhnrc/ndl

190

APPENDIX 2

Pressure-temperature-time history of tomato juice samples processed using pressure

assisted thermal processing (PATP) and thermal processing (TP)

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