Quality Evaluation of Vegetables Processed By

QUALITY EVALUATION OF VEGETABLES PROCESSED BY

MICROWAVE STERILIZATION/PASTEURIZATION

JING PENG

A dissertation submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY Department of Biological Systems Engineering

May 2014

To the Faculty of Washington State University:

The members of the Committee appointed to examine the dissertation of JING PENG find it satisfactory and recommend that it be accepted.

______Juming Tang, Ph.D, Chair

______Diane M. Barrett, Ph.D

______Shyam S. Sablani, Ph.D

______Joseph R. Powers, Ph.D

ACKNOWLEDGEMENT

Having read so many theses, it is finally my turn to write my own and express my gratitude to the people who supported me all the way throughout my Ph.D study here in Pullman. It feels so good!

First of all, I would like to express my deepest gratitude to my primary advisor, Dr. Juming

Tang, who is a role model in my professional development, gives me support and guidance, and keeps delivering positive energy to me whenever I need it.

I also greatly thank my committee members Drs. Diane M. Barrett (from UC Davis), Shyam S.

Sablani and Joseph R. Powers for their continuous and valuable advice and suggestions during my whole research study. I am so lucky to have these professionals and experts in their own areas of expertise on my committee. Thank you for all of your efforts to maintain multiple- campus communication and make meetings happen (in person or through conference calls). I really appreciate that!

I would also like to thank Drs. Boon Chew and John Fellman for providing advices and suggestions for my research and study. My great thanks as well go to Dr. Frank Liu, Dr.

Zhongwei Tang, Dr. Jae-Hyung Mah, Stewart Bohnet, Huimin Lin, Frank Younce, Peter Gray,

Feng Li, Vince Himsl, Jonathan Lomber and Galina Mikhaylenko, for their technical support during my research. I would like to extend my gratitude to my previous and current Food

Engineering Club colleagues who have been with me these years, Shunshan Jiao,

SumeetDhawan, Ofero Abagon Caparino, Fermin Jr. Pangilinan Resurreccion, Roopesh

Syamaladevi, BaluNayak, Yang Jiao, Wenjia Zhang, Donglei Luan, Ellen Rose Bornhorst,

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Rossana Villa Rojas, Kanishka Bhunia, Deepali Jain, Poonam Bajaj, Hongchao Zhang, Rajat

Tyagi and Ravi Tadapaneni.

I am also grateful for support and advice from my other friends Keke, Iris, Qianqian, Hong,

Liang, Jeff, Angie, Susan, Daisy and those who showed up in my life here in Pullman and who make it more beautiful.

Last but not least, I would like to thank my dearest parents and my young brother Xuanqi, who support me unconditionally and have been endless sources of encouragement and love for me. I would like to delicate this thesis to them, and to myself.

QUALITY EVALUATION OF VEGETABLES PROCESSED BY

MICROWAVE STERILIZATION/PASTEURIZATION

ABSTRACT

by Jing Peng, Ph.D. Washington State University May, 2014

Chair: Juming Tang

Microwave (MW) heating overcomes the disadvantages of slow conductive/convective heat transfer inherent in conventional thermal processes, and therefore has the potential to produce safe and high quality vegetable products. This research was conducted to evaluate the quality attributes of pre-packaged diced carrots after MW pasteurization and of diced tomatoes after

MW sterilization, in comparison with those subjected to conventional thermal processing. A systematic study of developing MW sterilization or pasteurization processes for tomato and carrot products, and evaluating their influence on product quality is presented in this thesis. A

MW-assisted sterilization thermal process (MAST) achieving a target F value of no less than 6 min was developed for processing diced tomatoes packaged in 8-oz pouches, which can deliver a

5D thermal treatment to Bacillus coagulans ATCC 8038 spores. For diced carrots, MW assisted pasteurization processes (MAP) with F90°C = 3 min and F90°C = 10 min were developed to achieve at least 6 D reductions of NP C. botulinum type E spores. Thermal resistance of the target

bacterium (B. coagulans spores) in tomatoes was characterized, and kinetics of texture degradation of carrots were investigated for developing thermal processes for these products.

Tomato/carrot dices with added salts (NaCl/CaCl2) at commonly used commercial levels were processed, and their dielectric properties were determined and used for computer simulation of heating patterns and cold spot locations in sample pouches. The quality related attributes of processed tomatoes (drained weight, soluble solids, color, texture, ascorbic acid, and lycopene content) and carrots (color, texture, pectin methylesterase activity, and carotenoids) were assessed. The results of quality evaluation of the processed products showed that the impact of

MW processing on the quality of vegetables depends on the characteristics of the vegetables and the specific quality parameters tested.

Table of Contents Chapter 1. Introduction ...... 1 1. Research background and problem statements ...... 1 2. Objectives ...... 5 3. Dissertation outline ...... 6 References ...... 9 Chapter 2. Literature Review-Thermal Pasteurization of Vegetables...... 14 1. Pathogens of concern and process design for thermal pasteurization ...... 14 1.1. Regulations and standards of pasteurization in the U.S...... 15 1.2. Regulations and standards of pasteurization in Europe ...... 18 2. Effect of thermal pasteurization on vegetable quality ...... 19 2.1. Color ...... 19 2.2. Texture ...... 20 2.3. Carotenoids ...... 22 2.4. Phenolics and antioxidant activity ...... 24 2.5. Vitamins ...... 25 2.6. Other components ...... 26 3. Enzyme, storage and shelf-life of pasteurized vegetables ...... 27 References ...... 30 Chapter 3. Thermal Inactivation Kinetics of Bacillus coagulans Spores in Tomato Juice ...... 55 1. Introduction ...... 56 2. Materials and Methods ...... 57 2.1. Microorganisms ...... 57 2.2. Preparation of B. coagulans spores ...... 58 2.3. Preparation of tomato juice ...... 58 2.4. Evaluation of cold-storage time on the viability of B. coagulans in sterile distilled water and its thermal resistance in tomato juice ...... 59 2.5. Preparation and pre-conditioning of a mixture of spore suspension and tomato juice ...... 59 2.6. Evaluation of heat resistance of B. coagulans spores using oil bath ...... 60 2.7. Evaluation of heat resistance of B. coagulans spores using a capillary tube setup ...... 61 3. Results and Discussion ...... 62

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3.1. Effect of cold-storage time on the viability of B. coagulans in sterile distilled water and its thermal resistance in tomato juice ...... 62 3.2. Effect of pH on the thermal resistance of B. coagulans ATCC 8038 spores ...... 64 3.3. Effect of pre-conditioning on the thermal resistance of B. coagulans ATCC 8038 spores ...... 64 3.4. Thermal resistance of B. coagulans ATCC 8038 spores in tomato juice using a conventional oil bath ...... 65 3.5. Thermal resistance of B. coagulans 185A spores at pH 4.3 using oil bath ...... 66 3.6. Thermal resistance of B. coagulans 8038 spores at pH 4.4 (OSU experiments) ...... 67 4. Conclusions ...... 67 References ...... 69 Chapter 4. Kinetics of Carrot Texture Degradation under Pasteurization Conditions ...... 81 1. Introduction ...... 82 2. Materials and Methods ...... 85 2.1. Sample preparation ...... 85 2.2. Determination of isotonic concentration of carrot tissue ...... 85 2.3. Thermal treatment ...... 86 2.4. Texture measurement ...... 87 2.5. Kinetic analysis ...... 87 3. Results and Discussion ...... 89 3.1. Determination of the isotonic solution for the carrot tissue and its effect on carrot texture ...... 89 3.2. Effects of preheating and calcium treatment on carrot texture ...... 90 3.3. Quality versus microbial/enzyme inactivation ...... 94 4. Conclusions ...... 95 References ...... 97 Chapter 5. Dielectric Properties of Tomatoes Assisting in the Development of Microwave Pasteurization and Sterilization Processes ...... 110 1. Introduction ...... 111 2. Materials and methods ...... 114 2.1. Sample preparation ...... 114 2.2. Moisture content, pH and total soluble solids ...... 114 2.3. Determination of dielectric properties ...... 115 2.4. Measurement of ionic conductivity ...... 115

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2.5. Determination of power penetration depth ...... 116 3. Results and discussion ...... 117 3.1. Physicochemical properties of pericarp, locular and placental tissues of raw tomatoes ...... 117 3.2. Dielectric properties of pericarp, locular and placental tissues of raw tomatoes ...... 118 3.3. Effect of NaCl ...... 120

3.4. Effect of CaCl2 ...... 121 3.5. Effect of ionic conductivity on dielectric loss factor ...... 122 3.6. Penetration depth ...... 123 4. Conclusions ...... 124 References ...... 126 Chapter 6. Developing Microwave Sterilization/Pasteurization Processes for Pre-packaged diced Tomatoes/Carrots ...... 143 1. Introduction ...... 143 2. Methods and materials ...... 147 2.1. Preparation of sample pouches ...... 147 2.2. MW heating system ...... 148 2.3. Measurement of dielectric properties of tomato/carrot samples ...... 149 2.4. Determination of the heating pattern and cold spot in sample pouches ...... 150 2.5. Heat penetration test...... 151 2.6. Incubation test for diced tomatoes ...... 154 3. Results and discussions ...... 155 3.1. Dielectric properties of tomato/carrot samples ...... 155 3.2. Heating pattern and cold spot in sample pouches ...... 157 3.3. Heat penetration results ...... 158 3.4. Incubation results for diced tomatoes ...... 159 4. Conclusions ...... 159 References ...... 161 Chapter 7. Quality Evaluation of Vegetable Products Thermally Processed with Microwave and Conventional Methods ...... 176 1. Introduction ...... 178 2. Materials and Methods ...... 181 2.1. Sample preparation ...... 181

2.2. Thermal treatments ...... 182 2.3. Color of carrot and tomato dices ...... 183 2.4. Texture of carrot and tomato dices...... 184 2.5. Pectin methylesterase (PME) activity of carrots ...... 185 2.6. Carotene analysis of carrots ...... 186 2.7. Determination of pH, soluble solids, drained weight of tomatoes ...... 187 2.8. Ascorbic acid content of tomatoes ...... 187 2.9. Lycopene content of tomatoes ...... 188 2.10. Statistical analysis ...... 189 3. Results and Discussion ...... 189 3.1. Color ...... 189 3.2. Texture ...... 191 3.3. PME activity of carrots ...... 192 3.4. Carotenoids of carrots ...... 193 3.5. pH, soluble solids, drained weight of diced tomatoes ...... 194 3.6. Ascorbic acid content of tomatoes ...... 195 3.7. Lycopene content of tomatoes ...... 195 4. Conclusions ...... 196 References ...... 198 Chapter 8. Conclusions and Recommendations ...... 212 1. Major conclusions ...... 212 2. Contributions to knowledge ...... 214 3. Recommendations for future research ...... 215

List of Tables

Chapter 2

Table 1. Commonly accepted growth boundaries of pathogenic microorganisms……….. …….37

Table 2. Summary table of regulations of thermal pasteurization of foods (milk, seafood, egg and juice products) in the United States…………………………………………………..…38

Table 3. Summary table of Standards of prepackaged chilled foods (pasteurized foods) in

Europe……………………………………………………………………………………………41

Table 4: Lethal rates for L. monocytogenes and necessary process to achieve 6-log reduction of L. monocytogenes………………………………………………...... 42

Table 5. Lethal rates for C. botulinum type B1and necessary process to achieve 6-log reduction of C. botulinum type B……………………………………………...... 43

Table 6. Effects of thermal pasteurization on the quality of vegetables…………………………45

Table 7. Major enzymes related to the quality of raw and processed vegetables………………..44

Table 8. Scientific publications of pasteurized vegetables related to storage and enzyme study………………………………………………………………………………………….…..51

Chapter 3

Table 1. The effect of 4˚Cstorage on the viability of vegetative cells and spores of

B. coagulans……………………………………………………………………………...... ….72

Table 2.Comparison of D- and z-values of B. coagulansATCC 8038 and 185A spores in commercial tomato juice………………………………………………………………………73

Table 3. The effect of heating method on the thermal resistance of spores of B. coagulans

ATCC 8038………………………………………………………………………………………74

Chapter 4

Table 1. Coefficients of determination (r2) from kinetic order (n) models for carrot texture degradation at four temperatures……………………………………………………….101

Chapter 5

Table 1. Moisture content, pH and soluble solids content of pericarp, locular and placental tissues in raw tomatoes……………………………………………………………….130

Table 2. Dielectric properties of tomato pericarp, locular and placental tissues with 0.2 g/100g of NaCl and 0.055 g/100g of CaCl2 at 915 and 2450 MHz…………………………….131

Table 3. Microwave penetration depth into tomato pericarp, locular and placental tissues at 915M Hz……………………………………………………………………………………..132

Chapter 6

Table 1. Processing conditions of carrot/tomato products for equivalent MW and HW processes with regard to microbial safety………………………………………………………163

Table 2. Microbial assay of raw whole tomatoes, diced tomatoes before and after process…………………………………………………….……………………………....164

Chapter 7

Table 1. Processing conditions for carrot and tomato products for equivalent MW and

HW processes with equivalent process severity..………………………………………………203

Table 2. CIE L*, a*, b* values, total color differences (∆E), and hue angle of carrot and tomato dices under different treatments………………………………………………...... 204

Table 3. pH, drained weight, color, soluble solids of tomato samples before and after processing…………………………………………………………………………………205

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List of Figures

Chapter 2

Figure 1. Vegetable color wheel…………………………………………………………………54

Chapter 3

Figure 1. Schematic of kinetics treatment chamber, with spore suspensions within sample in capillaries……………………………………………………………………………………...76

Figure 2. D100°C-values of B. coagulans ATCC 8038 spores exposed to different pH levels in tomato juice..………………………………………………………...... 77

Figure 3. Effect of pre-conditioning time on D100°C-value of B. coagulans ATCC 8038 spores in commercial tomato juice at pH 4.3…………………………………………….………78

Figure 4. Thermal survivor curves for B. coagulans ATCC 8038 spores at different temperatures in commercial tomato juice at pH 4.0….………………………………………….79

Figure 5. Thermal survivor curves for B. coagulans ATCC 8038 spores heated at different temperatures in tomato juice adjusted to pH 4.4 ………………………………………80

Chapter 4

Figure 1. Percent change in weight of excised carrot pericarp discs in 25 ml of aqueous solution at different mannitol concentrations…………………………………………………..102

Figure 2. Thermal degradation of texture of carrot dices in isotonic solution or distilled water at different temperatures………………………………………………………..………..103

Figure 3. Effect of preheating (60°C for 20 min) on the thermal texture degradation of carrots at different temperatures…………………………………………………….………….104

Figure 4. Plot of 1/C vs. time at different temperatures (n=2)………………………………….105

Figure 5. The final texture value (F∞/F0) of pretreated carrot dices as a function of

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Temperature in different solutions……………………...………………………………………106

Figure 6. Reaction rate k of preheated carrot dices as a function of temperature in different solutions………………………………………………………………………………107

Figure 7. Arrhenius plot of texture degradation rates of carrots immersed in different solutions with pretreatment……………………………………………………………………..108

Figure 8. TDT curves of target bacteria, enzymes vs. carrot texture…………………...... 109

Chapter 5

Figure 1. Illustration of different anatomical structures of tomato fruits...... 134

Figure 2. Schematic diagram of pressure-proof test cells used for dielectric properties measurement (from Wang et al., 2003)………………………………………………………...135

Figure 3. Dielectric properties of raw tomato locular tissue as a function of temperature and frequency…………………….…………………………………………………...... 136

Figure 4. Dielectric properties of raw pericarp (♦), locular (□) and placental (△) tissues at 915 (A) and 2450 (B) MHz…………………………………………………………………..137

Figure 5. Dielectric properties of tomato pericarp (♦), locular (□) and placental (△) tissues with 0.2 g/100g of NaCl at 915 (A) and 2450 MHz (B)……………………………………….138

Figure 6. Dielectric loss factor of tomato pericarp (♦), locular (□) and placental (△) tissues at 915 (A) and 2450 (B) MHz…………………………………………………………………..139

Figure 7. Ionic conductivity of tomato locular tissue as a function of temperature……………140

Figure 8. Measured ε" and calculated εσ" of tomato locular tissue (A raw sample; B with

NaCl; C with NaCl & CaCl2) as a function of frequency and temperature…………………….141

Chapter 6

Figure 1a. Front view diagram of four sections in the MATS system at WSU………...... 165

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Figure 1b. Front view of MAP system at WSU………………………………………………...165

Figure 2. Flowchart of determination of heating pattern in diced tomato pouches……………166

Figure 3. Preparation of sample pouch with Ellab sensor……………………………...... 167

Figure 4. Illustration of computer simulation…………………………………………………..167

Figure 5. Dielectric constant and loss factor of tomato puree added with salts (A) and carrot puree (B) as a function of temperature and frequency…………….…………………….168

Figure 6. Dielectric properties of processed products with temperatures at 915 MHz…………169

Figure 7. Simulation results of temperature profiles at the cold spot in sample pouches with a small tomato piece or a big piece for temperature measurement……………………….170

Figure 8. Heating pattern and cold spot location in tomato sample pouch……………………..171

Figure 9. Dielectric properties of tomato sample compared with those of five whey protein model foods……………………………………………………………………...……..172

Figure 10. Heating pattern and cold spot in carrot sample pouch……………………...... 173

Figure 11. Example of temperature-time profile at the cold spot in the diced tomato pouch under MW (A) and HW (B) processing………………………………………………………...174

Figure 12. Example of temperature-time profiles at the cold spot in the diced carrot pouch under MW (A) and HW (B) processing………………………………………………………...175

Chapter 7

Figure 1. Color parameter b*/a* of diced carrots (A) and a*/b* tomatoes (B) under different treatments……………………………………………………………………………………….206

Figure 2. Texture of carrot dices (A) and tomato dices (B) by MW and HW processing under different conditions…………………..…………………………………………………..207

Figure 3. Residual PME activity of carrot dices by MW and HW processing under

different conditions…………………….…………………………………………………….....208

Figure 4. Total carotenoids content, α- and β-carotene contents of diced carrots under different treatments……………………………………………………………………………..209

Figure 5. Ascorbic acid content of tomato samples under different conditions………………..210

Figure 6. Lycopene content of tomato samples under different condition……………………..211

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Chapter 1. Introduction

1. Research background and problem statements

Vegetables provide essential vitamins, minerals and dietary fiber for our bodies, and form an important part of a healthy diet. Abundant phytochemicals commonly found in vegetables, such as flavonoids, phenols, and carotenoids, bring us health benefits that prevent nutritional deficiencies and reduce the risk for various cancers and diseases (Van Duyn, 1999; Scheerens,

2001). The increased public awareness of a healthy balanced diet results in increasing vegetable consumption.

The highly perishable nature of vegetables requires efficient and appropriate preservation technologies to prolong shelf life while maintaining nutritional value and sensory quality.

Possible preservation methods include cold storage, heating, freezing, drying/dehydration, chemical preservation, preservation with sugar/acids, concentration, irradiation, or combinations of those different means of methods. Among these preservation methods, thermal processing is one of the most effective means. It is widely used to produce a number of vegetable products such as juice, puree, sauces, soups, jams, slices, dices and chunks. One of the recent trends in fruit and vegetable processing is using enhanced delivery of thermal energy (e.g. UHT, microwave, ohmic) combined with new packaging materials and technologies (e.g. aseptic, modified atmosphere packaging) to provide consumers with increased choices (Dauthy, 1995).

Compared to conventional heating, microwave (MW) heating provides a relatively short heating time due to its ability to generate volumetric heating within food materials, and thus has the potential to produce high quality self-stable food products.

Application of MW heating in foods has drawn increased attention over the past decades. Many researchers have reported its application in vegetable processing and its effect on vegetable quality, for example in carrot juice (Rayman and Baysal, 2011), carrot pieces (Lemmens et al.,

2009), Brussels sprouts (Vian et al., 2007; Olivera et al., 2008), potato (Alvarez and Canet, 2001;

Barba at el., 2008), peas and spinach (Hunter and Fletch, 2002), tomato (Begum and Brewer,

2001), Swiss chard and green beans (Villnaueva et al., 2000), asparagus (Sun et al., 2007), and sweet potato purees (Steed et al., 2008). However, in most of these publications, the research was carried out in a 2450 MHz domestic MW oven or a simply modified MW oven with specially installed temperature sensors. Different heat treatment levels were achieved by adjusting input power or heating time, normally several minutes for blanching or pasteurization. Limited information is available on quality changes of vegetables processed by pilot-scale MW sterilization/pasteurization systems. Sun et al. (2007) studied color, texture, rutin content and antioxidant activity of asparagus sterilized by a MW-circulated water combination heating system. Steed et al. (2008) investigated color, phenolic content, anthocyanins, antioxidant activities and rheological properties of sweet potato purees by a continuous flow MW-assisted processing. Koskiniemi et al. (2013) evaluated the quality of acidified vegetables (broccoli, red bell pepper, and sweet-potato) pasteurized by a continuous MW processing, and observed good retention of color and texture of acidified vegetable pieces after MW pasteurization. However, no information is available on quality changes in tomatoes or carrots processed by MW sterilization or pasteurization.

Tomatoes and carrots are two of the most commonly consumed vegetables in the United States.

Americans consume three-fourths of tomatoes in processed form, most of which are thermally

processed; and one-fourth of carrots in processed form, largely canned and frozen (Lucier and

Lin, 2007; Lucier and Glaser, 2009). For processed vegetables, texture is one of the primary marketable characteristics. The changes in texture of vegetables during processing result from the chemical composition and amount of cell wall and the middle lamella, which are closely related to enzymatic and non-enzymatic changes in pectin (a cell wall polysaccharide) (Bourne,

1989; Vu et al., 2004). Enzymatic degradation of cell wall pectin is catalyzed principally by pectin methylesterase (PME), polygalacturonase (PG) and pectate lyase (PL). PME catalyzes the de-esterification of pectin, yielding carboxylated pectin with release of methanol. The demethoxylated pectin can (1) crosslink with divalent cation (primarily Ca2+, naturally present in the tissue or added during processing) between the free carboxylic acid groups on the polygalacturonic acid backbone of the pectin to form cross-links between pectin chains (a firming effect) and (2) be a substrate for PG depolymerization (a softening effect). In addition,

PL can be expressed in plant tissues and depolymerize pectins (Sila et al., 2008). Pectin can also be depolymerized in a non-enzymatic way by β-elimination, a chemical reaction that takes place at higher pH levels (>4.5) and at temperatures higher than 80ºC (Keijbets and Pilnik, 1974; Sila et al., 2008)). Since the β-elimination reaction only occurs in pectins with methyl ester groups, reducing the extent of pectin methylesterification by activating PME in a low-temperature blanch could reduce the extent of this reaction (Krall and McFeeters, 1998). Anthon et al. (2005) observed a reduction of about 2/3 texture in diced tomatoes after 1 min heating at 100ºC with very little additional change over the next 4 min, and Greve et al. (1994ab) found that the firmness of carrot tissue was lost rapidly in the first 6 min and then more slowly over the next 15 min heating in boiling water. Studies shows that the early, rapid phase of texture loss in both tomatoes and carrots under high temperature heating is due to the turgor loss resulted from the

membrane disruption, while the 2nd slower, prolonged phase of softening occurred in carrots is mainly contributed to the breakdown of pectins through β-elimination reactions (Grant et al.,

1973; Greve et al., 1994ab; Anthon and Barrett, 2005). Because the pHs of the two vegetables are different (3.9-4.4 for the tomato while 5.2-5.8 for the carrot) and β-elimination takes place only at high pH (usually pH >4.5), the β-elimination in carrots under thermal processing is much more noticeable than in tomatoes. Therefore, tomatoes and carrots were chosen as the typical vegetables for MW processing in the current study. Although the texture degradation of carrots during thermal processing has previously been investigated in several studies, most of the authors immersed the carrot samples in either distilled water or high calcium solutions (usually

0.5% CaCl2), and none of them evaluated the kinetic models with different reaction order nor selected the best-fit one to estimate the related kinetic parameters (Huang et al., 1983; Bourne,

1989; Vu et al., 2004; Smout et al. 2005).

Successful exploration of MW application to foods relies on a thorough understanding of the interaction between microwaves and food materials, and on the ability to predict and provide a desired heating pattern in foods for specific applications (Tang et al., 2002). A good understanding of the dielectric properties of food materials is essential for developing MW processing. Information on dielectric properties of tomato or carrots in solutions with salt and calcium added at commercial product levels is limited and therefore was investigated in this study. In addition, a sterilization process is designed to kill all microorganisms while minimizing quality deterioration. Information on thermal resistance of target bacteria in food materials is needed for developing and validating thermal processes. In the current study, Bacillus coagulans spores and non-proteolytic Clostridium botulinum type E spores are considered as the target

bacteria for MW sterilization of tomatoes and MW pasteurization of carrots respectively, according to their acidity (tomato products 3.9-4.4; carrot products 5.2-5.8; FDA, 2001) and desired final products. Gaze and Brown (1990) studied the thermal resistance of NP C. botulinum type E spores in carrot from 75–90ºC and reported a z-value of 9.84ºC. For the B. coagulans spores, although their resistance in tomatoes was studied at acid pHs, information about the resistance of the spores at different acidic pH levels between 4.0 and 4.5 (the commonly controlled pH range for canned tomato process) is still limited, especially under high temperatures (York et al., 1975; Pirone et al., 1989; Mallidis et al., 1990; Rodrigo, 1990;

Sandoval et al., 1992; Palop et al., 1999).

2. Objectives

The overall objective of this dissertation was to develop MW sterilization/pasteurization processes for pre-packaged vegetables, and evaluate their quality in comparison with those processed by conventional heating. Two vegetables, tomatoes and carrots were chosen because of their popularity, differences in pH and the fact that β-elimination doesn’t proceed to a great extent in tomatoes at their pH under high-temperature processing.

The specific aims of this research were to (1) conduct thermal kinetic studies of target microorganism in tomatoes; (2) conduct kinetic studies of carrot texture degradation under pasteurization conditions; (3) determine dielectric properties of tomatoes and carrots; (4) develop

MW and conventional thermal processes for pre-packaged diced tomato and carrot products; (5) evaluate quality of MW and conventional heating processed products.

3. Dissertation outline

This dissertation contains seven chapters, as follows:

Chapter 1: Introduction. This chapter generally introduces the background of this research work, states the current problems, and outlines the objectives and structure of the dissertation.

Chapter 2: Literature review. This chapter is a review of fundament concepts, general methods, data and applications of thermal pasteurization of vegetables. In detail, it includes pathogens of concern and process design for thermal pasteurization; effect of thermal pasteurization on vegetable quality; and enzyme, storage and shelf-life of pasteurized vegetables.

Chapter 3: Thermal inactivation kinetics of Bacillus coagulans spores in tomato juice. This study was conducted to characterize the thermal resistance of three strains of Bacillus coagulans

(ATCC 8038, 7050 and 185A) spores and vegetative cells in tomato juice, and choose the one with the highest thermal resistance as the target microorganism for thermal processing of tomato products. Thermal inactivation kinetics of the target bacteria in tomato juice between 95°C and

115°C were determined. The effects of environmental factors, including cold-storage time, pH, and pre-conditioning on the thermal resistance of these bacterial spores were also investigated.

Chapter 4: Kinetics of carrot texture degradation under pasteurization conditions. This chapter describes the texture degradation of carrot dices in different solutions (distilled water, 0.1% and

1.4% CaCl2 solutions) under temperatures ranging from 80 to 110ºC. The effects of preheating before high temperature treatments on carrot texture were studied and kinetic parameters were estimated. Data obtained in this chapter were used to recommend processing conditions for carrot products that could control food pathogens and inactivate enzymes.

Chapter 5: Dielectric properties of tomatoes assisting in the development of MW pasteurization and sterilization processes. This chapter provides information on the dielectric properties of

tomatoes over a frequency range of 300–3000 MHz for temperatures between 22–120°C. Three tomato tissues, the pericarp tissue (including the skin), the locular tissue (including the seeds) and the placental tissue were studied separately. The effects of temperature, frequency and salts

(0.2g/100g of NaCl and 0.055g/100g of CaCl2) on the dielectric properties of three tissues were investigated, and their dielectric loss mechanisms were discussed in this chapter.

Chapter 6: Developing MW sterilization/pasteurization processes for pre-packaged diced tomatoes/carrots. In this chapter, a MW assisted thermal sterilization (MATS) process was developed for processing diced tomatoes packaged in 8-oz pouches using a semi-continuous,

915MHz single-mode MW system; while a MW assisted thermal pasteurization (MAP) process was developed for diced carrots using a 14-kW single-mode MW system. A 3-D computer simulation model that considered temperature dependent dielectric properties of food materials provided information about heating patterns and the cold spot location in the sample pouches.

The simulation results were validated with a chemical marker based computer-vision method.

Heat penetration tests were conducted to obtain temperature-time data for identifying the cold spot in diced tomatoes packaged in pouches, from which a MATS process was designed to achieve a 5D reduction in Bacillus coagulans ATCC 8038 spores (F105°C = 6.0 min). For diced carrots, two MAP processes were developed: a 6D reduction in non-proteolytic Clostridium botulinum type E spores (F90°C=3 min); and a F90°C=10 min process. Incubation test of the processed tomato products was conducted to validate the MATS processes.

Chapter 7: Quality evaluation of pilot-scale MW/conventional thermal processed vegetable products. This chapter presents the results of quality evaluation of diced tomato after MW sterilization and of diced carrots after MW pasteurization, in comparison with conventional thermal processing. Quality attributes of the processed products by MW/HW heating were

evaluated and compared. For diced tomatoes, quality related parameters included drained weight, soluble solids, color, texture, ascorbic acid and lycopene content; while for diced carrots, quality attributes of color, texture, pectin methylesterase activity and carotenoids were evaluated.

Chapter 8: Conclusions and recommendations. The main findings and contributions to knowledge of this research were summarized in this chapter; recommendations for future work were also given in this chapter.

Chapters 3, 4 and 5 have been published, and their formats followed the requirements of the target journals. The articles published and manuscripts prepared from this research are listed below:

1. Peng, J., Tang, J.,Barrett, D.M., Sablani, S.S., and Powers, J. 2014. Kinetics of carrot texture degradation under pasteurization conditions. J Food Eng. 122, 84‒91.

2. Peng, J., Tang, J., Jiao, Y., Bohnet, S.G., and Barrett, D.M. 2013. Dielectric properties of tomatoes assisting in the development of microwave pasteurization and sterilization processes.

LWT-Food Sci & Tech. 54, 367‒376.

3. Peng, J., Mah, J.H., Somavat, R., Mohamed, H., Sastry, S., and Tang, J. 2012. Thermal inactivation kinetics of Bacillus coagulans spores in tomato juice. JFood Prot. 75, 1236‒1242.

4. Peng, J., Tang, J.,Barrett, D.M., Sablani, S.S., and Powers, J. A review of thermal pasteurization of vegetables. In preparation.

5. Peng, J., Tang, J.,Barrett, D.M., Sablani, S.S., and Powers, J. Thermal pasteurization and quality evaluation of diced carrots pre-packaged in 8-oz pouches. In preparation.

References

Alvarez, M.D., and Canet, W. 2001. Kinetics of thermal softening of potato tissue heated by

different methods. Eur Food Res Technol. 212, 454–464.

Anthon, G.E., Blot, L., and Barrett, D.M. 2005. Improved firmness in calcified diced tomatoes

by temperature activation of pectin methylesterase. J Food Sci. 70(5), C342–C347.

Barba A.A., Calabretti, A., Amore, M., Piccinelli, A.L., and Rastrelli, L. 2008. Phenolic

constituents levels in cv. Agria potato under microwave processing. LWT-Food Sci & Tech.

41, 1919–1926.

Begum, S., and Brewer, M.S. 2001. Chemical, nutritive and sensory characteristics of tomatoes

before and after conventional and microwave blanching and during frozen storage. J Food

Quality. 24, 1–5.

Bourne, M.C. 1989. Applications of chemical kinetic theory to the rate of thermal softening of

vegetable tissue. In Quality Factors of Fruits and Vegetables, Vol. ACSSymp. Ser. 405 (J.J.

Jen, ed.). American Chemical Society, Washington, 98–110.

Dauthy, M.E. 1995. Fruit and vegetable processing. Food and Agriculture Organization of the

United Nations (ISBN 92-5-103657-8). Rome. Available at

http://www.fao.org/docrep/V5030E/V5030E00.htm#Contents.

Gaze, J.E., and Brown, G.D. 1990. Determination of the Heat Resistance of a Strain of Non-

proteolyticClostridium botulinum Type B and a Strain of Type E, Heated in Cod and Carrot

Homogenate Over the Temperature Range 70 to 92°C. Campden Food and Drink Research

Association Technical Memorandum N. 592. Chipping Campden, UK.

Grant, G.T., Morris E.R., Rees D.A., Smith P.J.C., and Thom D. 1973. Biological interactions

between polysaccharides and divalent cations: the egg-box model. FEBS Lett. 32, 195–8.

Greve, L.C., McArdle RN, Gohlke JR, Labavitch JM. 1994a. Impact of heating on

carrotfirmness. Changes in cell wall components. J Agric Food Chem. 42, 2900–6.

Greve, L.C, Shackel, K.A., Ahamdi, H., McArdle, R.N, Gohlke, J.R., and Labavitch, J.M. 1994b.

Impact of heating on carrot firmness: contribution of cellular turgor. J Agric Food Chem. 42,

2896–2899.

Huang, Y.T., and Bourne, M.C. 1983. Kinetics of thermal softening of vegetables. J Texture

Studies. 14, 1–9.

Hunter, K.J., and Fletch, J.M. 2002. The antioxidant activity and composition of fresh, frozen,

jarred and canned vegetables. Innov Food Sci & Emerg Tech.3, 399–406.

Keijbets, M.J.H., and Pilnik, W. 1974. Beta-elimition of pectin in the presence of anions and

cations. J Carbohydr Res. 33, 359–362.

Koskiniemi, C.B., Truong, V.D., Mcfeeters, R.F., and Simunovic, J. 2013. Quality evaluation of

packaged acidified vegetables subjected to continuous microwave pasteurization. LWT-Food

Sci & Tech. 54, 157–164.

Krall, S.M., and McFeeters, R.F. 1998. Pectin hydrolysis: Effect of temperature, degree

ofmethylation, pH, and calcium on hydrolysis rates. J Agric Food Chem. 46, 1311–5.

Lemmens, L., Tiback, E., Svelander, C., Smout, C., Ahrne, L., Langton, M., Alminger, M., Loey,

A.V., and Hendrickx, M. 2009. Thermal pretreatments of carrot pieces using different heating

techniques: Effect on quality related aspects. Innov Food Sci & Emerg Tech. 10, 522–529.

Lucier, G., and Glaser, L. 2009. Vegetables and melons: tomatoes. USDA Economic Research

Service. Available at http://www.ers.usda.gov/briefing/vegetables/tomatoes.htm

Lucier, G., and Lin, B.H. 2007. Factors affecting carrot consumption in the United States.

Outlook Report from the Economic Research Service/USDA. No. (VGS-31901): 1–21.

Mallidis, C., Frantzeskakis,P., Balatsouras G., and Katsabotxakis, G. 1990. Thermal treatment of

aseptically processed tomato paste. Int J Food Sci technol. 25, 442–448.

Olivera, D.F., Vina, S.Z., Marani, C.M, Ferreyra, R.M., Mugridge, A., Chaves, A.R., and

Mascheroni, R.H. 2008. Effect of blanching on the quality of Brussels sprouts (Brassica

oleracea L. gemmifera DC) after frozen storage. J Food Eng. 84, 148–155.

Palop, A., Raso, J., Pagán,R., Condón, S. and Sala, F.J. 1999. Influence of pH on heat resistance

of spores of Bacillus coagulans in buffer and homogenized foods. Int J Food Microbiol. 46,

243–249.

Pirone, G., Mannino,S., and Vicini, E. 1989. Termoresistenza di Bacillus coagulansin passato do

pomodoro. Ind Conserve. 64, 135–137.

Rayman, A., and Baysal, T. 2011. Yield and quality effects of electroplasmolysis and microwave

applications on carrot juice production and storage. J Food Sci. 76(4), C598–C605.

Rodrigo, M., A. Martinez, J. Sanchis, J. Trama, and V. Giner. 1990. Determination of hot-fill-

hold-cool process specifications for crushed tomatoes. J Food Sci. 55, 1029–1032.

Sandoval, A.J., Barreiro, J.A., and Mendoza, S. 1992. Thermal resistance of Bacillus coagulans

in double concentrated tomato paste. J Food Sci. 57, 1369–1370.

Smout, C., Sila, D.N., Vu, T.S., Van Loey A.M., and Hendrickx, M. 2005. Effect of preheating

and calcium pre-treatment on pectin structure and thermal texture degradation: a case study on

carrots. J Food Eng. 67, 419–425.

Scheerens, J.C. 2001. Phytochemicals and the consumer: factors affecting fruit and vegetable

consumption and the potential for increasing small fruit in the diet. HortTechnology. 11(4),

547–556.

Sila, D.N., Duvetter, T., Roeck, A.D., Verlent, I., Smout, C., Moates, G.K., Hills, B.P., Waldron,

K.K., Hendrickx, M., and Loey, A.V. 2008. Texture changes of processed fruits and

vegetables: potential use of high-pressure processing. Trends Food Sci Tech. 19, 309–319.

Steed, L.E., Truong, V.D., Simunovic, J., Sandeep, K.P., Kumar, P., Cartwright, G.D., and

Swartzel, K.R. 2008. Continuous flow microwave-assisted processing and aseptic packaging

of purple-fleshed sweetpotato purees. J Food Sci. 73(9), E455–E462.

Sun, T., Tang, J., and Powers, J.R. 2007. Antioxidant activity and quality of asparagus affected

by microwave-circulated water combination and conventional sterilization. Food Chem. 100,

813–819.

Tang, J., Feng, H., and Lau M. 2002. Microwave heating in Food Processing. Advances in

Bioprocessing Engineering. 1–44. River Edge, NJ: World Scientific.

Van Duyn, M.A.S. 1999. Year 2000 dietary guidelines: The case for fruit and vegetables first-A

scientific overview for the health professional. Produce for Better health Foundation,

Wilminton, Del.

Vian, S.Z., Olivera, D.F., Mariani, C.M., Ferreyra, R.M., Mugridge, A., Chaves, A.R., and

Mascheroni, R.H. 2007. Quality of Brussels sprouts (Brassica oleracea L. gemmifera DC) as

affected by blanching method. J Food Eng. 80, 218–225.

Villanueva M.O., Marquina, A.D., Vargas, E.F., and Abellan, G.B. 2000. Modification of

vitamins B1 and B2 by culinary processes: traditional systems and microwaves. Food Chem.

71, 417–421.

Vu, T.S., Smout, C., Sila, D.N., LyNguyen, B., Loey, A.V., and Hendrickx, M. 2004. Effect of

preheating on thermal degradation kinetics of carrot texture. Innov Food Sci & Emerg Tech. 5,

37–44.

York, G.K., Heil, J.R., Marsh, G.L., Ansar, A., Merson, R.L., Wolcott, T. and Leonard., S. 1975.

Thermobacteriology of canned whole peeled tomatoes. J. Food Sci. 40, 764–769.

Chapter 2. Literature Review-Thermal Pasteurization of Vegetables

1. Pathogens of concern and process design for thermal pasteurization

Known as important components for a balanced and healthy diet, vegetables provide essential vitamins, minerals and dietary fiber for our bodies. A range of phytochemicals commonly found in vegetables, such as flavonoids, phenols, and carotenoids, also prevent nutritional deficiencies and reduce the risk for various types of cancer, heart disease, diabetes, diverticulosis, stroke, hypertension, birth defects, cataracts, and obesity (Scheerens, 2001 and Van Duyn, 1999).

However, vegetables are highly perishable, they need efficient and appropriate preservation technology to prolong their shelf life while maintaining nutritional value and sensory quality.

With the increased public awareness of healthy diet and the needs for ready-to-eat foods, thermal pasteurization has regained our attention as an effective vegetable preservation method to provide safe convenient foods with high nutrients and good sensory quality.

The word “pasteurization” was originally named after the French scientist Louis Pasteur who invented the process of heating food, such as wine at 55°C for several minutes to kill disease- causing micro-organisms (pathogens). Thus, traditional pasteurization refers to a heat treatment of food (usually below 100°C) to destroy all organisms dangerous to health, or a heat treatment which destroys part but not all microorganism that cause food spoilage or that interfere with a desirable fermentation (Downing, 1996). Unlike sterilization, pasteurization process doesn’t kill all the micro-organisms in the foods, only destroying the vegetative pathogenic bacteria and lowering the level of spoilage organisms that will grow under refrigerated storage.

1.1. Regulations and standards of pasteurization in the U.S.

In recent years, the development of emerging technologies which can satisfy the goals of pasteurization, calls for a broadening of the definition of pasteurization. Therefore, the National

Advisory Committee on Microbiological Criteria for Foods (NACMCF) has determined the requisite scientific parameters for establishing equivalent alternative methods of pasteurization, and defined it as below (NACMCF, 2006):

“Any process, treatment, or combination thereof, that is applied to food to reduce the most resistant microorganism(s) of public health significance to a level that is not likely to present a public health risk under normal conditions of distribution and storage”.

This definition allows application of a broad range of technologies (one or in combination) to different food-pathogens for pasteurization such as ohmic heating, microwave heating, steam and hot water heating, pulsed electric field, chemical treatments, filtration, infrared, and high voltage arc discharge. In addition to the processing methods, three major points are made in this definition for developing a pasteurization process:

1. Determining the most resistant microorganism of public health concern for the food;

2. Assess the level of inactivation of target microorganism needed and validate it, to make sure

“not likely to present a public health risk”;

3. Consider the distribution and storage conditions, normally refrigerated.

According to the recommendations of NACMCF, more factors need to be considered for establishing a successful pasteurization process, such as the impact of food matrix on pathogen survival, developing specific Hazard Analysis Critical Control Point (HACCP) system and Good

Manufacturing Practices (GMPs) for the process, etc. In the current study, we will focus on the three major points that need to be addressed for process design.

First of all, determine the most resistant microorganism of public health concern for the process.

Table 1 lists the primary pathogens of concern and their general growth conditions (ECEF,

2006). Considering the normal distribution and storage temperatures for pasteurized foods

(<5°C) with some temperature margin, pathogens with a minimum growth temperature lower than 7.2°C should be considered as potential pathogens and be included in the hazard analysis to identify the target bacteria for the process. With this in mind, L. monocytogens, B. cereus, non- proteolytic C. botulinum, E. coli O 157:H7, Salmonella, Staphylococcus aureus, V. parahaemolyticus and Y. enterocolitica should to be taken into account for the hazard analysis.

There is no simple guideline for the target bacteria for a pasteurization process, because the presence of bacteria in the foods depends on the food characteristics and compositions, the resistance of bacteria may also vary under different processing technology. However, regulations and standards related to target bacteria and processing requirements of certain foods by pasteurization are given by the government in the U.S. for milk, seafood, egg and juice products

(Table 2).

The current processing of milk is governed by the FDA Milk Pasteurized Ordinance, and is based on two fundamental principles: 1) every particle must be heated to a specified minimum temperature for a specified time and 2) equipment is properly designed and operated. The first federal standard for milk pasteurization was established in 1924, requiring a 61.7°C, 30 min process targeting Mycobacterium tuberculosis (Meanwell, 1927). In 1956, Coxiella burnetii was

recognized as the most resistant bacteria of concern, and the current minimum pasteurization time and temperature combinations (63°C for 30 min or 72°C for 15 s) were established. Later,

Enright (1961) demonstrated a more rigorous pasteurization treatment was needed for three milk products including cream, chocolate milk and ice cream mix (Table 2).

The pasteurization of seafood in the US is governed by FDA “Fish and fisheries products hazards and control guidance” (FDA, 2011b). FDA considers a 6D process for target C. botulinum (type E and non-proteolytic types B and F) to be generally suitable for pasteurized seafood products. For the processing requirements, a minimum cumulative total lethality of F90°C

= 10 min is adequate for pasteurized fish and fishery products (Table 2). With regard to blue crabmeat, the National Blue Crab Industry Pasteurization and Alternative Thermal Processing

Standards requires a process of F85°C=31 min, which exceeds a 12-log reduction of C. botulinum type E spores. Some products like Dungeness crabmeat contain certain substances (e.g. lysozyme) that may enable the pathogen to more easily recover after heat damage. In this case, a longer intense heating is needed (F90°C = 57 min for Dungeness crabmeat, FDA, 2011b).

Under FSIS regulations and FDA criterion (2009 Egg Safety Action Plan, 21 CFR Parts 16 and

118), egg and egg products must be free of viable Salmonella. Thus, Salmonella is the target microorganism for pasteurized eggs and egg products. A process of 8.75-log reduction of

Salmonella is required for liquid eggs, and 5-log reduction is required for whole eggs. Specific processing conditions for the eggs and egg products are listed in Table 2.

The pasteurization of juice is regulated by the FDA Juice HACCP Hazards and Controls

Guidance. A process with minimum 5-log reduction of most resistant microorganism of public health significance under HACCP plan is required. The target bacteria are dependent on the juice product and process, including E. coli O157: H7, Salmonella, Cryptosporidium parvum or C. botulinum. For acidic juices (pH ≤ 4.6), E.coli O157:H7, Salmonella, and Cryptosporidium parvum may occur and cause serious foodborne illness outbreaks; while for low-acid juices such as carrot juice, C. botulinum may be present and produce toxin, and therefore becomes the pathogen hazard of concern.

In summary, the definition of pasteurization in the US is broad. Regulations and standards with regard to the pathogens of concern and processing conditions for pasteurization are specific, depending on the particular product, process conditions and packaging systems. There is no "one size fits all" approach to achieving microbiological safety.

1.2. Regulations and standards of pasteurization in Europe

The European Chilled Food Federation provides guidance for producing chilled foods in Europe

(ECFF, 2006). ECFF defines chilled food as “foods that for reasons of safety and/or quality rely on storage at refrigeration temperatures throughout their entire shelf-life.” According to ECFF recommendations, the common practice for heat-treated chilled food is to aim for a 6 log reduction of either (Table 3):

1) L. monocytogenes (this treatment will control other vegetative pathogens)

2) Cold growing C. botulinum (this treatment will not control other spore-forming pathogens such as B. cereus)

L. monocytogenes is the most heat-resistant vegetative pathogen while Type B C. botulinum is the most heat resistant form of non-proteolytic C. botulinum. Tables 4 and 5 give the commonly accepted lethal rates and temperature-time combinations to achieve 6-log reduction of L. monocytogenes and type B C. botulinum. It is generally accepted that a mild pasteurization of low acid food (F70°C=2.0 min) achieving 6 log reduction of L. monocytogenes is suitable for a shelf life of maximally 10 days at 5°C. A severe pasteurization process of F90°C=10.0 min aiming at a 6D process inactivation of non-proteolytic C. botulinum allows product a shelf life up to 6 weeks at 5°C (ECFF, 2006; CSIRO Food and Nutritional Sciences, 2010).

2. Effect of thermal pasteurization on vegetable quality

2.1. Color

Color plays a vital role in the consumer acceptance of a vegetable product, and is one of the most important characteristics of vegetables. This visual appeal mainly comes from pigments such as chlorophylls, anthocyanins, and carotenoids (lycopene), which provide health and nutrient benefits (Figure 1). The visual color of vegetables can be numerically expressed by color models. The CIE model is the most commonly used and its three color values L*(lightness), a*

(redness) and b* (yellowness) can be used individually or in combination in the form of hue, chroma, or total color difference value.

Published papers related to color changes by thermal pasteurization are listed in Table 6. Most authors found that the color degradation of vegetables by thermal pasteurization depends mainly on the heat intensity, duration, media, compounds responsible for the color, and storage time.

Lau et al. (2000) found that the surface color changes of asparagus followed a first order

Arrhenius reaction kinetics at temperatures from 70 to 98°C. Loong and Goh (2004) also reported first order kinetics of color degradation of a mixed vegetable juice (butterhead lettuce, celery, parsley, apple concentrate and kalamasi lime) between 80 and 100°C. Koskiniemi et al.

(2013) pasteurized three vegetables (broccoli, red bell pepper and sweet potato), and found that the green color of broccoli floret changed the most while the sweet potato color was stable over the course of processing. Among all these work, three included the studies of color changes during the storage period. The discoloration of pressurized vegetable and vegetable products can occur due to enzymatic browning. Both pectin methylesterase and peroxidase existed in mild thermal pasteurized carrots (P70°C= 2 min), but neither was present in severe thermal pasteurized carrots (P90°C= 10 min). The color values of most vegetables decreased in the storage period from

36 to 120 days (Koo et al., 2008; Koskiniemi et al., 2013; Rejano et al., 1997). Only in one study by Koskiniemi et al. (2013), did the color of broccoli florets not change during an extended storage or even during thermal processing. The reason was that addition of acid during equilibration process had a detrimental effect on the broccoli color, converting chlorophyll

(green) into pheophytin (olive green).

2.2. Texture

The texture of processed vegetables is another primary marketable characteristic for customers.

Mechanisms that contribute to the texture loss during heating of vegetables generally include turgor loss due to the breakdown of cellular membranes, cell wall degradation and disassembly resulting from enzymatic and non-enzymatic transformations in pectin structure and composition

(Anthon et al., 2005; Greve et al., 1994ab; Sila et al., 2008). For mild pasteurization in which

processing temperature is lower than 80°C, vegetable tissue softening due to pectin depolymerization by non-enzymatic degradation via β-elimination is negligible due to the relatively high temperature required for this reaction to take place.

Texture characteristics of vegetables can be evaluated by sensory and instrumental methods.

Sensory evaluation offers the opportunity to obtain a complete analysis of the textural properties of a food as perceived by the human sense, while instrumental measurements are more convenient, less expensive, and tend to provide consistent values when used by different people

(Bourne, 1982; Abbott, 2004). Evaluations of vegetable texture in the manuscripts listed in Table

1 were all assessed by instrumental methods, although the specific equipment and method are product-dependent. Most authors applied force-compression tests by a texture analyzer, only one study by Koo et al. (2008) used a rheometer to measure the texture of soybean sprouts.

Most authors observed a decreased texture of the processed vegetables in comparison to the raw materials. Lau et al. (2000) reported a first order reaction for the softening of green asparagus spears in a temperature range from 70-98°C. In regard to the storage effect on the vegetable texture, Koskiniemi et al. (2013) pasteurized sweet potato, red bell pepper and broccoli by continuous microwave (3.5 Kw) for 4 min after which the surface temperatures of vegetable packs upon exit of the MW cavity achieved 75-80°C, and the vegetables held in insulating molds for 30 min. They found that the largest texture degradation in the three vegetables occurred over a 60-day storage period at 30°C. For example, both the fracture peak force and total work of sweet potato decreased by 85% from post-process to the end of the 60-day storage. One possible reason they gave in the paper was the addition of NaCl and citric acid resulting in the tissue

softening during the storage time. Besides the compounds in the media, residual enzyme in the processed products may also degrade their texture through enzymatic reaction in the storage.

2.3. Carotenoids

Carotenoids are one of the predominant organic pigments present in vegetables, and include α- and β-carotenes (yellow/orange), lycopene (red/orange), xanthophyll (yellow), lutein and zeaxanthin (green/yellow). They are also one of the important bioactive compounds in carrots, and can act as antioxidants to reduce the risk of developing degenerative diseases. For example,

α- and β-carotenes are known as vitamin A precursors, responsible for the orange color such as carrot, sweet potato. Lycopene is considered to be a potential antioxidant and cancer-preventing agent, responsible for the red color in tomatoes. Lycopene, α- and β-carotenes may undergo isomerization, oxidation and other chemical changes during thermal processing and storage due to their highly unsaturated structure (Rodriguez-Amaya and Kimura, 2004; Shi et al., 2003).

The effect of thermal pasteurization on the carotenoids in vegetables depends on the heat intensity and the properties of the products. Total carotenoids found in vegetables are relative stable to mild pasteurization. Vervoort et al. (2012) heated carrot pieces from mild pasteurization

(P70°C = 2 min) to severe pasteurization (P90°C = 10 min) and found no considerable differences occurred in total carotenoid content after processing, or their individual α- or β-carotene concentration. The authors attributed the stability of carotenes to the protective food matrix, which preserves them from degradation during pasteurization. They concluded that the applied pasteurization conditions were not severe enough to cause a notable isomerization and/or oxidation of the carotenoids in carrots. Similar results were obtained by Lemmens et al. (2013), also for β-carotene content in carrots. However, Odriozola-Serrano et al. (2009) observed an

increase of total carotenoid content, lycopene and β-carotene after pasteurization of tomato juice at 90°C for 30 s or 60 s. One explanation given was that the homogenization and heat treatment condition disrupted cell membranes and protein-carotenoid complexes, increasing the extractability of the carotenoids. Rayman and Baysal (2011) reported a decrease in total carotenoid content of carrot juice after pasteurization at 100°C for 10 min.

With regard to the changes of carotenoids in vegetables during storage, Odriozola-Serrano et al.

(2008, 2009) reported a decreasing trend of total carotenoid content and lycopene content in pasteurized tomato juice during 56 days or 91 days storage at 4°C. The authors explained that the decrease of lycopene content throughout the storage may due to the oxygen availability in the headspace of the container. Rayman and Baysal (2011) reported an increase in the carotenoid content in carrot juice after 3 months of storage at 4°C, and attributed it to possible isomerization of β-carotene.

Thermal pasteurization may also influence the bio-accessibility of carotenoids in vegetables, depending on the thermal intensity and various other factors. In the paper mentioned above,

Lemmens et al. (2013) also investigated the bio-accessibility of β-carotene in carrot, and found mild thermal treatments of carrot could enhance the β-carotene bio-accessibility compared to raw carrots, but the differences were only significant on a less strict significance level. Higher β- carotene bio-accessibility is normally associated with intense thermal processes. Similar results were obtained by Knockaert et al. (2012), who reported an increased β-carotene bio-accessibility in carrot puree after thermal pasteurization.

2.4. Phenolics and antioxidant activity

Phenolics are important phytochemicals as bioactive compounds in vegetables. Most researchers have reported phenolics in relation to their antioxidant activity. Effects of thermal pasteurization on the total phenolics in vegetables were associated with the properties of the food material, package and storage conditions. Odriozola-Serrano et al. (2008) didn’t find significant changes in the total phenolic content between pasteurized and fresh tomato juice (90°C for 30/60 s), and noticed good maintenance of the phenolic compounds during storage which might be due to the inactivation of the enzymes responsible for its degradation. The same authors later reported no significant changes in the total phenolic content and individual phenolic concentration between tomato juices just after thermal pasteurization and fresh ones, but a decrease in later storage period (Odriozola-Serrano et al., 2009). Same processing conditions (90°C for 30/60 s) and same storage conditions (up to 56 days at 4°C) were applied in the latter study. The authors hypothesized that the degradation of phenolic compounds during storage was associated to the residual activity of peroxidase, but no enzyme activity test was carried out to support this assumption. Rayman and Baysal (2011) found a decrease in the total phenolic contents of carrot juice after pasteurization (100°C for 10 min) and a continued decrease during storage up to 4 months at 4°C.

Quercetin is one of the numerous flavonoids commonly found in vegetables. Roldán et al. (2008) evaluated the total quercetin content in pasteurized onion by-products and their frozen products, and found a lower value in all the pasteurized products compared to their corresponding frozen products, but higher than the sterilized ones. The differences in the quercetin content of onion

by-products among different treatments were associated to the onion cultivar and the specific product.

Pasteurization caused an almost 50% loss in the antioxidant activity in carrot juice, and storage further increased this loss, which related to the decrease in phenolic contents (Rayman and

Baysal, 2011).The antioxidant activity of fresh and pasteurized tomato juice, measured using the

DPPH stable radical assay, didn’t show significant difference from each other (Odriozola-

Serrano). However, the antioxidant capacity of tomato juice subjected to heat treatment decreased with storage time, with a 46.52% reduction of the initial value at 56 days storage for mild pasteurized juice (90°C for 30 s) and 35.7% for high pasteurized juice (90°C for 60 s). The authors studied the correlation among different bioactive compounds (lycopene, vitamin C and phenolic) and antioxidant capacity of tomato juice subjected to different treatments, and concluded the decrease of antioxidant capacity during storage could be attributed to the losses of vitamin C and lycopene.

2.5. Vitamins

It is known that vegetables are great sources of various essential vitamins. Vitamin C (ascorbic acid) is one of the numerous vitamins vegetables provide us. However, vitamin C is readily changed or brokendown in the presence of oxygen and light, and high temperature will accelerate this degradation process. Due to its thermolability, vitamin C in vegetables is used as an indicator for the loss of other vitamins and thermolabile nutrients during thermal processing

(Torregrosa et al., 2006). Thermal degradation of vitamin C in foods has been widely studied, and is generally reported as a first-order kinetic. Thermal pasteurization of gazpacho (a cold

vegetable soup) at 90°C for 1 min reduced the vitamin C level to 79.2% of its initial value (Elez-

Martínez and Martín-Belloso, 2007). Torregrosa et al. (2006) reported an 83% retention of ascorbic acid in pasteurized orange-carrot juice (98°C for 21 s). Pasteurizing soybean sprout at 70°C for 2 min reduced its vitamin C from 7.19 mg/100 g to 1.26 mg/100g, and to 1.17 mg/100 g after processing at 90°C for 10 min (Koo et al., 2008).

Most authors investigated the changes of vitamin C in pasteurized vegetables followed by a storage study, and reported a decrease of vitamin C during the storage period. The decreasing level depended on the storage conditions, such as temperature, oxygen content, light and packages. Odriozola-Serrano et al. (2008) found the contents of vitamin C in pasteurized tomato juice could be described by a first-kinetic model. For the effect of storage temperature,

Torregrosa et al. (2006) found that the ascorbic acid degradation rate in the orange-carrot juice stored at 2°C was less than those in the juice stored at 10°C, while Koo et al (2008) reported the decreased level of ascorbic acid in cook-chilled packaged sprouts stored at 3°C was much like those stored at 10°C.

One study by Barba (2012) evaluated the three types of vitamin E (α, Ƴ, and δ-tocopherol) and vitamin D in a pasteurized vegetable beverage (Table 6). The authors noticed a decrease in each vitamin E and also vitamin D. No storage study was conducted in that study.

2.6. Other components

Other nutrients or relevant quality aspects, such as sugars, dry matter content, fatty acid, isothiocyanates and furfural in certain processed vegetables have been reported. Vervoort et al.

(2012) pasteurized carrots pieces at two levels (F70°C= 2 min and P90°C = 10 min) and analyzed the dry matter content, sugar profile (glucose, fructose and sucrose), furfural and 5- hydroxymethylfurfural in the processed products. Pasteurization caused a significant reduction in dry matter content and all sugar concentrations, and an increasing intensity did not cause any further significant changes in those values. No furfural was detected in any of the pasteurized carrots.

3. Enzyme, storage and shelf-life of pasteurized vegetables

Thermal pasteurization of vegetables aims to inactivate pathogens and endogenous enzymes to provide safe and high quality products, with a relatively short shelf-life under expected storage and use conditions. The presence of residual endogenous enzymes in processed vegetable products may cause quality loss during storage. Therefore together with microbial growth, enzyme activity can considerably shorten the shelf life of the final products. The principal enzyme responsible for a specific quality loss is product-dependent. Major enzymes related to the quality of vegetables are listed in Table 7. When evaluating the quality of pasteurized vegetable products, most authors conducted a storage study to see the quality changes with storage time/temperature. Table 8 summarizes the published papers on pasteurized vegetables related to storage and enzyme study. Few studies investigated the enzyme activity after processing or during storage. We can also notice that storage temperatures and time in these work varied from product to product. For most of the pasteurized vegetables, a storage temperature of 3-5°C was used. For some pickled, high acidic vegetables, the storage temperature was much higher (23-30°C). The storage time varied from 21 days to 5 months, depending on the property of products and storage temperature. With regard to the shelf-life of

pasteurized foods, there does not appear to be a universally accepted standard for all the products. Torregrose et al. (2006) calculated the shelf-life of pasteurized orange-carrot juice as the time taken for the ascorbic acid concentration to reduce to 50% (Table 7). Most authors didn’t provide reasons for the selected storage conditions for their products.

The shelf life is defined as “the period of time for which a product remains safe and meets its quality specifications under expected storage and use conditions” (ECFF, 2006). Based on ECFF

(2006), the manufacturer is responsible for determining the shelf life and must take into account microbiological safety and stability, physical characteristics and organoleptic quality.

Microbiological safety and stability should always be priority for determining the shelf life when the acceptable shelf life for either physical condition or organoleptic quality exceeds that for microbial safety. The product shelf life are influenced by a number of factors, including raw material quality, product formulation (pH, aw), hygiene during manufacturing, scheduled heat or other preservation treatments, cooling methods applied to products, type of package, storage temperature and relevant hurdles (CAC, 1999). When determining the shelf life of the products,

Codex Alimentarius Commission (CAC) suggests taking into consideration the potential for temperature abuse which may occur during manufacture, storage, distribution, sale, and handling by the consumer. For example, fluid milk is most often held at marginal refrigeration temperatures of 6.1-7.2°C (43-45°F) instead of the ideal holding temperatures (≤ 3.3°C) to determine the potential shelf-life (Murphy, 2009). The authors believed these marginal refrigeration temperatures allow defects and sanitation deficiencies to become more evident.

Therefore, for a food producer determining the safe shelf-life for pasteurized vegetables, the following information needs to be collected based on ECFF recommendations:

1) Review relevant scientific information containing the characteristics of pathogens;

2) Use predictive modeling programs (e.g. ComBase, USFA Pathogen Modeling Program or

Growth Predictor) to estimate the growth of pathogens under the storage conditions;

3) Conduct challenge test with the relevant pathogens where predictive modeling on its own does not give sufficient confidence to set a safe shelf life;

4) Collect historical data for similar products;

5) Conduct storage trials, either by storing products at predetermined temperatures during specific time periods considering actual chill chain performance under HACCP or testing the product at minimum three time points for the relevant indicator and spoilage microorganisms as well as pathogens identified by HACCP.

The information above focuses on a safe shelf life. Desired quality should also be considered when determining the shelf-life of pasteurized products. This information also provides insights for researchers to conduct storage study for pasteurized products.

References

Abbott, J.A. 2004. Sensory and instrumental measurement of texture of fruits and vegetables.

HortScience. 39(4), 830.

Anonymous. 2013. Eat a colorful diet. Available at http://www.strive2bfitblog.com/eat-a-

colorful-diet/.

Anthon, G., and Barrett, D.M. 2002. Kinetic parameters for the thermal inactivation of quality-

related enzymes in carrots and potatoes. Journal of Agricultural and Food Chemistry. 50,

4119–4125.

Anthon, G.E., Blot, L., and Barrett, D.M. 2005. Improved firmness in calcified diced tomatoes

by temperature activation of pectin methylesterase. Journal of Food Science. 70(5), C342–

C347.

Barba, F.J., Esteve, M.J., and Frigola, A. 2012. Impact of high-pressure processing on vitamin E

(α-, γ-, and δ-Tocopherol), vitamin D (cholecalciferol and ergocalciferol) and fatty acid

profiles in liquid foods. Journal of Agricultural and Food Chemistry. 60, 3763–3768.

Bourne, M.C. 1982. Food texture and viscosity: concept and measurement. Academic Press,

New York.

Codex Alimentarius Commission. 1999. Codex code of hygienic practice for refrigerated

packaged foods with extended shelf life, CAC/RCP 46.

Code of Federal Regulations. 2011. 21 CFR 120.24 U.S. Government Printing Office.

Washington, D.C.

Code of Federal Regulations. 2012a. 9 CFR 590.570. U.S. Government Printing Office,

Washington, D.C.

Code of Federal Regulations. 2012b. 9 CFR 590.575. U.S. Government Printing Office,

Washington, D.C.

CSIRO Food and Nutritional Sciences. 2010. Make it safe: A guide to food safety. Collingwood:

CSIRO Publishing.

Downing, D.L. 1996. A complete course in canning and related processes 13th ed-Book II:

microbiology, packaging, HACCP & ingredients. CTI Publication, Inc. 463.

ECFF (European Chilled Food Federation). 2006. Recommendations for the production of

prepacked chilled food, 2nd ed. Available at

http://www.ecff.net/images/ECFF_Recommendations_2nd_ed_18_12_06.pdf.

Elez-Martínez, P. and Martín-Belloso, O. 2007. Effects of high intensity pulsed electric field

processing conditions on vitamin C and antioxidant capacity of orange juice and gazpacho, a

cold vegetable soup. Food Chemistry. 102, 201–209.

Enright, J.B., Sadler, W.W., and Thomas, R.C. 1957. Thermal inactivation of Coxiella Burnetti

and its relation to pasteurization of milk. Public Health Monograph. No. 47.

Enright, J.B. 1961. The pasteurization of cream, chocolate milk and ice cream mixes containing

the organism of Q fever. Journal of Milk and Food Technology. 24, 351–355.

Greve, L.C., McArdle, R.N., Gohlke, J.R., & Labavitch, J.M. (1994a). Impact of heating on

carrot firmness. Changes in cell wall components. Journal of Agricultural and Food

Chemistry.42, 2900–2906.

Greve L.C., Shackel K.A., Ahmadi H., McArdle R.N., Gohlke J.R., & Labavitch J.M. (1994b).

Impact of heating on carrot firmness: contribution of cellular turgor. Journal of Agricultural

and Food Chemistry.42, 2896–2899.

Howard, L.R., Burma, P., and Wagner, A.B. 1997. Firmness and cell wall characteristics of

pasteurized Jalapeño pepper rings as affected by preheating and storage. Journal of Food

Science. 62, 89─112.

Knockaert, G., Lemmens, L., Van Buggenhout, S., Hendrickx, M., and Van Loey, A. 2012.

Changes in β-carotene bioaccessibility and concentration during processing of carrot puree.

Food Chemistry. 133, 60─67.

Koo, K.M, Kim, H.W., Lee, D.S., Lyu, E.S., and Paik, H.D. 2008. Quality changes during

storage of cook-chilled soybean sprouts. Food Science and Biotechnology. 17, 540–546.

Koskiniemi, G.B., Truong, V.D., McFeeters, R.F., and Simunovic, J. 2013. Quality evaluation of

packaged acidified vegetables subjected to continuous microwave pasteurization. LWT-Food

Science and Technology. 54, 157–164.

Lau, M.H., and Tang, J. 2002. Pasteurization of pickled asparagus using 915 MHz microwaves.

Journal of Food Engineering. 51, 283–290.

Lau, M.H., Tang, J., and Swanson, B.G. 2000. Kinetics of textural and color changes in green

asparagus during thermal treatments. Journal of Food Engineering. 45, 231–236.

Lee, Y., and Howard, L. 1999. Firmness and phytochemical losses in pasteurized yellow banana

peppers (Capsicum annuum) as affected by calcium chloride and storage. Journal of

Agricultural Food and Chemistry. 47, 700─703.

Lemmens, L., Colle, I., Knockaert, G., Van Buggenhout, S., Van Loey, A., and Hendrickx, M.

2013. Influence of pilot scale in pack pasteurization and sterilization treatments on nutritional

and textural characteristics of carrot pieces. Food Research International. 50, 525–533.

Loong, M.N, and Goh, H.K. 2004. Colour degradation of acidified vegetable juice. International

Journal of Food Science and Technology. 39. 437─441.

Meanwell, L.J. 1927. An investigation into the effect of pasteurization on the bovine tubercle

Bacillus in naturally infected tuberculous milk. J. Hyg. 26, 392─402.

Murphy, S.C. 2009. Shelf-life of fluid milk products-microbial spoilage-The evaluation of shelf-

life. Dairy Foods Science Notes. Cornell Univ. Available at

http://foodscience.cornell.edu/cals/foodsci/extension/upload/CU-DFScience-Notes-Bacteria-

Milk-Shelf-Life-Evaluaton-06-09.pdf.

National Advisory Committee on Microbiological Criteria for Foods (NACMCF). 2006.

Requisite scientific parameters for establishing the equivalence of alternative methods of

pasteurization. Journal of Food Protection. 69, 1190–1216.

Odriozola-Serrano, I., Soliva-Fortuny R., and Martín-Belloso, O. 2008. Changes of health-

related compounds throughout cold storage of tomato juice stabilized by thermal or high

intensity pulsed electric field treatments. Innovative Food Science and Emerging

Technologies. 9, 272─279.

Odriozola-Serrano, I., Soliva-Fortuny R., Hernández-Jover, T., and Martín-Belloso, O. 2009.

Carotenoid and phenolic profile of tomato juices processed by high intensity pulsed electric

field compared with conventional thermal treatments. Food chemistry. 112, 258─266.

Ohba, R., Iio, M., and Sasaki, Y. 2002. Storage of a broccoli lactic acid bacteria drink. Food

Science and Technology Research. 8(2), 162─165.

Rayman, A., and Baysal, T. 2011. Yield and quality effects of electroplasmolysis and microwave

application on carrot juice production and storage. Journal of Food Science. 76, C598–C605.

Rejano, L., Sanchez, A.H., De Castro, A., and Montano, A. 1997. Chemical characteristics and

storage stability of pickled garlic prepared using different processes. Journal of Food Science.

62, 1120─1123.

Rodriguez-Amaya, D.B., and Kimura, M. 2004. HarvestPlus handbook for carotenoid analysis.

Washington D.C: HarvestPlus.

Roldán, E., Sánchez-Moreno, C., de Ancos, B., and Cano. M.P. 2008. Characterization of onion

(Allium cepa L.) by-products as food ingredients with antioxidant and antibrowning

properties. Food Chemistry. 108, 907–916.

Scheerens, J.C. 2001. Phytochemicals and the consumer: factors affecting fruit and vegetable

consumption and the potential for increasing small fruit in the diet. HortTechnology. 11(4),

547–556.

Shi, J., Maguer, M.L., Bryan, M., and Kakuda, Y. 2003. Kinetics of lycopene degradation in

tomato puree by heat and light irradiation. Journal of Food Process Engineering. 25, 485–

498.

Sila, D.N., Duvetter, T., Roeck, A.D., Verlent, I., Smout, C., Moates, G.K., Hills, B.P., Waldron,

K.K., Hendrickx, M., and Loey, A.V. 2008. Texture changes of processed fruits and

vegetables: potential use of high-pressure processing. Trends in Food Science and

Technology. 19, 309–319.

Terefe, N.S., Buckow, R., and Versteeg, C. 2012. Quality related enzymes in fruit and vegetable

products: effects of novel food processing technologies Part1: High pressure processing.

Critical Reviews in Food Science and Nutrition. DOI:10.1080/10408398.2011.566946.

Torregrosa, F., Esteve, M.J., Frígola, A., and Cortés, C. 2006. Ascorbic acid stability during

refrigerated storage of orange-carrot juice treated by high pulsed electric field and comparison

with pasteurized juice. Journal of Food Engineering. 73. 339─345.

Triska, J., Vrchotova, N., Houska, M., and Strohalm, J. 2007. Comparison of total

isothiocyanates content in vegetable juices during high pressure treatment, pasteurization and

freezing. High Pressure Research. 27, 147─149.

U.S. Department of Agriculture, Agricultural Research Service. 1969. Egg pasteurization

manual. ARS 74-78. U.S. Department of Agriculture, Agricultural Research Service, Albany,

Calif.

U.S. Food and Drug Administration, Department of Health and Human Services. 9 July, 2009.

21 CFR Parts 16 and 118. Prevention of Salmonella enteritidis in shell eggs during

production, storage, and transportation. Federal Register. 74. 33030-33101.

U.S. Food and Drug Administration, Department of Health and Human Services. 2011a

(Revision). Grade “A” Pasteurized Milk Ordinance. Available at

http://www.fda.gov/downloads/Food/GuidanceRegulation/UCM291757.pdf.

U.S. Food and Drug Administration, Department of Health and Human Services. 2011b. Fish

and fisheries products hazards and control guidance, 4rd ed. Available at

http://www.fda.gov/downloads/food/guidanceregulation/ucm251970.pdf.

U.S. Food and Drug Administration, Department of Health and Human Services. 3 March, 2004.

Guidance for Industry: Juice HACCP Hazards and Controls Guidance 1st ed; final guidance.

Available at

http://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/Ju

ice/ucm072557.htm

Van Duyn, M.A.S. 1999. Year 2000 dietary guidelines: The case for fruit and vegetables first-A

scientific overview for the health professional. Produce for Better health Foundation,

Wilminton, Del.

Van Buggenhout, S., Sila, D.N., Duvetter, T., Van Loey, A. and Hendrickx, M. 2009. Pectins in

Processed Fruits and Vegetables: Part III - Texture Engineering . Comprehensive Reviews in

Food Science and Food Safety. 8, 105–117.

Vervoort, L., Van der Plancken, I. Grauwet, T., Verlinde, T., Matser, A., Hendrickx, M., and

Van Loey, A. 2012. Thermal versus high pressure processing of carrots: a comparative pilot-

scale study on equivalent basis. Innovative Food Science and Emerging Technologie. 15, 1–

13.

Ward, D.R., Pierson, M.D., and Minnick, M.S. 1984. Determination of equivalent processes for

the pasteurization of crab meat in cans and flexible pouches. Journal of Food Science. 49,

1003-1004.

Table 1. Commonly accepted growth boundaries of pathogenic microorganisms (ECEF 2006)

Microorganism Min temp Min Min aw Aerobic/

(°C) pH anaerobic

L. monocytogens -0.4 4.3 0.92 Facultative

B. cereus 4 4.5 0.93 Facultative

Campylobacter jejuni 32 4.9 0.99 Microaerophilic

C. botulinum Mesophilic/proteolytic 10-12 4.6 0.93 Anaerobic

C. botulinum Pasyschrotrophic/non- 3.3 5.0 0.97 Anaerobic proteolytic (5% NaCl)

C. perfringens 12 5.5- 0.935 Anaerobic

5.8

E. coli 7-8 4.4 0.95 Facultative

E. coli O157:H7 6.5 4.5 0.95 Facultative

Salmonella 6 4.0 0.94 Facultative

Staphylococcus aureus 5.2 4.5 0.86 Facultative

V. cholera 10 5.0 0.97 Facultative

V. parahaemolyticus 5 4.8 0.94 Facultative

Y. enterocolitica -1.3 4.2 0.96 Facultative

Table 2. Summary table of regulations of thermal pasteurization of foods (milk, seafood, egg and juice products) in the United States.

Related regulations in the U.S. References Category I: The pasteurization of milk is governed by FDA, 2011a Milk FDA Milk Pasteurized Ordinance. The target microorganism original was Mycobacterium tuberculosisis (1924), now is Coxiella burnetii (since 1956). A process to eliminated 100, 000 guinea pig infectious doses is needed. Examples Target Processing requirements References Bacteria Milk, 1956- C. burnetii 63°C (145°F) for 30 min for batch Enright et al., present process 1957 Cream; C. burnetii 66°C (150°F) for 30 min for batch Enright, 1961 Chocolate milk process; 75°C(166°F) for 15 s for HTST Ice cream mix C. burnetii 69°C(155°F) for 30 min for batch Enright, 1961 process, 80°C (175°C) for 25 s for HTST Related regulations in the U.S. References Category II: For pasteurization of seafood is governed by FDA FDA, 2011b Seafood (Fish and fisheries products hazards and control guidance). FDA considers a 6D process for target C. botulinum (type E and non-proteolytic types B and F) to be generally suitable for pasteurized seafood products. Examples Target Processing requirements References bacteria Fish and fishery C. botulinum F90°C = 10 min, z value is 7°C for FDA, 2011b products type E and non- temperatures less than 90°C, 10°C generally (e.g., proteolytic for temperatures above 90°C. surimi-based types B and F products, soups or sauces) Blue crabmeat C. botulinum F85°C = 31 min, Z value is 9°C. FDA, 2011b type E and non- proteolytic types B and F Dungeness C. botulinum F90°C=57 min, Z value is 8.6°C. FDA, 2011b crabmeat type E and non- proteolytic types B and F

Related regulations in the U.S. References Category III: The current processing of egg products is governed CFR, 2012ab; Egg products by FSIS regulations and FDA criterion (2009 egg FDA, safety action plan, 21 CFR Parts 16 and 118). The 2009;USDA, target microorganism is Salmonella for pasteurized 1969 eggs and products.Aprocess of 8.75-log reduction of Salmonella is required for liquid eggs, and 5-log reduction is required for whole eggs. Examples Products Processing requirements References (Min temp & Min holding time) Liquid eggs Albumen (w/o use of 56.7°C (134°F) for 3.5 min CFR, 2012a chemicals) or 55.6 (132°F) for 6.2 min Whole egg 60°C (140°F) for 3.5 min CFR, 2012a Whole egg blends (<2% 61.1°C (142°F) for 3.5 min CFR, 2012a added nonegg or 60°C (140°F) for 6.2 ingredients); sugar min whole egg (2-12% sugar added); and plain yolk Fortified whole egg and 62.2°C (144°F) for 3.5 min CFR, 2012a blends (24-38% egg or 61.1°C (142°F) for 6.2 solids, 2-12% added min nonegg ingredients) Salt whole egg (≥2% salt 63.3°C (146°F) for 3.5 min CFR, 2012a added); sugar yolk (≥ 2% or 62.2°C (144°F) for 6.2 sugar added); and salt min yolk (2-12% salt added) Dried egg Spray-dried albumen 54.4°C (130°F) for 7 days CFR, 2012b whites Pan-dried albumen 51.7°C (125°F) for 5 days CFR, 2012b Related regulations in the U.S. References Category IV: The pasteurization of juice is governed by FDA CFR, 2011; Juice1 (2011. 21 CFR 120.24). A process of 5-log FDA, 2004 reduction of most resistant microorganism of public health significance under HACCP plan is required. The target bacteria are dependent on the juice product and process, including E. coli O157: H7, Salmonella, Cryptosporidium parvum or C. botulinum. Examples Target bacteria References Acidic juice E.coli O157:H7, Salmonella, and Cryptosporidium FDA, 2004 (pH≤ 4.6) parvum Low-acid juices C. botulinum FDA, 2004 (pH > 4.6)

1Juice is defined by FDA as the aqueous liquid expressed or extracted from one or more fruits or vegetables, or concentrates of such liquids or purees.

Table 3. Standards of prepackaged chilled foods (pasteurized foods) in Europe (CSIRO, 2010;

ECFF, 2006).

Products Target bacteria Processing Shelf-life

requirements

L. monocytogens 6D reduction. ≤ 10 days at 5°C

Common practice of

Heat-treated chilled F70°C=2.0 min is

foods1 considered suitable.

Non-proteolytic C. 6D reduction, Up to 6 weeks at

botulinum common practice of 5°C

F90°C =10.0 min is

universally accepted

1Chilled food: foods that for reasons of safety and/or quality rely on storage at refrigeration temperatures throughout their entire shelf life.

Table 4: Lethal rates for L. monocytogenes1 and necessary process to achieve 6-log reduction of

L. monocytogenes (ECFF, 2006).

Temperature (°C) Time (mins, secs) Lethal Rate 60 43’29” 0.046 61 31’44” 0.063 62 23’16” 0.086 63 17’06” 0.117 64 12’40” 0.158 65 9’18” 0.215 66 6’49” 0.293 67 5’01” 0.398 68 3’42” 0.541 69 2’43” 0.736 70 2’00” 1.000 71 1’28” 1.359 72 1’05” 1.848 73 0’48” 2.512 74 0’35” 3.415 75 0’26” 4.642 76 0’19” 6.310 77 0’14” 8.577 78 0’10” 11.659 79 0’06” 15.849 80 0’05” 21.544 81 0’04” 29.286 82 0’03” 39.810 83 0’02” 54.116 84 0’02” 73.564 85 0’01” 100.000

1L. monocytogens is the most heat-resistant vegetative pathogen. Values have been extrapolated assuming a linear z-value of 7.5°C and as a reference 70°C.

Table 5. Lethal rates for C. botulinum type B1and necessary process to achieve 6-log reduction of

C. botulinum type B (ECFF, 2006).

Temperature (°C) Time (mins) Lethal rate 80 270.3 0.037 81 192.3 0.052 82 138.9 0.072 83 100.0 0.100 84 71.9 0.139 85 51.8 0.193 86 37.0 0.270 87 27.0 0.370 88 19.2 0.520 89 13.9 0.720 90 10.0 1.000 91 7.9 1.260 92 6.3 1.600 93 5.0 2.000 94 4.0 2.510 95 3.2 3.160 96 2.5 3.980 97 2.0 5.010 98 1.6 6.310 99 1.3 7.940 100 1.0 10.000

1 Type B is the most heat resistant form of non-proteolytic C. botulinum. Values have been extrapolated assuming a linear z-value of 7°C below 90°C and 10°C above 90°C (reference is

90°C).

Table 7. Major enzymes related to the quality of raw and processed vegetables.

Category Enzymes Main effects References Texture- Pectin 1. Catalyzes the de-esterification of pectin Anthon and related methylesterase to create binding sites for divalent cations Barrett, enzymes (PME) on the polygalacturonic acid backbone of 2002; Terefe the pectin to form cross-links between et al., 2012; pectin chains (a firming effect); 2. Van demethoxylated pectin can also be a Buggenhout, substrate for PG depolymerization (a 2009 softening effect); 3. Causes cloud loss in juices Polygalacturon Catalyses the cleavage of polygalacturonic ase (PG) acid, resulting in pectin depolymerisation (softening effect) Peroxidase Involved in the oxidative cross-linking of (POG) cell wall polysaccharides

Color- Polyphenol Acts on phenols in the presence of oxygen, Terefe et al., related oxidase (PPO) catalyses browning 2012; enzymes POD Catalyses the oxidation of phenolics in the presence of hydrogen peroxide resulting in browning Anthocyanase Catalyses the hydrolysis of anthocyanins Chlorophyllase Catalyses the degradation of chlorophyll, causes the loss of green color Alliinase Hydrolyses the non-protein amino acid, involved in the discolouratioin of processed garlic products Lypoxygenase Causes the co-oxidation of carotenoids in (LOX) the presence of free fatty acids, affects the color intensity of foods

Off-flavor- LOX Catalyses the oxidation of polyunsaturated Terefe et al., related fatty acids, produces volatile off-flavor 2012; enzymes compounds Hydroperoxida One of the key enzyme in the “LOX se lyase (HPL) pathway” for producing volative compounds, the high concentration level of which results in off-flavor Cystine lyase Cleaves cystine producing ammonia, responsible for off flavor and off aroma in broccoli and cauliflower

Table 6. Effects of thermal pasteurization on the quality of vegetables

Quality Commodity/ Main focus related to the specific Thermal Processing conditions Source parameters products quality parameter technology 10 10 Color Carrot pieces Compare high pressure and thermal Steam P70°C = 2min or P90°C = Vervoort et treatments on an equivalent basis, and 10min al., 2012 characterize their overall impact on carrot quality attributes respectively. Color Broccoli (florets Evaluate the use of continuous MW Continuous 3.5 KW for 4 min, then Koskiniemi and stems) processing for pasteurization of microwave held in insulating molds for et al., 2013 acidified vegetable packages, and the 30 min. Surface changes in color and texture of the temperatures of vegetable products as affected by MW packs upon exit of the MW pasteurization. cavity were 75-80°C. Color Red bell pepper Evaluate the use of continuous MW Continuous 3.5 KW for 4 min, then Koskiniemi processing for pasteurization of microwave held in insulating molds for et al., 2013 acidified vegetable packages, and the 30 min. Surface

45 changes in color and texture of the temperatures of vegetable

products as affected by MW packs upon exit of the MW pasteurization. cavity were 75-80°C. Color Sweatpotato Evaluate the use of continuous MW Continuous 3.5 KW for 4 min, then Koskiniemi processing for pasteurization of microwave held in insulating molds for et al., 2013 acidified vegetable packages, and the 30 min. Surface changes in color and texture of the temperatures of vegetable products as affected by MW packs upon exit of the MW pasteurization. cavity were 75-80°C. Color Pickled garlic Evaluate the effects of blanching, Hot water 90°C for 8 min Rejano et al., preservation treatment and storage time bath 1997 on the quality of packaged blanched garlic. Color Soybean sprouts Apply soybean sprouts to sous vide and Heat (no P90°C= 10 min or P70°C= 2 Koo et al., cook-chill processing systems, and to information min 2008 evaluate the quality and microbial safty on the heating of the products during storage. media)

Table 6 (cont.)

Quality Commodity/ Main focus related to the specific quality Thermal Processing conditions Source parameters products parameter technology Color Asparagus Develop kinetic model to describe the Water bath 70-98°C for different time Lau et al., textural and color changes of green intervals 2000 asparagus spears during short time cooking and thermal pasteurization. Color Vegetable juice Determine the kinetics of green and total Oil batch 80, 90 and 100°C for 0, 20, Loong and (made from color degradation of acidified vegetable 40, and 60 s Goh, 2004 butterhead juice between 80-100°C. lettuce, celery, parsley, apple concentrate and kalamasi lime) 10 10 Texture Carrot pieces Compare high pressure and thermal Steam P70°C = 2min or P90°C = Vervoort et treatments on an equivalent basis, and 10min al., 2012

46 characterize their overall impact on

carrot quality attributes respectively. Texture Broccoli Evaluate the use of continuous MW Continuous 3.5 KW for 4 min, then held in Koskiniemi processing for pasteurization of acidified Microwave insulating molds for 30 min. et al., 2013 vegetable packages, and the changes in Surface temperatures of color and texture of the products as vegetable packs upon exit of affected by MW pasteurization. the MW cavity were 75-80°C. Texture Asparagus Investigate the effect of microwave Microwave 80°C for 10 s Lau and pasteurization on the heating uniformity or hot Tang, 2002 and textural quality of pickled asparagus water in glass bottles in comparison with the hot water pasteurization method. Texture Red bell pepper Evaluate the use of continuous MW Continuous 3.5 KW for 4 min, then held in Koskiniemi processing for pasteurization of acidified microwave insulating molds for 30 min. et al., 2013 vegetable packages, and the changes in Surface temperatures of color and texture of the products as vegetable packs upon exit of affected by MW pasteurization. the MW cavity were 75-80°C.

Table 6 (cont.)

Quality Commodity/ Main focus related to the specific Thermal Processing conditions Source parameters products quality parameter technology Texture Asparagus Develop kinetic model to describe the Water bath 70-98°C for different Lau et al., textural and color changes of green time intervals 2000 asparagus spears during short time cooking and thermal pasteurization. Texture Pickled garlic Evaluate the effects of blanching, Hot water bath 90°C for 8 min Rejano et preservation treatment and storage time al., 1997 on the quality of packaged blanched garlic. Texture Carrot pieces Investigate the effect of in pack thermal Steam Actual P70°C=1.85 min; Lemmens preservation processes in a retort or P90°C=9.67 min et al., 2013 system on particular carrot quality aspects (texture). Texture Soybean Apply soybean sprouts to sous vide and Heat (no P90°C= 10 min or P70°C= Koo et al.,

47 sprouts cook-chill processing systems, and to information on 2 min 2008

evaluate the quality and microbial safty the heating of the products during storage. media) Texture Sweatpotato Evaluate the use of continuous MW Continuous 3.5 KW for 4 min, then Koskiniemi processing for pasteurization of microwave held in insulating molds et al., 2013 acidified vegetable packages, and the for 30 min. Surface changes in color and texture of the temperatures of products as affected by MW vegetable packs upon pasteurization. exit of the MW cavity were 75-80°C. 10 10 Carotenoids Carrot pieces Compare high pressure and thermal Steam P70°C = 2min or P90°C = Vervoort et treatments on an equivalent basis, and 10min al., 2012 characterize their overall impact on carrot quality attributes respectively. Carotenoids Carrot juice Study the effect of electroplasmolysis MW heating or MW: flow rats 90-287 Rayman and microwave application on the yield traditional heat mL/min at 540, 720 and and Baysal, and quality of carrot juice during 900 W using a MW 2011 production and storage. oven; traditional heat: 100°C for 10 min

Table 6 (cont.)

Quality Commodity/ Main focus related to the specific quality Thermal Processing conditions Source parameters products parameter technology Carotenoids Tomato juice Evaluate and compare the effects of high Heat 90°C for 30 or 60 s Odriozola- intensity pulsed electric fields exchanger coil Serrano et processing and heat pasteurization on in hot water al., 2009 the quality of tomato juices. bath β-carotene Carrot puree Investigate the effect of different Steel tubes in P90°C=10min Knockaert et processing techniques of carrot puree on water bath al., 2012 β-carotene concentration, isomerisation and bioaccessibility (in vitro). β-carotene Carrot pieces Investigate the effect of in pack thermal Steam Actual P70°C=1.85 min; Lemmens et preservation processes in a retort system or P90°C=9.67 min al., 2013 on particular carrot quality aspects ( β- carotene bio-accessibility). Lycopene Tomato juice Evaluate and compare the effects of high Heat 90C for 30 or 60 s Odriozola-

48 intensity pulsed electric fields exchanger coil Serrano et

processing and heat pasteurization on in hot water al., 2008 the quality of tomato juices. bath Phenolics Carrot juice Study the effect of electroplasmolysis MW heating MW: flow rats 90-287 Rayman and and microwave application on the yield or traditional mL/min at 540, 720 Baysal, and quality of carrot juice during heat and 900 W using a 2011 production and storage. MW oven; Traditional heat: 100°C for 10 min Phenolics Tomato juice Evaluate and compare the effects of high Heat 90C for 30 or 60 s Odriozola- intensity pulsed electric fields exchanger coil Serrano et processing and heat pasteurization on in hot water al., 2008 the quality of tomato juices. bath Phenolics Onion by- Evaluate onion by-products stabilized by Steam 100°C for 11-17 min Roldán et products different treatment to show their al., 2008 (juice, paste bioactive, antioxidant, and antibrowning and bagasse) properties for the potential to be food ingredient.

Table 6 (cont.)

Quality Commodity/ Main focus related to the specific Thermal technology Processing Source parameters products quality parameter conditions Phenolics Tomato juice Evaluate and compare the effects of Heat exchanger coil 90°C for 30 or 60 s Odriozola- high intensity pulsed electric fields in hot water bath Serrano et processing and heat pasteurization on al., 2009 the quality of tomato juices. Vitamin C Tomato juice Evaluate and compare the effects of Heat exchanger coil 90C for 30 or 60 s Odriozola- high intensity pulsed electric fields in hot water bath Serrano et processing and heat pasteurization on al., 2008 the quality of tomato juices. Vitamin C Gazpacho (a cold Study the effects of high intensity Tubular heat- 90°C for 1 min Elez- vegetable soup) pulsed electric field on vitamin C and exchanger in hot Martínez antioxidant capacity of gazpacho, and water bath and Martín- compared with thermal pasteurization. Belloso, 2007

49 Vitamin C Orange-carrot Study the degradation of ascorbic acid Plate heat exchanger 98°C for 21 s Torregrosa

juice in orange-carrot juice treated by PEF or et al., 2006 thermal pasteurization to establish its shelf life. Vitamin C Soybean sprouts Apply soybean sprouts to sous vide and Heat (no information P90°C= 10 min or Koo et al. , cook-chill processing systems, and to on the heating P70°C= 2 min 2008 evaluate the quality and microbial safty media) of the products during storage. Vitamin D Vegetable juice Evaluate the impact of high-pressure Plate heat exchanger 90°C for 15 s Barba et al., (mainly made processing on vitamin E, vitamin D and 2012 from tomato, fatty acid profiles in vegetable green pepper, beverages, and in comparison with green celery, traditional pasteurization. cucumber, onion, carrot, lemon)

Table 6 (cont.)

Quality Commodity/ Main focus related to the specific quality Thermal Processing Source parameters products parameter technology conditions Vitamin E Vegetable juice Evaluate the impact of high-pressure Plate heat 90°C for 15 s Barba et al., (mainly made processing on vitamin E, vitamin D and exchanger 2012 from tomato, fatty acid profiles in vegetable beverages, green pepper, and in comparison with traditional green celery, pasteurization. cucumber, onion, carrot, lemon) Antioxida Gazpacho (a cold Study the effects of high intensity pulsed Tubular heat- 90°C for 1 min Elez-Martínez nt activity vegetable soup) electric field on vitamin C and antioxidant exchanger in and Martín- capacity of gazpacho, and compared with hot water bath Belloso, 2007 thermal pasteurization. Antioxida Carrot juice Study the effect of electroplasmolysis and MW heating MW: flow rats 90- Rayman and nt capacity microwave application on the yield and or traditional 287 mL/min at 540, Baysal, 2011

50 quality of carrot juice during production heat 720 and 900 W

and storage using a MW oven; Traditional heat: 100°C for 10 min Antioxida Onion by- Evaluate onion by-products stabilized by Steam 100°C for 11-17 Roldán et al., nt activity products different treatment to show their bioactive, min 2008 (juice, paste and antioxidant, and antibrowning properties for bagasse) the potential to be food ingredient. Antioxida Tomato juice Evaluate and compare the effects of high Heat 90°C for 30 or 60 s Odriozola- nt capacity intensity pulsed electric fields processing exchanger coil Serrano et al., and heat pasteurization on the quality of in hot water 2008 tomato juices. bath Quercetin Onion by- Evaluate onion by-products stabilized by Steam 100°C for 11-17 Roldán et al., products different treatment to show their bioactive, min 2008 (juice, paste and antioxidant, and antibrowning properties for bagasse) the potential to be food ingredient.

Table 8. Scientific publications of pasteurized vegetables related to storage and enzyme study.

Vegetables Pasteurization Storage Quality related Enzyme Microbial Reference (products) conditions conditions parameters studied test (temp & time) evaluated with storage time Soybean P90°C= 10 min or 3°C for 36 d; Color, texture, N/A Aerobic, Koo et al., sprouts P70°C= 2 min 10°C for 24 d and ascorbic acid, anaerobic and 2008 psychrophilic bacterial counts Carrot juice MW: flow rats 90- 4°C for 4 mo Total pectin PME N/A Rayman 287 mL/min at 540, contents, and 720 and 900 W carotenoids, Baysal, using a MW oven; phenolics, 2011 traditional heat: antioxidant

51 100°C for 10 min activities, titrable acidity values

Orange- 98°C for 21 s 2°C for 70 days; Ascorbic acid N/A N/A Torregrosa carrot juice 10°C for 59 d et al., 2006 Tomato juice 90°C for 30 or 60 s 4°C for 91 d Lycopene, N/A N/A Odriozola- vitamin C, total Serrano et phenolics, and al., 2008 antioxidant capacity

Tomato juice 90°C for 30 or 60 s 4 ± 1 °C for 56 d Carotenoids, N/A N/A Odriozola- phenolics, color, Serrano et pH and soluble al., 2009 solids content

Table 8 (Continued). Scientific publications of pasteurized vegetables related to storage and enzyme study.

Vegetables Pasteurization Storage Quality related Enzyme Microbial test Reference (products) conditions conditions parameters studied (temp & evaluated with time) storage time Acidified Continuous MW 30°C for 60 d Color, texture, N/A Visual signs of Koskiniem vegetables heating (3.5 KW) for 4 microbial stability spoilage (turbidity, i et al., (broccoli, red min, then held in mold growth, gas 2013 bell pepper, insulating molds for 30 production), and min. Surface metabolic products sweetpotato) temperatures of indicative of vegetable packs upon bacterial and yeast exit of the MW cavity growth (lactic acid were 75-80°C. and ethanol) Broccoli 70°C for 15 min 5°C For 21 d Volatile N/A Lactic acid bacteria Ohba et 52 lactic acid compounds, and al., 2002 bacteria drink organic acid Jalapeño Preheated at 50°C for 23°C For 5 Texture, pectic N/A N/A Howard et pepper rings 60 min, then held at mo substances, al., 1997 75°C for 5 min. methoxyl content Pickled garlic 90°C for 8 min 27 ± 2 °C for Color, texture, N/A Rejano et 4 mo Chemical al., 1997 characteristics (eg. pH, Acidity, sugar, ethanol) Yellow 74°C for 10 min 23°C for 124 Texture, fresh N/A N/A Lee and “banana” d weight, ascorbic Howard, pepper acid, quercetin, 1999 Luteolin, and capsaicin content

Table 8 (Continued). Scientific publications of pasteurized vegetables related to storage and enzyme study.

Vegetables Pasteurization Storage Quality related Enzyme Microbial Reference (products) conditions conditions parameters studied test (temp & evaluated with time) storage time 10 Carrot pieces P70°C = 2min or N/A N/A PME and N/A Vervoort et al., 10 P90°C = 10min POD 2012 Pickled asparagus 88°C for 10 s N/A N/A POD N/A Lau and Tang, 2002

Figure 1. Vegetable color wheel (Anonymous, 2013)

Chapter 3. Thermal Inactivation Kinetics of Bacillus coagulans Spores in

Tomato Juice

Abstracts: The thermal characteristics of three strains of Bacillus coagulans (ATCC 8038, 7050 and 185A) spores/vegetative cells in tomato juice were evaluated. B. coagulans 8038 was chosen as the target microorganism for thermal processing of tomato products due to its spores having the highest thermal resistance among the three strains. The thermal inactivation kinetics of B. coagulans 8038 spores in tomato juice between 95°C and 115°C were determined independently in two different laboratories using two different heating setups. The results obtained from both laboratories were in general agreement, with z-values of 8.3°C and 8.7°C, respectively. The z- value of B. coagulans 185A spores in tomato juice (pH 4.3) was found to be 10.2°C. The influence of environmental factors, including cold-storage time, pH, and pre-conditioning upon the thermal resistance of these bacterial spores were discussed. Results obtained showed that a storage temperature of 4°C was appropriate for maintaining the viability and thermal resistance of B. coagulans 8038 spores. Acidifying the pH of tomato juice decreased the thermal resistance of these spores. A1-h exposure at room temperature was considered optimal for pre-conditioning

B. coagulans ATCC 8038 spores in tomato juice.

Keywords: Thermal inactivation, kinetics, Bacillus coagulans, tomato juice

1. Introduction

Bacillus coagulans, a facultative anaerobic spore-forming bacterium, is acid tolerant and grows well in foods at pH 4.0 to 4.5 at ambient temperature. This is the single organism most frequently isolated from spoiled canned vegetables acidified to pH 4.0 to 4.5, and has been considered the primary cause of economically important spoilage in thermally processed tomatoes and tomato products (8, 20). It results in a type of spoilage commonly referred to as flat sour in tomato based products (14, 18, 20, 24). Although B. coagulans is a non pathogenic microorganism, it may cause a food safety hazard due to its ability to increase the pH of acidic foods, processed with a reduced treatment, to a level that can allow germination of surviving Clostridium botulinum (1,

2).

Thermal processing is the most common and effective method for inactivation of microorganisms and extending the shelf-life of tomato juice. Most published data related to the inactivation of B. coagulans spores in food media are based on studies performed with moderate heat only (18, 24), or combining heat treatment with other technologies, such as high pressure (5,

12, 22). For high temperature heating, Palop et al. (13, 14) studied heat resistance in food medium (pH 4 and 7) between 105°C and 130°C, using a strain isolated from canned asparagus, and Mallis et al. (8) obtained the z-value of B. coagulans spores in tomato serum (pH 4.24) at temperatures in a narrow range (95-105°C) . Several studies also reported on inactivation of B. coagulans spores by hydrodynamic cavitation (10), supercritical CO2 micro-bubble (4), and high-pressure with nisin (3).

The equilibrium pH of tomatoes varies from 4.0 to 4.7, depending on the variety and ripeness

(21). The practice of acidification of canned tomato juice to a pH lower than 4.5 before treatment

prevents the outgrowth of spores surviving heat treatment, particularly spores of Clostridium botulinum (11). Although the heat resistance of B. coagulans spores in tomatoes has been studied at acid pH (8, 14, 15, 17, 18, 24), information about heat resistance at different acidic pH levels between 4.0 and 4.5 (the commonly controlled pH range for canned tomato products) is still limited, especially under high temperatures. Since the thermal resistance of B. coagulans spores is strain-dependent, three strains of this microorganism (ATCC 8038, 7050 and 185A) were investigated in this study, although the first two strains have been used more frequently in previously published work (10, 16, 17, 23, 24). The objective of this study is to characterize the thermal resistance of B. coagulans spores in tomato juice product at pH between 4.0 and 4.4 using different heating setups in two different laboratories. The influence of some environmental factors, including cold-storage, pH, and pre-conditioning upon the thermal resistance of this microorganism was also investigated. The current study provides theoretical support for developing and validating thermal pasteurization processes of tomato products.

2. Materials and Methods

2.1. Microorganisms

Bacillus coagulans strains ATCC 7050 and 8038 were purchased from the American Type

Culture Collection (Manasses, VA, USA). Bacillus coagulans strain 185A was obtained from Dr.

V.M. Balasubramaniam at The Ohio State University (6). These strains were grown aerobically in Nutrient Broth (NB, Difco Laboratories Inc., Detroit, MI, USA) for 48 h at 37°C, and then resuspended in NB containing 20% glycerol. The stock culture was divided into sterile cryogenic vials (Fisher Scientific, Pittsburgh, PA, USA) and then stored in a freezer (-20°C) until further use.

2.2. Preparation of B. coagulans spores

To induce sporulation of vegetative cells of B. coagulans, the procedures described by Palop et al. (14) and modified by Milly et al. (10) were employed. Briefly, vegetative cells of B. coagulans were grown in NB aerobically for 48 h at 37°C and transferred into NB at least 3 times before spore preparation. Spores of B. coagulans were prepared by distributing 1 mL of actively growing vegetative cells (48 h, 37°C) onto a plate containing Nutrient Agar (NA, Difco;

Becton, Dickinson and Co., Sparks, MD, USA) fortified with 500 mg/L of dextrose (Bacto

Dextrose, Difco) and 3 mg/L manganese sulfate (Fisher Scientific, Pittsburgh, PA, USA). The inoculated plates were incubated at 50°C for 7 days, where more than 90% sporulation was obtained as verified by observing the refractive spores under phase-contrast microscopy. Spores were harvested by flooding plates with 5 ml of cold sterile deionized water, and dislodging spores from the agar surface with a sterile disposable inoculation loop. After harvesting, the spores were washed 3 times by centrifugation at 14,000 × g at 4°C for 10 min, resuspended in sterile deionized water and stored at 4°C until used.

2.3. Preparation of tomato juice

Two forms of tomato juice were used: commercial tomato juice (Campbell Soup Co., Camden,

NJ, USA) was used in the Washington State University (WSU) study involving oil bath heating; fresh Roma tomatoes bought from a local grocery store (Kroger Inc., Columbus, OH) were used in The Ohio State University (OSU) study. Tomatoes of bright red color (with an ‘a’ value of 20) were cut into quarters and then blended to prepare the tomato juice media.

2.4. Evaluation of cold-storage time on the viability of B. coagulans in sterile distilled water and its thermal resistance in tomato juice

Cultured B. coagulans spores (ATCC 8038 and ATCC 7050) were suspended in sterile distilled water and stored at 4°C. The viable numbers of vegetative cells and spores were counted after

10, 23 and 31 days of storage to study the effect of refrigerated storage on the viability of this microorganism. For enumeration, the spore suspensions were heat-shocked at 80°C for 15 min, cooled in a crushed ice water bath and checked microscopically to ensure the absence of vegetative cells. The spore count was obtained by preparation of 10-fold serial dilutions in sterile

0.1% peptone water. One hundred µL of each dilution was spread-plated onto NA and incubated for 7 days (ATCC strains) or 2 days (185A) at 37°C. The spore numbers were calculated from three replicates. The vegetative cells in the spore suspension were counted by the same procedures but without the heat-shocking step.

To further investigate the response of this microorganism to cold storage, the thermal resistance of prepared B. coagulansATCC8038 spores in tomato juice (pH 4.0) was measured at 100°C after 0, 10 and 28 days of cold storage. The thermal resistance of B. coagulans spores in tomato juice was determined following the procedures described subsequently.

2.5. Preparation and pre-conditioning of a mixture of spore suspension and tomato juice

The pH of tomato juice (Campbell Soup Co., Camden, NJ, USA), initially ranging from 4.0 to

4.1, was adjusted to different values by adding 1M sodium citrate or citric acid to evaluate the influence of pH of the heating medium on microbial heat resistance. To pre-condition spores in tomato juice (adjusted to pH 4.3),capillary tubes with a mixture of 50 µL tomato juice inoculated

with the spore suspension were placed at 4°C or room temperature for time periods of 1, 2, 3, and 4h. The D100-values of pre-conditioned mixtures were determined following the procedures described below.

2.6. Evaluation of heat resistance of B. coagulans spores using oil bath

Thermal resistance of test microorganisms was determined by thermal death time (TDT) tests and reflected by D- and z-values. D-value is defined as the time required at a certain temperature for 1-log reduction of the target microorganisms and z-value is the change in temperature required for a 10-fold reduction of D-values. Since the thermal destruction of B. coagulans generally follows a first-order reaction based on most published data (14, 18, 20), D values of

B.coagulans spores were obtained by taking the negative reciprocal of the slope from linear regression of the survivor curves. The z-value was estimated by plotting the log10 D-values versus heating temperatures and taking the negative reciprocal of the slope from linear regression. Fifty µL of tomato juice inoculated with spore suspension (initial spore concentration was 108 CFU/mL) was injected into a glass capillary tube with an inner diameter of 1.8 mm and an outer diameter of 3 mm (Corning Inc., Corning, NY, USA) using a pipette, and the open ends of the tubes were heat sealed. The tubes were immersed completely in a circulating oil bath

(Thermo Electron Corporation, Waltham, MA, USA) and heated between 95°C and 115°C for different time intervals. The come-up times (the time for sample to reach within 0.5°C of the target temperature) was around 5 sec. After heating, the tubes were removed from the oil bath, cooled immediately in a crushed ice water bath, and washed in 70% ethyl alcohol. Both tube ends were cut aseptically and the suspension was flushed out with 3 mL of sterile 0.1% peptone water. The treated samples were then 10-fold serially diluted in sterile 0.1% peptone water and

spread-plated onto NA medium. Based on our preliminary test results, ATCC strains were incubated for 7 days and the 185A strain for 2 days at 37°C, and then colonies were manually counted, as described previously.

2.7. Evaluation of heat resistance of B. coagulans spores using a capillary tube setup

This part of the study was performed in the Department of Food, Agricultural and Biological

Engineering at Ohio State University (OSU) using B. coagulans ATCC 8038 spores prepared in the same way as described previously. Tomato juice (inherent pH ranging from 4.1 to 4.3) was adjusted to a standard value of 4.4 using sodium citrate to eliminate the varying acidity from affecting the thermal resistance of the organism. Tomato juice samples inoculated with B. coagulans spores were heated in conventional capillary cells (19) and treated at temperatures ranging from 95 to 110°C for different time intervals. The come-up times were 158, 170, 180 and

192 sec when heating from room temperature to 95, 100, 105, and 110 °C, respectively. All zero- time samples were allowed to reach the process temperature, and then immediately cooled to provide initial count data. Two sample-containing capillary cells mounted on each capillary tube holder were used for each holding time. All tests were replicated three times.

For system design, capillary tubes used at OSU containing 37 μl of tomato juice inoculated with

B. coagulans spores were plugged at both ends with nonconductive capillary tube sealant. The capillary tubes were placed inside an external ohmic heating device to enable heating under pressurized conditions. The samples in capillaries had insulating gel plugs at the end to prevent their heating ohmically; thus all capillaries heated by heat transfer from the external, ohmically heated medium. To hold capillary cells in place, they were mounted on cell holders (two cells per

holder) attached to the treatment chamber (Fig. 1). Temperatures were measured in selected, thermocouple containing capillary cells. The system also facilitated rapid post-treatment cooling through pulling of the treated samples into the cooling section with the help of an attached thread. A detailed description of the setup and procedures is provided by Somavat et al. (19).

To enumerate spore survival, treated capillary cells were washed with cold 1400 ppm hypochlorite solution and rinsed with cold sterile water. The capillary washing protocol developed by Somavat et al. (19) was followed to eliminate any residual hypochlorite from affecting the final plate count. The clean capillary cells were then crushed inside sterile polypropylene tubes containing 0.1% peptone water using sterile glass rods. A heat shock of

80°C for 15 min was given to inactivate all vegetative cells. Tenfold serial dilutions in peptone water were prepared and spread-plated onto TSA plates. Inoculated plates were incubated for 48 hours at 37°C and colonies enumerated.

3. Results and Discussion

3.1. Effect of cold-storage time on the viability of B. coagulans in sterile distilled water and its thermal resistance in tomato juice

To investigate whether storage at 4°C influences the viability of B. coagulans, viable numbers of vegetative cells and spores in sterile distilled water were counted over a period of one month.

Table 1 shows that there were few changes in the viable numbers of both vegetative cells and spores of B. coagulans ATCC 8038 during 4°C storage. In contrast, viable vegetative cells of

ATCC 7050 experienced a one-log reduction after 10 days of cold-storage and no viable vegetative cells could be detected after 23 days of storage (detection limit: ≤ 5 Log CFU/mL).

Since spores produced by ATCC 8038 strain (ca. 108 CFU/ml) were much more numerous than those obtained from ATCC 7050 strain (below the detection limit, ≤ 5 Log CFU/mL) when grown under the same conditions, and also due to the stable viability of ATCC 8038 stored in sterile distilled water at 4°C, B. coagulans ATCC 8038 was chosen for further study.

In addition, since the sporulation temperature of 50°C was relatively high and the sporulation time of 7 days was relatively long, maintaining the moisture content of sporulation medium was taken into consideration. The viability of spores produced in a moist chamber was compared with those cultured without a moist chamber, along with the viability of their vegetative cells

(Table 1). It was found that maintaining the moisture content of sporulation medium decreased the viability of both spores and vegetative cells, and the reduction of viability was more evident with spores than with vegetative cells. Thus, B. coagulans was grown on sporulation medium without the use of a moist chamber in order to produce high-viability spores.

To further investigate the effect of cold storage on this microorganism, B. coagulans ATCC 8038 spores stored at 4°C for up to 28 days were used to inoculate tomato juice (pH 4.0) after which

D100°C-values were determined. D100°C values of 2.56 ± 0.00, 2.09 ± 0.34, and 2.87 ± 0.03 were found after 0, 10 and 28 days storage, respectively. No significant difference (P>0.05) in D- values was found. These results demonstrate the relative stability of thermal resistance of those spores under cold-storage conditions. Therefore, a temperature of 4°Cwas used for storing B. coagulans ATCC 8038 spores. Similar results were observed in our previous study (7) which showed that storing Clostridium sporogenes PA 3679 spores at 4°C is satisfactory for maintaining the viability and heat resistance of those spores during short term storage.

3.2. Effect of pH on the thermal resistance of B. coagulans ATCC 8038 spores

According to published data, most authors have revealed that acidification of the heating medium causes a decrease in microbial heat resistance. Palop et al. (14) investigated the thermal resistance of B. coagulans spores in homogenized tomato and asparagus at pH 7 and 4 at temperatures between 105°C and 130°C, and found that the spores were less heat resistant in both food media at pH 4. Similar results were obtained by Mazas et al. (9) who reported sharp D- value reductions for spores of three Bacillus cereus strains by lowering the pH of the heating medium from 7.0 to 4.0. In the current study, the effect of pH on the thermal resistance of B. coagulans ATCC 8038 spores in commercial tomato juice was examined by determining the

D100°C-values of the spores in commercial tomato juice adjusted to pH 3.8, 4.0 and 4.3 by adding sodium citrate or citric acid. Significant differences in D100°C-values were found at different pH levels (P<0.05). As shown in Fig. 2, D100°C-values increased from 2.85 min at pH 3.8 to 3.85min at pH 4.3, which indicates that the thermal resistance of B. coagulans ATCC 8038 spores is influenced by tomato juice pH, decreasing with increased acidification.

3.3. Effect of pre-conditioning on the thermal resistance of B. coagulans ATCC 8038 spores

The influence of pre-conditioning time of commercial tomato juice on the thermal resistance of B. coagulans ATCC 8038 spores was evaluated by exposing the spores to tomato juice for 1, 2, 3 and 4h at room temperature and 4°C, respectively. As shown in Fig. 3, there were no significant differences (P>0.05) of D100°C-values for different pre-conditioning times at each treatment temperature. However, a significant difference (P<0.05) in D100°C-values was found between the two temperatures. D100°C-values of B. coagulans ATCC 8038 spores exposed to tomato juice at room temperature were higher than those exposed to 4°C (3.5 min vs 2.86 min, respectively).

Therefore, a 1-h exposure at room temperature was considered to be optimum for pre- conditioning B. coagulans ATCC 8038 spores in tomato juice.

3.4. Thermal resistance of B. coagulans ATCC 8038 spores in tomato juice using a conventional oil bath

Thermal resistance of B. coagulans ATCC 8038 spores at different temperatures in commercial tomato juice heated in an oil bath was determined by means of D-value measurements. Fig. 4 shows typical thermal survivor curves of B. coagulans ATCC spores in tomato juice (pH 4.0).

The D-values of B. coagulans ATCC 8038 spores decreased with increasing heating temperature.

D-values of 7.05min at 95°C, 2.56 min at 100°C, 1.18 min at 105°C, and 0.20 min at 110˚C were obtained. The calculated z-value of B. coagulans ATCC 8038 spores in commercial tomato juice at pH 4.0 was 10.0°C. This value is higher than that obtained by Milly et al. (10) who obtained a z-value of 8°C when the spores were treated in tomato juice at pH 4.1. Since only the D100°C value and z-value were reported in that study, the difference in z-values between the two studies might be due to the different pH of tomato juice used in both studies. Sandoval et al. (18) determined D values of a strain of B. coagulans spores in double concentrated tomato paste (pH

4.0) at 75, 80, 85 and 90°C and reported a corresponding z value of 9.5°C. The difference of z value obtained by Sandoval et al. could be due to the use of different strains, sporulation temperatures, and water activity.

The D- and z-values of spores in commercial tomato juice at pH 4.3 are shown in Table 2, along with the thermal inactivation data at pH 4.0. The D-value of B. coagulans ATCC 8038 spores in tomato juice at pH 4.3 was 4.56 min at 100°C, 1.20 min at 105°C, 0.27 min at 110°C, and 0.07

min at 115°C, with a corresponding z-value of 8.3°C. As seen in Table 2, all the D-values obtained at pH 4.3 under the same heating temperature were higher than those corresponding values obtained at pH 4.0, which demonstrated that lowering the pH of the heating medium could reduce the thermal resistance of bacterial spores. This is in agreement with our previous results shown in Fig. 2. Meanwhile, D values of the spores in tomato juice dropped from 4.56 min (pH 4.3) to 2.56 min (pH 4.0) at 100°C, whereas this value decreased only from 0.27 min to

0.20 min at 110°C. This illustrates the reduced influence of acidification of heating medium to depress the thermal resistance of bacterial spores at higher temperatures. However, an increase in z-values of B. coagulans ATCC 8038 spores with acidification was observed from 8.3°C at pH

4.3 to 10.0°C at pH 4.0. Similar trends were observed by Palop et al. (14) who studied the heat resistance of B. coagulans spores (STCC 4522) in homogenized tomato at pH 7 and 4.

3.5. Thermal resistance of B. coagulans 185A spores at pH 4.3 using oil bath

Thermal resistance of B. coagulans 185A can only be found associated with thermal-assisted pressure processing (6) among published research. In the present study, the D- and z-values of B. coagulans 185A spores were determined following the same procedures as for B. coagulans

ATCC 8038 spores and are shown in Table 2. Similar to ATCC strains, the D-values of strain

185A spores decreased with increasing heat treatment temperature, showing D-values of 1.41 min at 100°C, 1.53 min at 105°C and 0.14 min at 110°C. The D100°C value of strain 185A spores subjected to heat only was higher than that of spores processed by combining heat with high pressure (D100°C =0.5 min under 600 Mpa) (6). The D115°C-value was not measured due to the exceptionally brief treatment time required. The z-value of strain 185A spores was calculated to be10.2°C.

As shown in Table 2, when exposed to the same pH and treatment temperature, B. coagulans

ATCC 8038 spores had a much greater thermal resistance than 185A spores, with corresponding

D-values 1-3 times greater. Therefore, B. coagulans ATCC 8038 spores were chosen as the major target bacterium for further thermal inactivation experiments.

3.6. Thermal resistance of B. coagulans 8038 spores at pH 4.4 (OSU experiments)

The thermal survivor curves (D-values) for B. coagulans ATCC 8038 spores in tomato juice (pH

4.4) from experiments conducted at OSU are shown in Fig.5. A comparison of the D- and z- values for the OSU and Washington State University (WSU) data are shown in Table 3. Within each selected temperature, D-values obtained from oil bath and electrical heating methods showed no significant difference (P>0.05). D-105°C values were 1.20 and 1.32 min obtained from oil bath and electrical heating methods, respectively; and the corresponding D-110°C values were

0.27 and 0.16 min. Although the D-100°C values obtained from the two heating methods deviated from one another somewhat, the agreement is quite remarkable, given that the data were obtained independently using two different heating methods in two different laboratories. The obtained z- values for the spores were 8.3 and 8.7°C for the WSU and OSU data, respectively. Two key reasons for differences appear to be the slightly different pH levels (4.4 at OSU vs. 4.3 at WSU) and the source of juice (commercial juice at WSU vs. freshly blended tomatoes at OSU).

4. Conclusions

The results of this study show that B. coagulans ATCC 8038 strain can produce consistent heat- stable spores during refrigerated storage. Compared to strain 185A, B. coagulans ATCC 8038 spores have greater thermal resistance (D-values) and are therefore considered ideal target

bacteria for developing and validating thermal processes of tomato products. A storage temperature of 4°C is appropriate for maintaining viability and thermal resistance of B. coagulans ATCC 8038 spores during short term storage. Both pH and pre-conditioning temperature influence D-values of these spores. Thermal resistance data for B. coagulans ATCC

8038 determined independently at two different laboratories were in general agreement, with differences explainable by slightly different pH levels and juice sources.

Acknowledgements

This work was supported in part by USDA-CSREES-NRICGP Grant No. 2009-55503-05198, titled: Quality of Foods Processed Using Selected Alternative Processing Technologies. Salaries and research support provided in part by the Ohio Agricultural Research and Development

Center, The Ohio State University. The senior author acknowledges fellowship supports from

Chinese Scholarship Council.

References

1. Anderson, R.E. 1984. Growth and corresponding elevation of tomato juice pH by

Bacillus coagulans. J. Food Sci. 49:647–649.

2. Fields, M.L., A.F. Zamora, and M. Bradsher. 1977. Microbiological analysis of home-

canned tomatoes and green beans. J. Food Sci. 42:931–934.

3. Gao, Y., and X. Ju. 2011. Inactivation of Bacillus coagulans spores subjected to

combinations of high-pressure precessing and nisin. Trans. ASABE. 54:385–392.

4. Ishikawa, H., M. Shimoda, K. Tamaya, A. Yonekura, T. Kawano, and Y. Osajima. 1997.

Inactivation of Bacillus spores by the supercritical carbon dioxide micro-bubble method.

Biosci. Biotech. Biochem. 61:1022–1023.

5. Islam, M.S., A. Inoue, N. Igura, M. Shimoda, and I. Hayakawa. 2006. Inactivation of

Bacillus spores by the combination of moderate heat and low hydrostatic pressure in

ketchup and potage. Int. J. Food Microbiol. 107:124–130.

6. Johnson C., and V.M. Balasubramaniam. 2010. Inactivation of Bacillus coagualns spores

by pressure-assisted thermal processing. Oculus. 1:35–38.

7. Mah, J.H., D.H. Kang, and J. Tang. 2009. Comparison of viability and heat resistance of

Clostridium sporogens stored at different temperatures. J. Food Sci. 74:23–27.

8. Mallidis, C., P. Frantzeskakis, G. Balatsouras, and G. Katsabotxakis. 1990. Thermal

treatment of aseptically processed tomato paste. Int. J. Food Sci. technol. 25:442–448.

9. Mazas, M., M. López, I. González, J. González, A. Bernardo, and R. Martín. 1998. Effect

of pH heating medium on the thermal resistance of Bacillus stearothermphilus spores. J.

Food Safety.18:25–36.

10. Milly, P. J., R.T. Toledo, M.A. Harrison, and D. Armstead. 2007. Inactivation of food

spoilage microorganisms by hydrodynamic cavitation to achieve pasteurization and

sterilization of fluid foods. J. Food Sci. 72:M414–M422.

11. Odlaug, T.E., and I.J. Pflug. 1978. Clostridium botulinum and acid foods. J. Food Prot.

41:566–573.

12. Olivier, S.A., M.K. Bull, G. Stone, R.J. Diepenbeek, F. Kormelink, L. Jacops, and B.

Chapman. 2011. Strong and consistently synergistic inactivation of spores of apoilage-

associated Bacillus and Geobacillus spp. by high pressure and heat compared with

inactivation by heat alone. Appl Environ Microbiol. 77:2317–2324.

13. Palop, A., J. Raso, S. Condón, and F.J. Sala. 1996. Heat resistance of Bacillus subtilis and

Bacillus coagulans: effect of sporulation temperature in foods with various acidulants. J.

Food Prot. 59:487–492.

14. Palop, A., J. Raso, R. Pagán, S. Condón, and F.J. Sala. 1999. Influence of pH on heat

resistance of spores of Bacillus coagulans in buffer and homogenized foods. Int. J. Food

Microbiol. 46:243–249.

15. Pirone, G., S. Mannino, and E. Vicini. 1989. Termoresistenza di Bacillus coagulans in

passato do pomodoro. Ind. Conserve. 64:135–137.

16. Riazi, S., R.E. Wirawan, V. Badmaev, and M.L. Chikindas. 2009. Characterization of

lactosporin, a novel antimicrobial protein produced by Bacillus coaguans ATCC 7050. J

Appl Microbiol. 106: 1364–5072.

17. Rodrigo, M., A. Martinez, J. Sanchis, J. Trama, and V. Giner. 1990. Determination of

hot-fill-hold-cool process specifications for crushed tomatoes. J. Food Sci. 55:1029–

1032.

18. Sandoval, A.J., J.A. Barreiro, and S. Mendoza. 1992. Thermal resistance of Bacillus

coagulans in double concentrated tomato paste. J. Food Sci. 57:1369–1370.

19. Somavat, R., H. Mohamed, Y.K. Chung, A.E. Yousef, and S.K Sastry.

2012. Acceleration of inactivation of Geobacillus stearothermophilus spores by ohmic

heating. J. Food Eng. 108:69–76.

20. Stumbo, C.R. 1973. Thermobacteriology in Food Processing, 2nd ed. Academic Press,

New York.

21. U.S. Food & Drug Administration. 2010. Draft guidance for industry: acidified foods (44

FR 16230 at 16231).

22. Wang, B., B. Li, Q. Zeng, J. Huang, Z. Ruan, Z. Zhu, and L. Li. 2009. Inactivation

kinetics and reduction of Bacillus coagulans spores by the combination of high pressure

and moderate heat. J. Food Proc. Eng. 32:692–708.

23. Wiencek, K.M, N.A. Klapes, and P.M. Foegeding. 1990. Hydrophobicity of Bacillus and

Clostridium spores. Appl Environ Microbiol. 56:2600–2605.

24. York, G.K., J.R. Heil, G.L. Marsh, A. Ansar, R.L. Merson, T. Wolcott, and S. Leonard.

1975. Thermobacteriology of canned whole peeled tomatoes. J. Food Sci. 40:764–769.

TABLE 1. The effect of 4˚Cstorage on the viability of vegetative cells and spores of B.

coagulans

Strains Storage time (days) (sporulation Vegetative cells (Log CFU/ml) Spores (Log CFU/ml) environment) 0 10 23 31 0 10 23 31 ATCC 8038a 8.94±0.00 8.49±0.06 8.71±0.25 8.77±0.16 8.81±0.05 8.82±0.03 9.35±0.08 9.02±0.14 ATCC 8038b 8.57±0.07 8.10±0.10 8.07±0.66 8.27±0.05 7.84±0.18 7.96±0.04 8.68±0.03 8.55±0.04 ATCC 7050a 6.69±0.12 5.63±0.43 NDc ND ND ND ND ND ATCC 7050b 6.54±0.09 5.60±0.46 ND ND ND ND ND ND a Without moist chamber, plates sealed with Parafilm (Pechiney Plastic Packaging, Menasha, WI,

USA) only.

bIncubated in moist chamber: plates sealed with Parafilm and Petri dish containing water were

put into a plastic bag to prevent moisture loss of the sporulation medium.

c Colonies not detectable, detection limit 5 Log CFU/ml. Data are the mean ± SD of replicates.

TABLE 2. Comparison of D- and z-values of B. coagulans ATCC 8038 and 185A spores in commercial tomato juice

D-values (min) z-value Strains pH 95°C 100°C 105°C 110°C 115°C (°C)

7.05±0.14 2.56±0.15 1.18±0.02 0.20±0.01 -a 10.0 4.0 ATCC 8038 - 4.56±0.25 1.20±0.01 0.27±0.01 0.07±0.01 8.3 4.3

185A - 1.41±0.30 0.53±0.10 0.14±0.00 - 10.2 4.3

Data are the mean ± SD of replicates. a Not tested because of too long or too short heat treatment time.

TABLE 3. The effect of heating method on the thermal resistance of spores of B. coagulans

ATCC 8038

D-values (min)

Heating method pH z-value 95˚C 100˚C 105˚C 110˚C 115˚C

Oil bath 4.3 -a 4.56 ± 0.25 1.20 ± 0.01 0.27± 0.01 0.07 ± 0.01 8.3

Electrical 4.4 10.13 ± 0.31 2.52 ± 0.14 1.32 ± 0.02 0.17 ± 0.05 - 8.7

Data are the mean ± SD of replicates. a Not tested because of too long or too short heat treatment time.

Figure legends

FIGURE 1. Schematic of kinetics treatment chamber, with spore suspensions within sample in capillaries. Gel plugs in the miniature heater are non-conductive, so that the sample heats by heat transfer from an outer ohmic heater. Samples are withdrawn into a cooling chamber after processing.

FIGURE 2. D100°C-values of B. coagulans ATCC 8038 spores exposed to different pH levels in tomato juice. Data are the mean ± SD of three replicates. Data obtained at WSU.

FIGURE 3. Effect of pre-conditioning time on D100°C-value of B. coagulans ATCC 8038 spores in commercial tomato juice at pH 4.3. D100°C-value of control (spores in tomato juice without pre-conditioning): <3 min. Data are the mean ± SD of duplicates. Data obtained at WSU.

FIGURE 4. Thermal survivor curves for B. coagulans ATCC 8038 spores at different temperatures in commercial tomato juice at pH 4.0. Data are the mean ± SD of three replicates.

Data obtained at WSU.

FIGURE 5. Thermal survivor curves for B. coagulans ATCC 8038 spores heated at different temperatures in tomato juice adjusted to pH 4.4. Data are the mean ± SD of three replicates. Data obtained at OSU.

Figure 1. Schematic of kinetics treatment chamber, with spore suspensions within sample in capillaries. Gel plugs in the miniature heater are non-conductive, so that the sample heats by heat transfer from an outer ohmic heater. Samples are withdrawn into a cooling chamber after processing.

Figure 2. D100°C-values of B. coagulans ATCC 8038 spores exposed to different pH levels in tomato juice. Data are the mean ± SD of three replicates. Data obtained at WSU.

Figure 3. Effect of pre-conditioning time on D100°C-value of B. coagulansATCC 8038 spores in commercial tomato juice at pH 4.3. D100°C-value of control (spores in tomato juice without pre- conditioning): <3 min. Data are the mean ± SD of duplicates. Data obtained at WSU.

Figure 4. Thermal survivor curves for B. coagulans ATCC 8038 spores at different temperatures in commercial tomato juice at pH 4.0. Data are the mean ± SD of three replicates. Data obtained at WSU.

Figure 5. Thermal survivor curves for B. coagulans ATCC 8038 spores heated at different temperatures in tomato juice adjusted to pH 4.4. Data are the mean ± SD of three replicates. Data obtained at OSU.

Chapter 4. Kinetics of Carrot Texture Degradation under Pasteurization

Conditions

Abstract: Texture degradation of carrot dices in different solutions (distilled water, 0.1% and

1.4% CaCl2 solutions) under temperatures ranging from 80 to 110ºC was investigated. The effects of preheating (60ºC for 20 min) before high temperature treatment on carrot texture were studied and kinetic parameters were estimated. It was found that preheating enhanced the texture of the final products, and the improvement in texture became more apparent when CaCl2 was added. High temperature increased the texture degradation rate. The isotonic solution of carrot tissue was used to avoid possible ion leakage of carrot tissue during heating, but no significant differences were found between the texture of carrots immersed in isotonic solution and distilled water after thermal treatments. The texture degradation of preheated carrot dices under the investigated pasteurization conditions follows a 2nd order reaction. Kinetic results obtained were used to recommend processing conditions for carrot products that could control food pathogens and inactivate enzymes.

Keywords: carrot texture; preheating; isotonic concentration; calcium; microbial curve

1. Introduction

Carrots are one of the most commonly consumed vegetables in the United States, with one- fourth of all carrots consumed in processed form, largely canned and frozen (Lucier and Lin,

2007). In processed vegetables, texture is a primary marketable characteristic for the customer.

The texture of processed products is mainly controlled by the chemical composition, physical structure and amount of cell wall and middle lamella (Bourne, 1989). The various mechanisms of texture loss during heating of vegetables include turgor loss due to the breakdown of cellular membranes and cell wall degradation and disassembly resulting from enzymatic and non- enzymatic transformations in pectin structure and composition (Anthon et al., 2005; Greve et al.,

1994ab; Sila et al., 2008). Pectinmethylesterase (PME) and polygalacturonase (PG) are the two principle enzymes related to the enzymatic degradation of cell wall pectin. PME catalyzes the de-esterification of pectins, creating binding sites for divalent cations (primarily Ca2+, naturally present in the tissue or added during processing) on the polygalacturonic acid backbone of the pectin to form cross-links between pectin chains which improves the texture. Pectin may undergo nonenzymatic degradation through β-elimination, a chemical reaction that takes place at higher pH levels (>4.5) and at temperatures higher than 80ºC (Keijbets and Pilnik, 1974; Sila et al.,

2008).

Texture degradation of carrots during thermal processing has previously been investigated in several studies. Huang et al. (1983) and Bourne (1989) observed a rapid initial softening followed by a much slower rate of softening during the retort process of diced carrots. The authors proposed that carrot texture degradation consisted of two simultaneous first order reactions at different reaction rates during the thermal softening process. Rizvi and Tong (1997)

re-determined the kinetic parameters using the fractional conversion technique based on the published data supporting two substrate mechanisms of tissue softening. They suggested fractional conversion as an alternate technique which was more accurate and reliable to describe the overall trends for texture degradation of vegetables. Vu et al. (2004) investigated the kinetic degradation of sliced carrots in distilled and demineralized water in a temperature range from 80 to 110ºC, and estimated the kinetic parameters using a fractional conversion model. Later, Smout et al. (2005) studied the thermal texture degradation of carrot cylinders in a 0.5% CaCl2 solution using different preheating conditions followed by treatments at two heating temperatures (90 and

100°C) and also applied a fractional conversion model. In the current study, the concepts of

“equilibrium texture” and the fraction of texture changes were used to evaluate the kinetic data of texture degradation of diced carrots. Kinetic models with different reaction order were evaluated and the best-fit one was selected to estimate the related kinetic parameters.

When heating cut vegetables in aqueous solutions, differences in osmotic pressures within and outside the cells may result in ion leakage of the higher salt concentration within the cell and loss of cell integrity, which may influence mechanical properties (e.g. texture) (De EscaladaPla et al.,

2006; Gonzalez et al., 2010). Thus, an isotonic solution may be helpful in reducing the additional stress that a hypotonic bathing solution places on the already perturbed vegetable membranes.

Gonzalez et al. (2010) found that isotonic solutions help maintain membrane integrity in fresh onion tissues, and reported that the onion cell membranes ruptured between 50-60ºC. However, no published literature reported the rupture temperature of carrot cell membranes, nor the impact of osmotic solutions on carrot texture. In the current study, the isotonic concentration of carrot

tissue was determined and the effects of immersing carrot slices in the isotonic solution on the texture of the tissue at elevated temperatures were studied.

It is known that blanching vegetables at low-temperatures (generally 50-70ºC) prior to high- temperature processing may improve the texture of the final products (Anthon and Barrett, 2006;

Bartolome and Hoff, 1972; Vu et al., 2004; Wu and Chang, 1990). Preheating at these conditions activates pectin methylesterase (PME), resulting in extensive pectin de-esterification. This increases the chances for formation of ionically cross-linked pectin complexes and reduces the β- elimination reaction. Vu et al. (2004) reported that preheating carrots in distilled and demineralized water at 50–70°C for 20–40 min prior to high temperature heating could slow texture degradation, increase the final value of hardness and lower the activation energy of texture degradation. In the current study, one preheating condition (60°C for 20 min) was selected. According to published literature, preheating carrots at 60°C for 20 min prior to high heat treatment should enhance the vegetable texture (Stanley et al., 1995; Vu et al., 2004). The preheating step also mimics the microwave processing in our further study for pre-packaged carrot dices, where we always preheat the samples to a certain temperature before microwave heating (Tang et al., 2008). Since calcium salt is a commonly used firming agent, the effects of calcium on carrot texture were also investigated in this study. The calcium solution concentration used was chosen based on the FDA regulation for canned carrot products (0.036% Ca in the final products), which is far lower than that used in the published literature (Rastogi, et al., 2008;

Smout et al., 2005).

In addition to investigating the kinetics of texture degradation of carrot dices in solutions with different calcium levels, the goal of microbial/enzyme inactivation versus texture retention of carrots during thermal processing predicted by the degradation models was also discussed. This study provides useful information for determining thermal processing parameters for pre- packaged diced carrots, and for predicting quality changes related to texture during processing.

2. Materials and Methods

2.1. Sample preparation

Fresh carrots (Bolthouse Farms, Inc., Bakersfield, CA) purchased from a local grocery store were diced into 12.7×12.7×6 mm pieces. In order to prepare consistent samples, carrots with similar length and diameter were selected (the portion between 4-6 cm from the root tip and 4-8 cm from the stem), only those dices that contained a core (xylem) size of 4-7 mm diameter and 6 mm height were used in the study. A specially designed cylindrical aluminum test cell with a net inner space of 50 mm in diameter and 8 mm in depth was used to hold meaningful sample sizes for texture analyses while minimizing the come-up time during heating. Eight carrot dices (6.5 ±

0.2 g) were placed in the test cell, then 6 mL solution was added and the test cell was sealed. An o-ring fitting placed in the groove between the base and lid was used to provide a hermetic seal.

2.2. Determination of isotonic concentration of carrot tissue

The concentration of isotonic solution was determined according to the method of Saltveit

(2002). Briefly, fresh cut carrot dices were rinsed twice in distilled water for about 1 min each time, blotted dry, and 20 randomly selected pieces were transferred to each tared Petri-dish. The

Petri-dishes were placed into a plastic tub lined with wet paper towels and held overnight (ca.

18h) at room temperature. Twenty-five mL of mannitol solution (0–0.4 M) was added to each

dish and shaken at 60 cycles/min for a time period of either 20, 60, 120 or 240 min, and then the solutions were vacuum aspirated off. The weight gain or loss by the carrot pieces bathed in the mannitol solutions was recorded. The concentration of mannitol where there was no net weight gain or loss of the carrot pieces after the initial weight gain was taken to be the isotonic concentration of the carrot tissue. Experiments were done in triplicates.

2.3. Thermal treatment

Carrot dices immersed in different solutions in each test cell were heated in a thermostated oil bath (Model HAAKE DC 30, Thermo Electron Corp., Waltham, MA, USA) at 80, 90, 100 and

110°C for different time intervals. The temperatures were selected based on the heat-sensitivity of carrot texture and pasteurization conditions. Four solutions were investigated in this study:

1) Double distilled water.

2) Isotonic mannitol solution.

3) 0.1% CaCl2 solution (equivalent to containing 0.035% calcium).

4) 1.4% CaCl2 solution (equivalent to containing 0.5% calcium).

For the two calcium levels, the former was chosen according to the FDA regulation which allows addition of “up to 0.036% calcium to canned carrots” while the latter was within the range of the most commonly used calcium concentrations (0.5-2.0% CaCl2) added to vegetable products in published reports (Rastogi, et al., 2008; Smout et al., 2005). Since the diffusion of calcium into carrot tissues before heating may affect their texture, the time that was sufficient for sample preparation which resulted in little texture change was pre-determined as the equilibration time to keep the consistency of the initial carrot texture. According to our preliminary tests, all samples in the test cells were equilibrated in solutions for 10 min before heating.

To evaluate the effects of preheating on carrot texture, test cells containing carrot dices with different solutions were preheated in a thermostated water bath (Model HAAKE DC 30, Thermo

Electron Corp., Waltham, MA, USA) at 60°C for 20 min, then immediately transferred to an oil bath (Model HAAKE DL 30, Thermo Electron Corp., Waltham, MA, USA) and followed by a high heat treatment with preset temperatures ranging from 80 to 110°C. After heating, samples were cooled in ice water for 2 min, drained, equilibrated to room temperature and texture analysis was conducted. Unless otherwise stated, the zero-time samples were the samples at the end of the come-up time for the high heat.

2.4. Texture measurement

The firmness of treated carrot dices was determined using a TA.XT2 Texture analyzer (Stable

Micro Systems Ltd., Godalming, UK) fitted with a 25mm diameter aluminum cylinder probe following the methods described by Lemmens et al. (2009). The samples were compressed to

70% strain at a cross head speed of 1 mm/s. For each test, one piece of sample was placed under the probe. The peak force of the first compression cycle of the sample was marked the maximum force and recorded as the indicator of firmness. At least 6 replicates were measured for each treatment condition. Statistical analysis (Student’s t-test) was performed using Matlab 7.0 (The

MathWorks, Inc., 2004), and the significance level α was set as 0.05.

2.5. Kinetic analysis

A general form of the reaction equation is expressed as:

(1)

where C is a quality index or concentration of a chemical compound, t is the reaction time, k is the rate constant and n is the order of reaction.

Following the integration of both sides of Eq. (1), it becomes:

For n=1, (2)

For n≠1, ( ) (3)

The texture property is presented as the fraction of texture change C, which provides an accurate way to know the extent of quality change at any time t and can be expressed as

∞ (4) ∞ where F0 is the initial firmness at time 0; Ft is the firmness at time t; F∞ is the firmness at equilibrium or the nonzero maximum retainable firmness after prolonged heating (Rizvi and

Tong, 1997). In the current study, the F∞ value was obtained by measuring the texture of carrot after 24 hrs heating at 80 and 90°C, 12 hrs heating at 100 and 110°C, that the firmness was no longer changed with respect to time (within the standard deviation) (Rizvi and Tong, 1997).

A graph of t against (n≠1) or lnC (n=1) was plotted and linear regression was performed.

The best-fitted reaction order was determined by comparing the coefficient of determination (r2) for all the treated temperatures. The rate constant (k) of samples at each temperature was determined accordingly.

The temperature dependence of the reaction rate constant can by represented by the Arrhenius equation:

(5)

where A is a pre-exponential factor, Ea is activation energy (J/mole), R is the universal gas constant (8.314 J/K·mole), and T is the temperature (K). Thus, by plotting lnk against 1/T should result in a straight line, and the activation energy (Ea) can be calculated by the slope of the line

(Ea=8.314×Slope).

3. Results and Discussion

3.1. Determination of the isotonic solution for the carrot tissue and its effect on carrot texture

Apparent weight gain of carrot dices was observed after the first 20 min in all mannitol solutions, from 0.0–0.4 M (Figure 1A). After 60 min, carrot dices in the 0.3–0.4 M solutions began to lose weight; the higher the mannitol concentration, the more the weight loss. For those in 0.0–0.1 M solutions, the carrot dices kept gaining weight for up to 4h. Only in the 0.2 M solution, carrot dices did not have any weight change after the initial weight gain. The initial weight gain was due to the rapid ion diffusion of carrot tissue when it was immersed in the aqueous solution, until the rate of ion leakage reached a relatively constant point where there was neither weight gain nor loss. A better understanding of the isotonic concentration of carrot tissue can be seen in

Figure 1B, the weight change with regard to solution concentration. For the 0.2 M solution, the weight gain of carrot dices maintained 10% when compared to the fresh samples for up to 4h without any change. Therefore the concentration of the solution was considered to be isotonic

with respect to the carrot tissue. Saltveit (2002) also found the concentration of isotonic mannitol solution for mature green tomato tissue to be 0.2 M.

The texture of carrot dices heated in isotonic solution under temperatures ranging from 80–

110°C up to 2 h was compared with those heated in double distilled water (Figure 2). At each temperature, no significant difference was found in the texture between samples immersed in isotonic solution and distilled water at the corresponding heating time. It is likely that carrot cell membranes were completely ruptured at the test temperatures (80-110ºC) in the thermal treatments. Gonzales et al. (2010) observed complete loss of membrane integrity of onion cells at

60ºC and above.

3.2. Effects of preheating and calcium treatment on carrot texture

The effect of preheating on carrot texture was investigated by heating at 60°C for 20min before subjecting the samples to high temperatures ranging from 80 to 110°C in distilled water or calcium solutions (0.1% and 1.4%). As shown in Figure 3, preheating carrot dices at 60°C for 20 min changed their initial texture very little, with texture expressed by maximum force at 260 ±

22 N in distilled water compared to those fresh ones at 274 ± 23 N. However, the preheating step retarded the texture degradation of carrots in each solution at the subsequently high temperature; and the higher calcium concentration, the greater the decrease in degradation rate. The most possible reason is that PME activity greatly increased at the mild preheating temperature, resulting in the increase of demethylation of pectins and the number of calcium-binding sites.

This allows increased calcium cross-linking of the pectin chains and improved texture. A more apparent texture improvement due to preheating was observed at 90 and 100°C. At 110°C,

carrots lost most of their texture during the first 5-10 minutes. Nearly 80% loss in firmness took place within the come-up time (5min) in those immersed in DI water or 0.1% calcium solution, and around 70% texture loss in firmness occurred within 10 min heating in those immersed in

1.4% calcium solution. Anthon et al. (2005) also observed the texture of diced tomatoes was reduced to about 1/3 of the original level after 1 min at 100ºC with very little additional change over the next 4 min.

The reaction order was determined in order to select the texture degradation model with the best fit. According to the maximum force-time curve of carrot texture which is clearly non-linear, the reaction order n in equation (1) was set to 1, 1.5 and 2, and the coefficients of determination (r2) were obtained according to the methods described previously and listed in Table 1. One can observe that at 80°C, the kinetic model of the three reaction orders all work well for carrots immersed in each solution, but the 1storder reaction didn’t fit well at high temperatures.

Considering each temperature and immersion solution, the 2nd order reaction had the highest r2 values in all cases, thus it is the best fitting model for the degradation of carrot texture. The plot of 1/C vs. time of preheated carrots at different temperature (Figure 4) shows a well-fitting linear regression of the model when n=2 and demonstrates the degradation of carrot texture followed a

2nd order reaction. This indicates complex chemical reaction mechanisms that resulted in the texture changes of carrots during heating, which could have been affected by many factors such as the amount of cell wall and middle lamella, physical structures, and enzymes. The 2nd order reaction model of this study is different from those obtained by Smout et al. (2005) and Vu et al.

(2004 & 2006) who used a modified 1st order reaction model to analyze carrot texture degradation. However, in those studies, they didn’t assess the suitability of other reaction orders.

In addition, they didn’t experimentally determine values but rather estimated those values through regression analyses.

The estimated relative final values of the texture parameter ( /F0) are shown in Figure 5. The

/F0 values decreased with increasing temperature, from 0.037 ± 0.006 to 0.008 ± 0.002 for samples immersed in distilled water and from 0.129 ± 0.025 to 0.023 ± 0.004 for those immersed in 1.4% CaCl2 solution, due to decreasing values with increasing temperature. The carrots immersed in 1.4% CaCl2 solution had the highest /F0 while samples in distilled water exhibited the lowest values at each corresponding temperature. This can be explained by the fact that the value increased with increasing calcium concentration due to the firming effects of calcium. Smout et al. (2005) also reported a general decreasing trend of the final texture values of carrot discs with higher temperature processing in distilled and demineralized water.

The reaction rate constant (k) of carrot texture degradation behaved in the opposite way, as illustrated in Figure 6. As the temperature increased, the degradation rate constant increased from 0.035 min-1 to 1.453 min-1 for samples immersed in distilled water, and from 0.018 min-1 to

-1 0.360 min for those immersed in 1.4% CaCl2 solution. The degradation rate constant started to increase sharply when the temperature was higher than 90ºC for samples without added calcium, while for samples with added calcium, this sharp increase occurs when the temperature exceeded

100ºC. It is likely that higher temperature facilitated the breakdown of the pectins, which resulted in texture softening. However, increasing temperature could increase the calcium diffusion into the carrot tissue which might have a firming effect on texture. The degradation rate constant of samples at each temperature ranged in the following order: lowest for those

immersed in 1.4% CaCl2 solution, followed by 0.1% CaCl2 solution, and lastly the highest ones were in distilled water. This increase of the degradation rate constant among samples in the three solutions was more evident at high temperatures than at low temperatures. At 110ºC, the

-1 degradation rate constant of carrots in 1.4% CaCl2 is 1.453 min , almost 4 times smaller than those in distilled water, and 3 times smaller than those in 0.1% CaCl2 solution. The reason again is due to the firming effects of calcium, which positively correlates to the calcium concentration within a certain level depending on the free calcium-binding sites in the carrot pectin chains, and therefore reduces the degradation of carrot texture.

The Arrhenius equation accurately described the temperature dependence of the reaction rate constants, and can be used to correlate the reaction rate constant in food systems over typical temperature ranges associated with preservation processes and storage of food products. The

Arrhenius plot (ln k vs 1/T, the reciprocal absolute temperature) of carrot texture degradation is given in Figure 7. The activation energy (Ea), which represents the least amount of energy needed for a chemical reaction to take place, was calculated by the Arrhenius plot based on Eq.

(5). The calculated Ea was 138.9 kJ/mol for carrots immersed in distilled water, 118.3 kJ/mol for samples in 0.1% CaCl2 solution and 108.0 kJ/mol for samples in 1.4% CaCl2 solution. Vu et al.

(2004) reported activation energies for texture degradation of carrots in distilled and demineralized water of 117.56 kJ/mol using the conversion fraction model, while Paulus and

Saguy (1980) obtained Ea values of 92–117 kJ/mole for three different carrot varieties during thermal softening.

3.3. Quality versus microbial/enzyme inactivation

Kinetic models for quality degradation are required to predict quality changes for different processes targeted to achieve an equivalent level of microbial safety, thus helping optimizing process conditions. For pasteurization of diced carrot products, target bacterium and processing requirements are associated with product storage temperature and their shelf-life. For mild pasteurization of low-acid foods to provide a shelf life of 10 days maximum at 5ºC, traditionally a 6 log or 6 D reduction of Listeria monocytogenes (L. monocytogenes) is recommended; while for a longer shelf life (up to 6 weeks at 5ºC), a 6 log reduction of non- proteolytic Clostridium botulinum (NP C. botulinum) type E spores is required (ECFF, 2006;

Vervoort et al., 2012).

In addition to pathogen inactivation, pasteurization also aims to inactivate enzymes that cause quality loss during storage. For carrots, polygalacturonase (PG) is the most heat resistant texture- related enzyme which is involved in the degradation of pectins and results in texture loss

(Anthon and Barrett, 2002). Anthon and Barrett (2002) reported an Ea-value of PG in carrot juice as 411 kJ/mol and a reaction rate (k) of 0.0087 s-1 at 80ºC.

Gaze et al. (1989) determined the heat resistance of two strains of L. monocytogenes in carrots and obtained a z-value of 6.70–7.04ºC; later they studied the thermal resistance of NP C. botulinum type E spores in carrot from 75–90ºC and reported a z-value of 9.84ºC (Gaze and

Brown, 1990).

The processing times to achieve 4 and 6 log reduction of NP C. botulinum type E spores and L. monocytogenes in carrots and 90% inactivation of PG under different processing temperatures were calculated based on the thermal kinetic data obtained from the publications mentioned

above, and are presented in Figure 8. For the quality of carrots, texture is selected as the parameter and the quality retention in this study focuses on carrot dices immersed in 0.1% CaCl2 solution. The times needed to achieve 20%, 50% and 80% texture loss under each temperature were calculated from the kinetic texture degradation model obtained previously and are illustrated in Figure 8. Since the texture of carrots degraded very quickly at 110ºC (nearly 80% texture loss during the come-up time), the temperature of 110ºC was not considered as a processing temperature for pasteurization and was not included in Figure 8.

Appropriate processing conditions may be chosen based on the data in Figure 8 using a graphic approach as suggested by Holdsworth and Simpson (2008). That is, process conditions for carrots can be selected above the dashed lines in Figure 8 to ensure adequate reduction of target bacteria (NP CB type E spores or LM) or 90% of inactivation of PG, but below the solid lines to avoid a chosen level of carrot texture degradation. It is clear from Figure 8A that very short process times (e.g, 2-10 min at 90oC or 0.2-5 min at 100oC) should be used to achieve 6 log reduction in NP C. botulinum type E spores while still retaining 50% of the original texture in diced carrots. For control of LM, up to 20 minutes can be used at 80oC to retain 80% texture while achieving over 90% of PG inactivation (Figure 8 B and C). More curves for other quality parameters could be added to this figure to give a comprehensive quality retention- microbial/enzyme inactivation chart to facilitate the selection of appropriate process conditions.

4. Conclusions

Thermal degradation of carrot texture with pretreatments (preheating and calcium addition) under investigated pasteurization conditions follows a 2nd order reaction. Data presented in this

paper also show that carrot dices immersed in isotonic solution during preheating treatment

(60°C for 20 min) followed by high temperature heating didn’t help maintain their texture, compared to those immersed in distilled water. The obtained kinetic model of carrot texture was used to draw the temperature-time plots for texture retention of carrots during thermal processing, along with its microbial/enzyme inactivation curves. These provide a useful graphic approach for selecting appropriate processing conditions for pasteurization processes of carrot products, and also for predicting texture retention of thermally processed carrots.

Acknowledgements

This research was supported by USDA-NIFA Grant No. 2011-5116-68003-20996, titled: Control of Food-borne Bacterial & Viral Pathogens using Microwave Technologies. The senior author would like to thank the Chinese Scholarship Council for fellowship support.

References

Anthon, G., & Barrett, D.M. (2002). Kinetic parameters for the thermal inactivation of quality- related enzymes in carrots and potatoes. J Agric Food Chem, 50, 4119–4125.

Anthon, G. & Barrett, D.M. (2006). Characterization of the temperature activation of pectin

methylesterase in green beans and tomatoes. J Agric Food Chem, 54, 204–211.

Anthon, G.E., Blot, L., & Barrett, D.M. (2005). Improved firmness in calcified diced tomatoes

by temperature activation of pectin methylesterase. J Food Sci, 70(5), C342–C347.

Bourne, M.C. (1989). Applications of chemical kinetic theory to the rate of thermal softening of

vegetable tissue. In Quality Factors of Fruits and Vegetables, ACS Symp. Ser. 405 (J.J. Jen,

ed.). American Chemical Society, Washington, 98–110.

Bartolome, L.G., & Hoff, J.E. (1972). Firming of potatoes: biochemical effects of preheating. J

Agr Food Chem, 20(2), 266–270.

De EscaladaPla, M., Delbon, M., Rojas, A.M., & Gerschenson, L.N. (2006). Effect of immersion

and turgor pressure change on mechanical properties of pumpkin (Cucumismoschata, Duch.).

J Sci Food Agr, 86, 2628–2637.

ECFF (European Chilled Food Federation). (2010). Recommendations for the production of

prepackaged chilled food. Retrieved on Feb 6, 2013, from

http://www.chilledfood.org/Resources/Chilled%20Food%20Association/Public%20Resources

/ECFF_Recommendations_2nd_ed_18_12_06.pdf

Gaze, J.E., Brown, G.D., Gaskell, D.E., & Banks, J.G. (1989). Heat resistance of Listeria

monocytogenes in homogenates of chicken, beef steak and carrot. Food Micro, 6, 251–259.

Gaze, J.E., & Brown, G.D. (1990). Determination of the Heat Resistance of a Strain of Non-

proteolytic Clostridium botulinum Type B and a Strain of Type E, Heated in Cod and Carrot

Homogenate Over the Temperature Range 70 to 92°C. Campden Food and Drink Research

Association Technical Memorandum N. 592. Chipping Campden, UK.

Gonzalez, M.E., Anthon G.E., & Barrett D.M. (2010). Onion cells after high pressure and

thermal processing: comparison of membrane integrity changes using different analytical

methods and impact on tissue texture. J Food Sci, 75, E426–E432.

Greve, L.C., McArdle, R.N., Gohlke, J.R., & Labavitch, J.M. (1994a). Impact of heating on

carrot firmness. Changes in cell wall components. J Agric Food Chem, 42, 2900–2906.

Greve L.C., Shackel K.A., Ahmadi H., McArdle R.N., Gohlke J.R., & Labavitch J.M. (1994b).

Impact of heating on carrot firmness: contribution of cellular turgor. J Agric Food Chem, 42,

2896–2899.

Holdsworth, D., & Simpson, R. (2008). Thermal processing of packaged foods. 2nd ed. New

York: Springer.

Huang, Y.T., & Bourne, M.C. (1983). Kinetics of thermal softening of vegetables. J Texture

Studies, 14, 1–9.

Keijbets, M.J.H., & Pilnik, W. (1974). Beta-elimination of pectin in the presence of anions and

cations. J Carbohydr Res, 33, 359–362.

Lemmens, L., Tiback, E., Svelander, C., Smout, C., Ahrne, L., Langton, M., Alminger, M., Loey,

A.V., & Hendrickx, M. (2009). Thermal pretreatments of carrot pieces using different heating

techniques: effect on quality related aspects. Innov Food Sci & Emerg Tech, 10, 522–529.

Lucier, G., & Lin, B.H. (2007). Factors affecting carrot consumption in the United States.

Outlook Report from the Economic Research Service/USDA. No. (VGS-31901): 1–21.

Paulus, K., & Saguy, I. (1980). Effect of heat treatment on the quality of cooked carrots. J Food

Sci, 45, 239 –241.

Rastogi, N.K., Nguyen, L.T., & Balasubramaniam, V.M. (2008). Effect of pretreatments on

carrot texture after thermal and pressure-assisted thermal processing. J Food Eng, 88, 541–

547.

Rizvi, A.F., & Tong, C.H. (1997). Fractional conversion for determining texture degradation

kinetics of vegetables. J Food Sci, 62, 1–7.

Saltveit, M.E. (2002). The rate of ion leakage from chilling-sensitive tissue does not immediately

increase upon exposure to chilling temperature. Postharv Bio & Technol, 26, 295–304.

Sila, D.N., Duvetter, T., Roeck, A.D., Verlent, I., Smout, C., Moates, G.K., Hills, B.P., Waldron,

K.K., Hendrickx, M., & Loey, A.V. (2008). Texture changes of processed fruits and

vegetables: potential use of high-pressure processing. Trends Food Sci Tech, 19, 309–319.

Smout, C., Sila, D.N., Vu, T.S., Van Loey A.M., & Hendrickx, M. (2005). Effect of preheating

and calcium pre-treatment on pectin structure and thermal texture degradation: a case study on

carrots. J Food Eng, 67, 419–425.

Stanley, D.W., Bourne, M.C., Stone, A.P., & Wismer, W.V. (1995). Low temperature blanching

effects on chemistry firmness and structure of canned green beans and carrots. J Food Sci, 60,

327–333.

Tang, Z., Mikhaylenko, G., Liu, F., Mah, J.M., Pandit, R., Younce, F., & Tang, J. (2008).

Microwave sterilization of sliced beef in gravy 7-oz tray. J Food Eng, 89, 375–383.

Vervoort, L., Van der Plancken, I. Grauwet, T., Verlinde, T., Matser, A., Hendrickx, M., & Van

Loey, A. (2012). Thermal versus high pressure processing of carrots: a comparative pilot-

scale study on equivalent basis. Innov Food Sci & Emerg Tech, 15, 1–13.

Vu, T.S., Smout, C., Sila, D.N., LyNguyen, B., Loey, A.V., & Hendrickx, M. (2004). Effect of

preheating on thermal degradation kinetics of carrot texture. Innov Food Sci & Emerg Tech, 5,

37–44.

Vu, T.S., Smout, C., Sila, D.N., Van Loey, A.M.L., & Hendrickx, M. (2006). The effect of brine

ingredients on carrot texture during thermal processing in relation to pectin depolymerization

due to β-elimination reaction. J Food Sci, 71, E370–E375.

Wu, A., & Chang, W.H. (1990). Influence of precooking on the firmness and pectic substances

of three stem vegetables. Intl J Food Sci & Tech, 25, 558–565.

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Table 1. Coefficients of determination (r2) from kinetic order (n) models for carrot texture degradation at four temperatures.

r2 for different kinetic orders (n) Temp, °C Solution with preheating treatment 1 1.5 2 DI water 0.921 0.979 0.989 80 0.1% CaCl2 0.928 0.985 0.990 1.4% CaCl2 0.997 0.971 0.939 DI water 0.846 0.955 0.975

90 0.1% CaCl2 0.887 0.961 0.977 1.4% CaCl2 0.918 0.961 0.981

DI water 0.784 0.975 0.990 100 0.1% CaCl2 0.787 0.938 0.980 1.4% CaCl2 0.800 0.887 0.936 DI water 0.513 0.930 0.979 110 0.1% CaCl2 0.669 0.922 0.992

1.4% CaCl2 0.809 0.970 0.992

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Figure 1. Percent change in weight of excised carrot pericarp discs in 25 ml of aqueous solution at different mannitol concentrations. The percent change in weight is related to (A) time in solution, or (B) solution concentration. Data are the means ± S.D. (n≥3).

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Figure 2. Thermal degradation of texture of carrot dices in isotonic solution or distilled water at different temperatures. Data are the means ± S.D. (n≥6).

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Figure 3. Effect of preheating (60°C for 20 min) on the thermal texture degradation of carrots at different temperatures. For samples without preheating, time-zero equals raw materials before heating; for preheated samples, time-zero represents the time pre-heated samples were beginning to be subjected to high temperature heat. Data are the means ± S.D. (n≥6).

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Figure 4. Plot of 1/C vs. time at different temperatures (n=2). A: 80°C; B: 90°C; C: 100°C; D:

110°C; all the samples were preheated at 60°C for 20min. The line is the regression to the 2nd order model.

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Figure 5. The final texture value (F∞/F0) of pretreated carrot dices as a function of temperature in different solutions. Data are the means ± S.D. (n≥6).

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Figure 6. Reaction rate k of preheated carrot dices as a function of temperature in different solutions.

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Figure 7. Arrhenius plot of texture degradation rates of carrots immersed in different solutions with pretreatment.

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Figure 8. TDT curves of target bacteria, enzymes vs. carrot texture. A: Non-proteolytic C. botulinum type E spores; B: L. monocytogenes; C: Polygalacturonase enzyme. Data for NP CB type E spores are from Gaze and Brown (1990), LM are from Gaze et al. (1989) and PG are from

Anthon and Barrett (2002). The dash lines represent either 4D (6D) reduction on microbial load, or 90% inactivation of enzyme activity.

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Chapter 5. Dielectric Properties of Tomatoes Assisting in the Development of

Microwave Pasteurization and Sterilization Processes

Abstract: Dielectric properties of tomatoes crucially affect their dielectric behaviors in an electromagnetic field and are essential for developing microwave pasteurization and sterilization processes for different tomato products. An open-ended coaxial probe technique was used to determine the dielectric properties of tomatoes over a frequency range of 300–3000 MHz for temperatures between 22–120°C. Three tomato tissues, the pericarp tissue (including the skin), the locular tissue (including the seeds) and the placental tissue were studied separately. The effects of NaCl (0.2g/100g) and CaCl2 (0.055g/100g) on the dielectric properties of tomatoes were also investigated. The dielectric loss factors were significantly different among the three tomato tissues, and among the samples with and without salt. However, no significant differences were found in their corresponding dielectric constants. The loss factors of the three tomato tissues decreased with increasing frequency and increased with salts added. Increasing temperature increased the loss factors of the three tomato tissues at 915 MHz, but initially decreased then increased their corresponding values at 2450 MHz. The differences in the loss factors of the three tomato tissues were related to their different ionic conductivity. Penetration depths of the three tomato tissues exhibited similar trends with temperature at a given microwave frequency.

Keywords: Dielectric properties, tomato, microwave, salt, pasteurization/sterilization

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1. Introduction

The tomato is one of the most popular vegetables in the United States, second only to potato in terms of crop yield and consumption. The U.S. is one of the world’s leading producers of tomatoes, with an annual production of 12 to 15 million metric tons valued around $10 billion dollars over the last decade (USDA, 2010). Three-fourths of these tomatoes are consumed in processed form, most of which are thermally processed (Lucier and Glaser, 2009). The U.S. consumption of processed tomatoes began a steady climb that accelerated in the late 1980s with the rising popularity of pizza, pasta, and salsa (Lucier and Glaser, 2009). Even with the increase in consumption of fresh tomatoes in recent years, the demand for processed tomatoes remains relatively stable and consistent.

As one of the advanced processing technologies, microwave heating provides a relatively short heating time due to its ability to generate volumetric heating within food materials, and thus has the potential to be an alternative thermal treatment method for processing tomato products. Two frequency bands are allocated by the U.S. Federal Communication Commission (FCC) for microwave heating applications: the 915 MHz band for industrial use and the 2450 MHz band for both industrial and domestic uses. Dielectric properties of food materials which reflect the interaction between the foods and electromagnetic energy are essential for successful design of microwave pasteurization and sterilization processes. The dielectric properties of biological materials include the dielectric constant (ε') which is related to a material’s ability to store electric energy when subjected to an electromagnetic field, and dielectric loss factor (ε") which influences the conversion of electromagnetic energy into thermal energy. They are two elements of material’s complex relative permittivity (ε*) presented as ε* = ε' ─ jε", where √ . The

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dielectric properties of a material can also be used to estimate the thermal energy converted from electric energy at microwave frequencies. If heat loss is negligible, the increase in temperature

(∆T) of the material can be calculated from (Nelson, 1996):

3 where is the specific heat of the material (J/kgºC), ρ is the density of the material (kg/m ), ∆t

-12 is the time (s), ε0 (8.8542 × 10 F/m) is the permittivity of free space or vacuum, E is the strength of electric filed (V/m), and f is the frequency (Hz).

Several papers have reported the dielectric properties of fruits and vegetables (Seaman and Seals,

1991; Nelson et al., 1994; Ikediala et al., 2000; Feng et al., 2002; Birla et al., 2008; Wang et al.,

2011). There is very limited published information related to dielectric properties of tomatoes in a wide temperature range for the two microwave frequencies. Experimental data for those properties are, however, needed for proper design of microwave pasteurization and sterilization processes. Reyes et al. (2007) obtained the dielectric constant and loss factor of osmotically dehydrated cherry tomatoes measured at 2450 MHz and 20°C. Ghanem (2010) studied the dielectric properties and penetration depth of tomato juice from 25 to 45°C at 2450 MHz. Kumar et al. (2008) measured ε' and ε" of tomato particulates and puree for salsa con queso in a temperature range of 20–130°C at 915MHz. However, there is no published data on the dielectric properties of different tomato tissues. In the commercial processing of tomato paste, whole peel or dice products, the entire tomato containing all three tissue types, is used. For the Roma tomato, the average wet weight percentage of the percarp tissue (including skin), locular tissue

(including seeds) and placental tissue was 74 ± 6 g/100g, 13 ± 2 g/100g and 13 ± 4 g/100g, based on our measurements. The differences in the physicochemical properties of different tomato

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tissues may affect their dielectric properties, which would result in different heating rates and behaviors in microwave heating. Thus, it is helpful to know their individual dielectric properties to develop microwave pasteurization and sterilization processes for specific tomato products.

It is known that many factors may influence the dielectric properties of a given food, including frequency, temperature, moisture content, salts and other food constituents (Tang, 2005). NaCl and CaCl2 are the two salts most commonly added to canned tomato products; the former is for improved taste while the latter is a firming agent to retain texture. Several publications have discussed specific correlations between dielectric properties of foods and salt levels, frequency and food matrix (Goedeken et al., 1997; Guan et al., 2004; Ahmed et al., 2007; Zhang et al.,

2007; Wang et al., 2011). Little information is available on the influence of salts on the dielectric properties of tomatoes. Only one analysis conducted by Reyes et al. (2007) studied the dielectric spectroscopy of cherry tomatoes dehydrated with sucrose, NaCl and calcium lactate solutions at

2450 MHz. However, a very high salt concentration was used in Reyes’s study (1–20 g/100g of

NaCl, and 1–2 g/100g of calcium lactate) in order to create osmotic conditions for dehydration.

In the current study, the effect of NaCl and CaCl2 on the different tomato tissues will be discussed for typical levels found in commercially canned tomato products.

The objectives of the current study were: (1) measuring the dielectric properties of the three different tomato tissues (pericarp, locular and placental tissues) in a temperature range of 22–

120°C, over 300–3000MHz; (2) studying the effects of tomato compositions, temperature, frequency, NaCl and CaCl2 addition on their dielectric properties, particularly at the microwave

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industrial frequencies of 915 and 2450MHz; (3) investigating their loss mechanism; (4) determining the microwave penetration depths for the three tomato tissues.

2. Materials and methods

2.1. Sample preparation

Fresh Roma tomatoes were purchased from a local grocery store (Safeway, Pullman, WA, USA).

After washing, tomatoes were quartered and the three tissues were separated: pericarp tissue

(including skin), locular tissue (including seeds in the locular cavity) and placental tissue (Figure

1). Each tissue was collected and blended into a homogenate individually. A total of 25 ml tomato sample made from 2–3 tomatoes was used for each measurement. Separate samples were prepared with 0.2 g/100g of NaCl and 0.055 g/100g CaCl2 (equivalent to containing 200 mg/kg

Calcium) to evaluate the effects of salt addition on their dielectric properties. The salt concentrations were chosen based on common practices in the tomato canning industry.

2.2. Moisture content, pH and total soluble solids

Physiochemical properties of the three tomato tissues including moisture content, pH and soluble solid content were analyzed immediately after samples were prepared as described above.

Determination of moisture content was carried out in a vacuum oven following AOAC method

920.151 (AOAC, 2005). pH was measured using a Fisher Scientific Accumet pH meter. Total soluble solids were assessed by optical refractometer (Atago Co. LTD, Japan) and expressed as

°Brix. All measurements were conducted in triplicates.

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2.3. Determination of dielectric properties

The dielectric properties of tomato samples were measured using an open-ended coaxial probe connected to an HP 8752C network analyzer (Hewlett Packard Corp., Santa Clara, CA, USA) with system accuracy within 5% of error. An Agilent 85032B type N calibration kit which included open/short circuits and a 50 ohm load was used to calibrate the network analyzer. Then, the open-ended coaxial probe was calibrated by an Agilent 85070E dielectric probe kit, with air, short-circuit, and deionized water (25°C). After the calibration of the analyzer and the probe, tomato samples were added and tightly sealed in a test cell (Figure 2). The test cell was designed to hold the sample against the probe while allowing the sample temperature to be raised by a fluid (circulated from an oil bath) in the jacket wall to the designated temperature (Wang et al.,

2003). To avoid air bubble formation which could influence the probe sensor reading and cause error of a measurement, tomato samples were vacuum degassed (KOCH Packaging Supplies

Inc., Kansas City, MO) before each filling. Each measurement was repeated three times. The dielectric properties (ε' and ε") were determined over a frequency range of 300–3000 MHz for temperatures ranging 22–120°C in 20°C increments. Statistical analysis was performed using

Matlab 7.0 (The MathWorks, Inc., 2004), employing a student’s t-test (α=0.05).

2.4. Measurement of ionic conductivity

The dielectric loss mechanisms of biological materials in electromagnetic energy fields mainly include polar, electronic, atomic and Maxwell-Wagner responses (Metaxas and Meredith, 1983).

At microwave frequency ranges (915 and 2450 MHz), the dominant loss mechanisms in foods are dipole dispersion and conductive (ionic) charge migration, and can be expressed as

(Ryynänen, 1995):

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(1)

where represents contributions of dipole dispersion to a material’s dielectric loss factor and represents contributions of ionic conduction to dielectric loss factor. Since

(2)

We can substitute equation (2) into equation (1) and take logarithms of both sides to get

(3)

-12 where ε0 is the permittivity of free space or vacuum (8.854×10 F/m), σ is the ionic conductivity (S/m) of a given material, and f is frequency (Hz).

Ionic conductivity of pureed tomato tissues was measured at 22, 40, 60 and 80°C, using an electrical conductivity meter (Cole-Parmer Con 500 conductivity meter, Chicago, IL) with a direct current of 500 mA. Thirty-five ml tomato homogenate was poured into a Corning tube

(Corning Incorporated, NY) and the probe was placed in the center of the sample. The tube was sealed with Parafilm, and placed in a water bath (Thermo Electron Corporation, Waltham, MA,

USA) to heat to the desired test temperature. A Type-T thermal couple (accuracy ± 0.5°C) was inserted into the center of the sample to check the temperature. Experiments were done in triplicate.

2.5. Determination of power penetration depth

The penetration depth of microwaves is a measure of how deep microwave radiation can penetrate into a material. It is defined as the depth where the dissipated power is reduced to 1/e of the initial power entering the surface, and can be calculated by the following equation (Von

Hippel, 1954)

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(4)

√ √

8 where dp is the penetration depth (m) and c is the speed of light in free space (3×10 m/s).

Microwave power penetration depth is generally used to select appropriate thickness of food inside packages to ensure a relatively uniform heating along the depth of a food during dielectric heating processes (Wang et al., 2003; Wang et al., 2008). The penetration depth of microwaves into tomato samples was calculated at 915 MHz, at temperatures of 22, 40, 60, 80, 100, and

120°C.

3. Results and discussion

3.1. Physicochemical properties of pericarp, locular and placental tissues of raw tomatoes

The moisture content, pH and soluble solids content of the three tissues of raw tomato samples used in this study are reported in Table 1. The moisture content in the three tissues was very high and varied from 93–95 g/100g. Both the pH and soluble solids content in the locular tissue were the highest among the three tissues, with a value of 4.4 and 4.57 °Brix, respectively. Moretti et al. (1998) studied the chemical composition and physical properties in different tomato tissues and provided information about other components, including vitamin C, total carotenoids and chlorophyll. However, due to their large molecular weight and low content in the total sample, those components have little influence on the dielectric loss factor and thus were not analyzed in the current study. Since the moisture content in the three tomato tissues was similar and at a high value, one possible reason for the difference in their dielectric loss factors might be the different ionic conductivity due to the different amount and mobility of charged ions in the test tissues.

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This was confirmed by our measurement, in which we obtained an average ionic conductivity of

5.81, 7.42 and 5.01 mS/cm (1 mS/cm=0.1 S/m) for pericarp, locular and placental tissues, respectively.

3.2. Dielectric properties of pericarp, locular and placental tissues of raw tomatoes

Figure 3 shows typical trends for changes in dielectric constant and loss factor of tomato locular tissue over temperature and frequency. The dielectric constant decreased linearly with increasing temperature, about a 3–5 unit reduction for every 20°C temperature increase at the same frequency. In general, the dielectric constant also decreased with increasing frequency. Unlike the dielectric constant, the effect of temperature on the loss factor behaved in an opposite manner, increased with increasing temperature. The loss factor of tomatoes also decreased with increasing frequency, more sharply at lower frequencies (300–1500 MHz). At low temperatures

(22 and 40°C), a slight increase in loss factor was observed at the higher frequencies after the value decreased to the minimum at around 2000 MHz. These same trends noted for effects of temperature and frequency on dielectric properties of the locular tissues were also observed in tomato pericarp and placental tissues. These trends in tomato tissues are in agreement with those observed in other fresh fruits and vegetables with high moisture content (Seaman and Seals,

1991; Nelson et al., 1994; Ikediala at el., 2000; Feng et al., 2002).

The dielectric properties of different tomato tissues at 915 and 2450 MHz are shown in Figure 4.

At 915 MHz, the three tomato tissues had the same dielectric constant at each temperature, with the value decreasing from around 78 at 22°C to 57 at 120°C. However, the dielectric loss factor of the three tomato tissues differed from each other at each temperature, from 10–17 at 22°C to

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21–38 at 120°C. The placental tissue had the lowest dielectric loss factor while the locular tissue showed the highest value at each temperature. About a 3–5 unit increment of the loss factor of the three tomato tissues was observed at the same temperature, from placental tissue, locular tissue to pericarp tissue, accordingly. The same trends were observed in dielectric constant of the three tomato tissues at the frequency of 2450 MHz, with a slightly lower value at each temperature compared to those at 915 MHz. For the dielectric loss factor of the three tomato tissues at 2450MHz, their values were still arranged in the same order, with the lowest being the placental tissue and the highest locular tissue. However, the changes in their dielectric loss factors at 2450 MHz were different. Raising the temperature at this frequency, the dielectric loss factor initially decreased then increased when temperature reached or exceeded 80°C, exhibited a

U–shaped trend. At higher temperatures, the differences among the three tomato tissues were more apparent. The differences in dielectric loss factor of tomatoes with temperature between the two frequencies (915 and 2450 MHz) might result from the differences in their dominant loss mechanism. At the lower frequencies, dielectric loss due to ionic conductivity is more important while at higher frequencies, dipolar rotation of free water is the dominant contributor (Ryynänen,

1995; Calay et al., 1995). The dielectric loss factor of tomatoes continued increasing with increasing temperatures at 915 MHz because conductive loss played the dominant role and it increased with temperature increase. But at 2450 MHz, dipolar loss became dominant, which decreased with temperature increase. Raising temperature initially decreased the overall dielectric loss factor in tomatoes due to the dominant dipolar loss, and then increased because conductive losses took over at higher temperatures.

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3.3. Effect of NaCl

Values for the dielectric constants and loss factors of the three tomato tissues with addition of 0.2 g/100g NaCl at 915 and 2450 MHz are summarized in Figure 5. For the dielectric constant, the three tissues gave the same results as those without NaCl addition at each frequency, with higher values at 915 MHz than those at 2450 MHz. At each frequency, although the dielectric constant of the locular tissue was slightly higher than the other two tissues, no significant difference was found among these values of the three tomato tissues at each temperature. However, a significant difference was found in their corresponding dielectric loss factors. At 915 MHz, the dielectric loss factor was greatly increased compared to the samples without NaCl addition (Figure 4), from 10–17 to 17–24 at 22°C, and 21–38 to 41–55 at 120°C. The increase in the loss factor of the three tomato tissues is more pronounced at high temperatures. The same changes in dielectric loss factor of the three tomato tissues with NaCl addition were observed at 2450 MHz frequency.

These results indicate that 0.2 g/100g NaCl didn’t influence the dielectric constant of tomato samples, but appparently increased their dielectric loss factor. The increase of loss factor of the three tomato tissues may be explained by an increase of ionic conductivity due to dissolved ions coming from NaCl. This positive effect on the loss factor with increasing salt level was previously found in many foods, such as salmon fillets with 0–0.5 g/100g NaCl (Wang et al.,

2009), potato purees with 0–7 g/100g NaCl ( Guan et al., 2004; Wang et al., 2011), meat with 0–

5 g/100 salt (Lyng et al., 2005; Tanaka et al., 2000; Zhang et al., 2007), surimi with 0–6 g/100g

NaCl (Yaghmaee and Durance, 2001), and butter with 0–0.6 g/100g Na+ (Ahmed et al., 2007).

However, salting a product may also reduce the free water content due to the binding of free water molecules by the dissolved ions, therefore depressing the dielectric constant (Ahmed et al.,

2007;Calay et al., 1995; Zhang et al., 2007). Since the NaCl concentration used in the current

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study was only 0.2 g/100g, in such a high moisture-content food these binding effects on reducing the overall dielectric constant of the tomato tissue maybe negligible. A similar obsersvation was made by Ikediala et al. (2002), who showed that up to 2 g/100g NaCl addition to saline water produced little change in its dielectric constant at 915 MHz.

3.4. Effect of CaCl2

Calcium is often added as a firming agent to retain tomato texture. The effect of calcium on the dielectric properties of the three tomato tissues was investigated. According to the FDA regulation on calcium addition to canned tomato products (≤ 800 mg/kg calcium by weight in the finished product) and typical commercial processing practices, a concentration of 200 mg/kg calcium (equivalent to 0.055 g/100g CaCl2) was added to each tomato sample, along with the 0.2 g/100g NaCl. Dielectric properties of tomato pericarp, locular and placental tissues with these two added salts are summarized in Table 2. Again, at a specific frequency (915 or 2450 MHz), no significant difference was found in the value of the measured dielectric constant for the three tomato tissues, while an apparent difference existed in their dielectric loss factor at each temperature. The dielectric loss factor of each tissue at the frequency of 915 MHz varied from

20–26 at 22°C, to 50–64 at 120°C. Similar to the situation with NaCl, addition of CaCl2 only influenced the dielectric loss factor of tomato samples. Ikediala et al. (2002) also reported that increasing the concentration of CaCl2 solution from 0.1 g/100g to 2.0 g/100g at the frequency of

915 MHz sharply increased its loss factor, but resulted in little change in its dielectric constant.

A better understanding of the effects of NaCl and CaCl2 on the dielectric loss factor of different tomato tissues can be seen in Figure 6. Although the changing trends of the dielectric loss factors

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of three tomato tissues with temperatures were different at the two frequencies (915 and 2450

MHz), the effects of either salt addition were the same. Both salts increased the dielectric loss factor. Figure 6 also shows the increase in loss factor at high temperatures was more evident than at low temperatures. Besides, adding salts sharply increased loss factor in tomatoes with higher temperatures at 2450 MHz, lowering the turning point of temperature where their loss factor started to increase with increasing temperature.

3.5. Effect of ionic conductivity on dielectric loss factor

As shown in Eqs (1)–(3), two major dominant contributors to the value of dielectric loss in food materials at microwave frequencies are ionic loss which results from migration of ions, and dipole loss which results from water dipole dispersion. The ionic conductivity is a function of the concentration and type of ions present, and the temperature. Generally, the ionic conductivity of a very dilute aqueous solution is proportional to the amount of dissolved ions it contains (Gray,

2004). For high moisture foods, ionic conductivity normally increased with higher temperatures due to reduced viscosity and increased mobility of the ions (Tang et al., 2002). The values of ionic conductivity of tomato locular tissue at 22, 40, 60 and 80°C are shown in Figure 7. A sharp increase in measured ionic conductivity was observed after the addition of salt (NaCl or CaCl2), and the increase was more pronounced at high temperatures.

The dielectric loss factor value of tomato locular tissue contributed by ionic conduction (εσ")was calculated according to Eq. (2) and shown in Figure 8, along with the corresponding overall dielectric loss factor values (ε") measured by the network analyzer. Tomato locular tissue had similar trends, with or without salt. There is good agreement between measured ε" and calculated

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εσ" values at all temperatures in the frequency range below 700 MHz. This phenomenon demonstrated that the dielectric loss factor of tomato locular tissue was governed mainly by ionic conduction in the lower end of the studied frequency range. The increase in the dielectric loss factor with increasing temperature was indeed caused by the increase in ionic conductivity

(Figure 7). When the frequency increased above 700MHz, the measured ε" values started to shift above the calculated εσ" values. The deviation was caused by dipole dispersion which took place in the tomato samples at the high frequency range. The results are also in agreement with Figure

6, which indicates that the increase in measured ε" of each tissue was not proportional to the increase of total molar concentration of ions from added salts. The contribution of dipole rotation to the overall dielectric loss factor became more important when moving towards higher frequencies. The peak value of ε" due to dipole water at room temperature with respect to frequency occurs between 16–20 GHz (Mashimo et al., 1987; Tang et al., 2002). Raising temperature would move this peak towards higher frequency bands. Figure 8 also shows that this shifting at the higher frequencies was more pronounced at low temperatures than at high temperatures, which means the dipole loss had a larger influence at low temperatures. At high temperatures ionic conductivity increases and contributes more to the overall loss factor which results in a less shifting from the measured ε" to the calculated εσ". These resultsagree with our previous results of ε" (tomatoes) vs. temperature profiles (Figure 4–6), which were linear at 915

MHz while a U–shaped curve existed at 2450 MHz.

3.6. Penetration depth

The penetration depth of microwaves in the three tomato tissues (pericarp, locular, and placental tissue) at the two microwave frequencies is summarized in Table 3. Fresh samples had the

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highest penetration depths, while the samples with the two salts added had the lowest values under the conditions studied. All of the three tomato tissues showed higher penetration depth at

915 MHz than their corresponding values at 2450 MHz, with average values varied from 7.0–

43.3 mm for the former and 5.8–17.6 mm for the latter. Although the penetration depth of the three tomato tissues varied from each other, they exhibited very similar trends in their changes with salt addition, decreasing with increasing salt. For temperature changes, increasing temperature decreased their penetration depth at 915 MHz; while initially increased then decreased their penetration depth at 2450 MHz. For every 20°C temperature increment at 915

MHz, the penetration depth decreased around 2–4 mm under the same condition; while the penetration depth changed around 0.2–2 mm at 2450 MHz. The tendency of change in penetration depth with increasing temperature is opposite to those in their loss factor, which is easy to understand because according to Equation (4) ε" is inversely related to dp. The changes in

ε" affected dp more than the influence from ε' in tomato samples used in our studies. A similar change in the penetration depth with temperature at 915 MHz has been reported for whey protein gel, macaroni and cheese by Wang et al. (2003), and pink salmon fillets by Wang et al. (2009).

4. Conclusions

Our results showed that dielectric loss factor of three tomato tissues (the pericarp, the locular and the placental tissues) were significantly different from each other, either with or without salt.

However, no significant differences were found in their corresponding dielectric constant. Salt addition at the typical commercial canned tomato product level (0.2 g/100g NaCl or 0.055 g/100gCaCl2) sharply increased the loss factor of the three tomato tissues, but didn’t affect their dielectric constant at the microwave frequencies (915 and 2450 MHz). Similar trends for changes

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in dielectric loss factor were observed in the three tomato tissues, decreasing with increased frequency, and increasing with salt addition. For the effects of temperature, increasing temperature continued increasing their dielectric loss factor at 915 MHz while initially increased then decreased their corresponding values at 2450 MH, resulting from their different dominant loss mechanism at the two frequencies. Furthermore, a positive correlation was found between the loss factor of the tomato tissue and their ionic conductivity. At a specific frequency (either

915 or 2450 MHz), the penetration depth of the three tomato tissues varied from each other, but again exhibited similar change tendency. Results obtained in this study may be used for developing microwave pasteurization and sterilization processes for different tomato products, and also add new information to the database for computer simulation.

Acknowledgements

This research was supported in part by USDA-CSREES-NRICGP Grant No. 2009-55503-05198, titled: Quality of Foods Processed Using Selected Alternative Processing Technologies and

USDA-NIFA Grant No. 2011-5116-68003-20996, titled: Control of Food-borne Bacterial& Viral

Pathogens using Microwave Technologies. The senior author would like to thank the Chinese

Scholarship Council for fellowship support.

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References

Ahmed, J., Ramaswamy, H.S., & Raghavan, V.G. (2007). Dielectric properties of butter in the

MW frequency range as affected by salt and temperature. Journal of Food Engineering, 82,

351–358.

AOAC. (2005). AOAC official method 920.151: solids (total) in fruits and fruit products.

Official Methods of Analysis of AOAC International. 18th ed. Washington, DC.

Birla, S.L., Wang, S., Tang, J., & Tiwari, G. (2008). Characterization of radio frequency heating

of fresh fruits influenced by dielectric properties. Journal of Food Engineering, 89, 390–398.

Calay, R.K., Newborough, M., Probert D., & Calay P. (1995). Predictive equations for the

dielectric properties of foods. Journal of Food Science and Technology, 29, 699–713.

Feng, H., Tang, J., & Cavalieri, R.P. (2002). Dielectric properties of dehydrated apples as

affected by moisture and temperature. Transactions of the ASAE, 45(1), 129–135.

Ghanem, T.H. (2010). Dielectric properties of liquid foods affected by moisture contents and

temperatures. Misr Journal of Agricultural and Engineering, 27(2), 688–698.

Goedeken, D.L., Tong, C.H., & Virtanen, A.J. (1997). Dielectric properties of a pregelatinized

bread system at 2450 MHz as a function of temperature, moisture, salt and specific volume.

Journal of Food Science, 62, 145–149.

Gray, J.R. (2004). Conductivity analyzers and their application. In Down, R.D. & Lehr, J.H.

Environmental Instrumentation and Analysis Handbook. Wiley, 491–510.

Guan, D., Cheng, M., Wang, Y., & Tang, J. (2004). Dielectric properties of mashed potatoes

relevant to microwave and radio-frequency pasteurization and sterilization processes. Journal

of Food Science, 69(1), 30–37.

126

Ikediala, J.N., Hansen, J.D., Tang, J., Drake, S.R., & Wang, S. (2002). Development of a saline

water immersion technique with RF energy as a postharvest treatment against codling moth in

cherries. Postharvest Biology and Technology, 24, 209–221.

Ikediala, J.N., Tang, J., Drake, S.R., & Neven, L.G. (2000). Dielectric properties of apple

cultivars and codling moth larvae. Transactions of the ASAE, 43(5), 1175–1184.

Kumar, P., Coronel, P., Simunovic, J., & Sandeep, K.P. (2008). Thermophysical and dielectric

properties of salsa con queso and its vegetable ingredients at sterilization temperatures.

International Journal of Food Properties, 11, 112–126.

Lucier, G., & Glaser, L. (2009). Vegetables and melons: tomatoes. USDA Economic Research

Service. Retrieved from http://www.ers.usda.gov/briefing/vegetables/tomatoes.htm (18 Feb

2013).

Lyng, J.G., Zhang, L., & Brunton, N.P. (2005). A survey of the dielectric properties of meats and

ingredients used in meat product manufacture. Meat Science, 69, 589–602.

Mashimo, S., Kuwabar, S., & Higasi, K. (1987). Dielectric relaxation time and structure of

bound water in biological materials. The Journal of Physical Chemisty, 91, 6337–6338.

Metaxas, A.C., & Meredith, R.J. (1983). Industrial Microwave Heating. Peter Peregrinus,

London.

Moretti, C.L., Sargent, S.A., & Huber, D.J. (1998). Chemical composition and physical

properties of pericarp, locule and placental tissues of tomatoes with internal bruising. Journal

of the American Society for Horticultural Science, 123(4), 656–660.

Nelson, S., Forbus, J.W., & Lawrence, K. (1994). Permittivities of fresh fruits and vegetables at

0.2 to 20 GHz. Journal of Microwave Power and Electromagnetic Energy, 29(2), 81–93.

127

Nelson, S. (1996). Review and assessment of radio-frequency and microwave energy for stored-

grain insect control. Transactions of the ASAE, 39, 1475–1484.

Reyes, R.D., Heredia, A., Fito, F., Reyes, E.D., & Andres, A. (2007). Dielectric spectroscopy of

osmotic solutions and osmotically dehydrated tomato products. Journal of Food Engineering,

80, 1218–1225.

Ryynänen, S. (1995). The electromagnetic properties of food materials: a review of the basic

principles. Journal of Food Engineering, 29, 409–429.

Seaman, R., & Seals, J. (1991). Fruit pulp and skin dielectric properties for 150 MHz to 6400

MHz. Journal of Microwave Power and Electromagnetic Energy, 26(2), 72–81.

Tang, J., Feng, H., & Lau M. (2002). Microwave heating in Food Processing. Advances in

Bioprocessing Engineering, 1–44. River Edge, NJ: World Scientific.

Tang, J. (2005). Dielectric properties of foods. The Microwave Processing of Food. H. Schubert

and M. Regier (Ed.), CRC Press, Woodhead Publishing Limited, Cambridge, London, pp. 22-

40.

Tanaka, F., Mallikarjunan, P., Kim, C., & Hung Y.C. (2000). Measurement of dielectric

properties of chicken breast meat. Journal of Japanese Society of Agricultural Machinery,

62(4), 109–119.

U.S. Department of Agriculture, Economic Research Service. (2010). U.S. tomato statistics

(92010). Retrieved from

http://usda.mannlib.cornell.edu/MannUsda/viewDocumentInfo.do?documentID=1210htm (18

Feb 2013).

Von Hippel, A.R. (1954). Dielectric properties and waves. New York: John Wiley.

128

Wang, Y., Tang, J., Rasco, B., Kong, F., & Wang, S. (2008). Dielectric properties of salmon

fillets as a function of temperature and composition. Journal of Food Engineering, 87, 236–

246.

Wang, Y., Wig, T., Tang, J., & Hallberg, L. (2003). Dielectric properties of foods related to RF

and microwave pasteurization and sterilization. Journal of Food Engineering, 57, 257–268.

Wang, Y., Tang, J., Rasco, B., Wang, S., Alshami, A.A., & Kong, F. (2009). Using whey protein

gel as a model food to study dielectric heating properties of salmon

(Oncorhynchusgorbuscha) fillets. LWT Food Science and Technology, 42, 1174–1178.

Wang, R., Zhang, M., Mujumdar, A.S., & Jiang, H. (2011). Effect of salt and sucrose content on

dielectric properties and microwave freeze drying behavior of re-structured potato slices.

Journal of Food Engineering, 106, 290–297.

Yaghmaee, P., & Durance, T.D. (2001). Predictive equations for dielectric properties of NaCl,

D-sorbitol and sucrose solutions and surimi at 2450 MHz. Journal of Food Science, 67(6),

2207–2111.

Zhang, L., Lyng, J.G., & Brunton, N.P. (2007). The effect of fat, water and salt on the thermal

and dielectric properties of meat batter and its temperature following microwave or radio

frequency heating. Journal of Food Engineering, 80, 142–151.

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Table 1. Moisture content, pH and soluble solids content of pericarp, locular and placental tissues in raw tomatoes.

Moisture content, (g/100g) pH Soluble solids, °Brix

Pericarp tissue 94.6 ± 0.4 4.15 ± 0.01 4.03 ± 0.06

Locular tissue 93.8 ± 0.5 4.41 ± 0.08 4.57 ± 0.06

Placental tissue 94.5 ± 0.4 4.19 ± 0.01 4.30 ± 0.14

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Table 2. Dielectric properties of tomato pericarp, locular and placental tissues with 0.2 g/100g of

NaCl and 0.055 g/100g of CaCl2 at 915 and 2450 MHz.

Temp (°C) 915MHz 2450MHz

ε' ε" ε' ε"

22 77.9 ± 0.5 22.5 ± 0.2 76.0 ± 0.7 17.1 ± 0.1

40 74.6 ± 0.9 26.3 ± 0.0 73.2 ± 0.9 16.1 ± 0.2

Pericarp 60 70.6 ± 0.8 31.6 ±0.6 69.4 ± 0.8 16.4 ± 0.2

tissue 80 66.3 ± 0.7 38.5 ± 0.4 65.5 ± 0.6 18.0 ± 0.2

100 61.7 ± 0.8 47.3 ± 0.3 60.9 ± 0.6 20.4 ± 0.1

120 57.7 ± 0.7 55.9 ± 0.3 56.9 ± 0.9 23.1 ± 0.2

22 77.7 ± 0.2 25.9 ± 0.4 75.1 ± 0.4 18.5 ± 0.2

40 75.2 ± 0.3 30.4 ± 0.2 73.1 ± 0.1 18.0 ± 0.2

Locular 60 71.2 ± 0.2 36.7 ± 0.3 69.7 ± 0.3 18.9 ± 0.3

tissue 80 67.2 ± 0.5 44.6 ± 0.2 66.0 ± 0.6 20.5 ± 0.4

100 63.0 ± 0.4 54.4 ± 1.0 61.6 ± 0.4 23.5 ± 0.5

120 59.1 ± 0.4 63.3 ± 1.7 58.1 ± 0.2 26.2 ± 0.5

22 76.5 ± 0.1 20.9 ± 0.2 74.3 ± 0.4 16.8 ± 0.1

40 74.0 ± 0.4 24.2 ± 0.2 72.0 ± 0.2 15.7 ± 0.1

Placental 60 69.9 ± 0.3 28.9 ± 0.3 68.5 ± 0.4 15.9 ± 0.3

tissue 80 65.7 ± 0.0 34.8 ± 0.0 64.6 ± 0.1 16.7 ± 0.2

100 61.3 ± 0.3 41.8 ± 0.5 60.3 ± 0.1 18.7 ± 0.1

120 57.3 ± 0.7 49.3 ± 0.4 56.4 ± 0.4 20.7 ± 0.2

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Table 3. Microwave penetration depth into tomato pericarp, locular and placental tissues at

915M Hz.

Penetration Depth (mm) 915MHz 2450MHz Temp (°C) Fresh with 0.2 g/100g with Fresh with 0.2g/100g with 0.2 NaCl 0.2g/100g NaCl NaCl g/100g NaCl & & 0.055 g/100g 0.055 g/100g

CaCl2 CaCl2 22 33.1 ± 0.3 22.0 ± 0.5 20.7 ± 0.1 12.4 ± 0.3 11.0 ± 0.2 10.0 ± 0.2

40 29.1 ± 0.3 19.2 ± 0.7 17.4 ± 0.1 14.0 ± 0.5 11.5 ± 0.2 10.5 ± 0.2

Pericarp 60 23.9 ± 0.3 15.6 ± 0.4 14.2 ± 0.2 14.5 ± 0.7 11.1 ± 0.1 10.0 ± 0.0

tissue 80 19.3 ± 0.6 12.4 ± 0.3 11.5 ± 0.1 13.9 ± 0.8 10.1 ± 0.0 8.8 ± 0.1

100 15.6 ± 0.7 10.3 ± 0.0 9.2 ± 0.1 12.6 ± 0.8 8.7 ± 0.1 7.6 ± 0.1

120 12.4 ± 0.8 8.6 ± 0.1 7.8 ± 0.1 10.5 ± 0.8 7.4 ± 0.0 6.5 ± 0.0

22 27.1 ± 0.9 19.9 ± 0.5 18.0 ± 0.3 11.4 ± 0.1 10.0 ± 0.0 9.2 ± 0.1

40 24.0 ± 1.0 17.1 ± 0.2 15.2 ± 0.1 12.3 ± 0.1 10.4 ± 0.0 9.3 ± 0.1

Locular 60 19.6 ± 0.7 14.0 ± 0.0 12.4 ± 0.1 12.5 ± 0.2 9.9 ± 0.1 8.7 ± 0.1

tissue 80 16.1 ± 0.6 11.3 ± 0.0 10.1 ± 0.1 11.8 ± 0.1 8.9 ± 0.1 7.8 ± 0.1

100 13.1 ± 0.1 9.4 ± 0.1 8.2 ± 0.1 10.4 ± 0.1 7.7 ± 0.1 6.6 ± 0.2

120 11.0 ± 0.1 7.9 ± 0.0 7.0 ± 0.1 9.2 ± 0.1 6.7 ± 0.0 5.8 ± 0.2

22 43.3 ± 0.4 26.2 ± 0.7 22.0 ± 0.2 13.5 ± 0.1 11.2 ± 0.5 10.1 ± 0.1

40 41.0 ± 0.1 22.7 ± 0.8 18.8 ± 0.1 15.9 ± 0.1 12.1 ± 0.6 10.6 ± 0.1

Placental 60 33.9 ± 0.5 18.6 ± 0.4 15.4 ± 0.1 17.4 ± 0.1 12.2 ± 0.5 10.2 ± 0.2

tissue 80 28.1 ± 0.0 15.0 ± 0.4 12.6 ± 0.0 17.6 ± 0.7 11.2 ± 0.6 9.5 ± 0.1

100 23.4 ± 0.4 12.2 ± 0.2 10.3 ± 0.1 16.7 ± 0.4 9.9 ± 0.4 8.2 ± 0.0

120 18.6 ± 0.5 10.2 ± 0.2 8.6 ± 0.0 14.8 ± 0.2 8.6 ± 0.3 7.2 ± 0.1

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List of Figure Captions

Figure 1. Illustration of different anatomical structures of tomato fruits.

Figure 2. Schematic diagram of pressure-proof test cells used for dielectric properties measurement (from Wang et al., 2003).

Figure 3. Dielectric properties of raw tomato locular tissue as a function of temperature and frequency (♦ 22°C; □ 40°C; ▲ 60°C; × 80°C; ● 100°C; ○ 120°C). Data are the means± S.D.

(n=3).

Figure 4. Dielectric properties of raw pericarp (♦), locular (□) and placental (△) tissues at 915

(A) and 2450 (B) MHz. Data are the means± S.D. (n=3).

Figure 5. Dielectric properties of tomato pericarp (♦), locular (□) and placental (△) tissues with

0.2 g/100g of NaCl at 915 (A) and 2450 MHz (B). Data are the means± S.D. (n=3).

Figure 6. Dielectric loss factor of tomato pericarp (♦), locular (□) and placental (△) tissues at 915

(A) and 2450 (B) MHz. Added NaCl was 0.2 g/100g and CaCl2 was 0.055 g/100g. Data are the means± S.D. (n=3).

Figure 7. Ionic conductivity of tomato locular tissue as a function of temperature (♦ raw sample;

□ added with 0.2 g/100g of NaCl; △ added with 0.2 g/100g of NaCl and 0.055 g/100g of CaCl2).

Data are the means± S.D. (n=3).

Figure 8. Measured ε" and calculated εσ" of tomato locular tissue (A raw sample; B with NaCl; C with NaCl & CaCl2) as a function of frequency and temperature ( 22ºC, measured;

40ºC, measured; 60ºC, measured; 80ºC, measured; 22ºC, calculated;

40ºC, calculated; 60ºC, calculated; 80ºC, calculated). Data are the means± S.D.

(n=3).

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Figure 1. Illustration of different anatomical structures of tomato fruits.

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Figure 2. Schematic diagram of pressure-proof test cells used for dielectric properties measurement (from Wang et al., 2003), dimensions are in mm.

135

Figure 3. Dielectric properties of raw tomato locular tissue as a function of temperature and frequency (♦ 22°C; □ 40°C; ▲ 60°C; × 80°C; ● 100°C; ○ 120°C). Data are the means ± S.D.

(n=3).

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Figure 4. Dielectric properties of raw pericarp (♦), locular (□) and placental (△) tissues at 915

(A) and 2450 (B) MHz. Data are the means ± S.D. (n=3).

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Figure 5. Dielectric properties of tomato pericarp (♦), locular (□) and placental (△) tissues with

0.2 g/100g of NaCl at 915 (A) and 2450 MHz (B). Data are the means ± S.D. (n=3).

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Figure 6. Dielectric loss factor of tomato pericarp (A1 & B1), locular (A2 & B2) and placental

(A3 & B3) tissues at 915 (A) and 2450 (B) MHz (♦ raw sample; □ added with 0.2 g/100g of

NaCl; △ added with 0.2 g/100g of NaCl and 0.055 g/100g of CaCl2). Data are the means ± S.D

(n=3).

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Figure 7. Ionic conductivity of tomato locular tissue as a function of temperature(♦ raw sample;

□ added with 0.2 g/100g of NaCl; △ added with 0.2 g/100g of NaCl and 0.055 g/100g of CaCl2).

Data are the means ± S.D. (n=3).

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Figure 8. Measured ε" and calculated εσ" of tomato locular tissue (A raw sample; B with NaCl; C with NaCl & CaCl2) as a function of frequency and temperature ( 22ºC, measured;

40ºC, measured; 60ºC, measured; 80ºC, measured; 22ºC, calculated;

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40ºC, calculated; 60ºC, calculated; 80ºC, calculated). Arrows show the influence of increasing temperature. Data are the means ± S.D (n=3).

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Chapter 6. Developing Microwave Sterilization/Pasteurization Processes for

Pre-packaged diced Tomatoes/Carrots

Abstract: Microwave (MW) processing as one of the few emerging food preservation technologies takes relatively short heating time due to its volumetric heating generated within food materials, and therefore has the potential to produce high quality shelf-stable or chilled food products. In the current study, a MW assisted thermal sterilization (MATS) process was developed for processing diced tomatoes packaged in 8-oz pouches using a semi-continuous,

The simulation results were validated with a chemical marker based computer-vision method.

Heat penetration tests were conducted to obtain temperature-time data for the cold spot in diced tomatoes packaged in pouches, from which a MATS process was designed to achieve a 5D reduction in Bacillus coagulans ATCC 8038 spores (F105°C = 6.0 min). For diced carrots, two

MAP processes were developed: a 6D process for non-proteolytic Clostridium botulinum type E spores (F90°C=3 min); and a F90°C=10 min process. Incubation tests of the processed tomato products verified the successful development of MATS processes.

Keywords: Microwave; sterilization; pasteurization; pre-packaged; tomato; carrot

1. Introduction

Applications of MW energy in food processes have drawn increased attention over the past decades, due to the rapid heating (usually several minutes) of MW processes by generating

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volumetric heating within food materials. MW heating overcomes the disadvantages of slow conductive/convective heat transfer in conventional thermal processes. Therefore, MW processing has the potential to produce high quality self-stable food products. Tomatoes and carrots are two of the most commonly consumed vegetables in the US. Three-fourths of the tomatoes consumed by Americans are in processed forms, most of which are thermally processed; while one-fourth of carrots are consumed in a processed form, largely canned and frozen (Lucier and Glaser, 2009; Lucier and Lin, 2007). However, there is no published paper on developing systematic microwave sterilization or pasteurization processes for the diced tomato or carrot products.

Non-uniformity in temperature distribution remains the challenge for developing a MW assisted thermal process for food products to meet the stringent food safety regulation and standards. The uneven heating results in the hot and cold spots in the MW processed foods. The cold spot is the area which receives the lowest thermal energy and the hot spot is the area of highest thermal energy reception. Several factors contribute to the non-uniform heating during the MW processing, mainly including uneven distribution of the electric field due to electromagnetic energy dispersion, different dielectric properties of food materials, package geometry, and heat loss on the boundary. Thus, it is critical to determine the heating pattern, especially the location of cold spots inside the foods for developing a successful MW process. Kim and Taub (1993) developed a chemical marker method at the U.S. Army Natick Research Center. This method correlated heating intensity with brown color caused by the formation of the Maillard browning product chemical marker M-2 (4-hydroxy-5-methyl-3(2H)furanone). The thermal intensity can be detected by the yield of M-2 in the homogenous foods through HPLC analysis. Pandit et al.

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(2007) then improved this chemical marker method for heating pattern determination by developing a rapid computer vision method to establish a linear correlation between the M-2 marker yield and the cumulative thermal lethality (F0). Considering the processing temperature and the gelling temperature, whey protein gels containing D-ribose and salt have been used in our laboratory as a model food to determine heating patterns in foods during high temperature

MATS processes (Lau et al, 2003; Tang, 2008; Wang et al., 2009) while gellan gels, egg whites and whole eggs were investigated for low temperature MAP processes (Zhang et al., 2013).

Besides the chemical marker methods in experimentation, computer simulation is another effective way to predict the heating pattern and numerically solve the problems in MW heating.

Chen et al. (2007) successfully developed a commercial electromagnetic software combined with a customer-built heat transfer model to simulate the coupled electromagnetic-thermal MW system. They also validated the simulation model with a pilot-scale MW system using direct temperature measurement data and indirect color patterns in whey protein gels via formation of the thermally induced chemical marker M-2 (Chen et al., 2008). Resurreccion et al. (2013) further improved this simulation model for the MATS system. The model considered the coupled electromagnetic-thermal phenomena in food packages moving in multiple MW cavities. In the current study, the chemical-marker and computer simulation methods were also used to detect the heating patterns and cold spots of the foods during MW processing.

When using the model food to simulate the real products and determining the heating patterns and cold spots, this requires that the dielectric properties of the real foods match that of the model foods. The dielectric properties of food materials reflect the interaction between the foods and electromagnetic energy. They include the dielectric constant (ε') and dielectric loss factor

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(ε"). The former is related to a material’s ability to store electric energy when subjected to an electromagnetic field, while the latter influences the conversion of electromagnetic energy into thermal energy. Therefore, the dielectric properties of the diced tomato and carrot products were also measured in the current study.

In addition to determination of heating pattern and cold spots, temperature profiles of foods during processing are also essential for a successful design of MW processing to ensure food safety. Temperature sensors are usually placed at the cold spot inside the foods to trace the temperature of the cold spot during thermal processing, and the information obtained can be used to calculate the thermal lethality levels (F value). Using fiber-optic thermometers is the most reliable and accurate way for direct temperature measurement in electromagnetic fields, and they are often used in MW and radio frequency heating (Tang et al., 2008). However, this method is costly, and not convenient in a continuous process. Luan et al. (2013) investigated the feasibility of using mobile metallic temperature sensors (Ellab sensors) in continuous MATS systems and verified that the metallic temperature sensor could be used to capture temperature profiles in a

MATS system when placed in a suitable orientation. The same Ellab sensors were used in the current study to record the temperature profile of the cold spots of diced tomato and carrot products. Besides this, an incubation test of the MATS processed tomato pouches was used for microbial validation of the processing procedures developed.

The objectives of this study were to develop a pilot-scale MATS process for diced tomatoes, and a MAP process for diced carrots prepackaged in 8 oz pouches. To achieve these goals, the following steps were taken: 1) measuring dielectric properties of the desired products to match

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those of the model foods; 2) determining heating pattern and cold spots in the food pouches; 3) conducting heat penetration tests to develop MATS/MAP processes for desired products; and 4) verifying microbial safety of the MATS processed foods by incubation test. This study should provide useful information for future commercial MATS/MAP applications in vegetable processing.

2. Methods and materials

2.1. Preparation of sample pouches

Roma tomatoes were purchased from a local grocery store (Safeway, Pullman, WA) and stored at 4ºC. Tomatoes with a Hunter color “a” value (redness) of 22-25 were used. Tomato pericarp

(with skin) was quartered and diced into pieces (12.7× 12.7× (6-8) mm), the remainder was blended into a puree. NaCl and CaCl2 were added into the tomato puree during blending. A total of 137.5 ± 0.5 g diced tomato and 89.5 ± 0.5 g puree with 0.2% NaCl, 0.055% CaCl2 (w/w, total samples in each pouch) were filled into each 8-oz laminate pouch (18.5 × 13.2 ×1.6 cm,

Printpack Inc., Atlanta, GA).

Fresh carrots with their peel (Cultivar Imperator, from Bolthouse Farms, Inc., Bakersfield, CA) were purchased from the same grocery store as the tomatoes and stored at 4ºC. Carrots were peeled, cut into dices using a Hallde Flexi RG-7 dicer (Hicksville, NY) the same day just prior to processing. A total of 136.5 ± 0.5 g diced carrots and 90.5 ± 0.5 g CaCl2 solution (0.1% or 1.4%, w/w, total samples in each pouch) were filled into each 8-oz laminate pouches.

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All filled pouches were sealed with an UltraVac 250 vacuum sealer (KOCH Packaging Supplies

Inc., Kansas City, MO). Prepared sample pouches were then loaded into the MW heating system to process immediately.

2.2. MW heating system

2.2.1. Microwave assisted thermal sterilization (MATS) system

A single-mode 915 MHz MATS system was used to process the diced tomato pouches. Figure 1a shows a schematic view of the pilot scale MATS system developed by our laboratory. The system consisted of preheating, MW heating, holding and cooling sections. Each section was filled with circulation water from a water conditioning unit. The circulation water at pre-set temperature provides thermal energy from the outside of food packages, while MW sources caused volumetric heating inside the food packages. Pre-packaged foods were loaded into pockets on a mesh belt conveyor that transported samples through each section during thermal processing. More details about the system can be found in Resurreccion et al. (2013).

2.2.2. Microwave assisted thermal pasteurization (MAP) system

Considering the processing temperature (90°C) and pressure (normal atmosphere), the diced carrots were processed by a new pilot scale MAP system developed in our laboratory. This pilot scale MAP system also used single mode 915 MHz cavities. It had four sections, including preheating, MW heating, holding and cooling (Fig. 1b). The preheating and cooling section also play the roles of loading and unloading, respectively. Each section has a separated water circulation system to control water flow at a constant speed and temperature. The MAP had two

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MW heating cavities, with a total MW power of 14 kW. Food packages were preloaded in carriers that were transported on rotary wheels through the four sections at a constant speed.

2.2.3. Conventional hot water processing

For the purpose of comparison, conventional hot water (HW) heating was also conducted on the tomato/carrot pouches using the MATS/MAP systems. During the HW processes, the system pressure and supply water temperature setting were the same as for the MW processing, only the

MW power was turned off. The holding time of food pouches in the holding section was adjusted to achieve the same target process.

2.3. Measurement of dielectric properties of tomato/carrot samples

The dielectric properties of tomato/carrot samples were measured using an open-ended coaxial probe connected to an HP 8752C network analyzer (Hewlett Packard Corp., Santa Clara,

CA, USA, CA, USA). The whole sample pouch of tomatoes was blended into a homogenate before measurement. For carrot products, drained carrot dices were blended into puree before test, and the dielectric properties of the two solutions (0.1% and 1.4% CaCl2 in distilled water) were also determined. An Agilent 85032B type N calibration kit which contains open, short and load probes was used to calibrate the impedance analyzer. Then, the open-ended coaxial probe was calibrated by an Agilent 85070E dielectric probe kit, with air, short-circuit, and deionized water (25°C). After the calibration of the analyzer and the probe, around 25 ml sample was added into and tight sealed in a test cell. The test cell was designed to hold the sample against the probe while allowing sample temperature to be raised by a fluid (circulated from an oil bath) in the jacket wall. A detailed description of the system and calibration procedures is provided in

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Chapter 5. The dielectric properties (dielectric constant and loss factor) were determined over a frequency range of 300-3000 MHz for temperatures ranged between 22 and 120°C. Each measurement was replicated three times.

2.4. Determination of the heating pattern and cold spot in sample pouches

The cold spot location in the sample pouch needed to be determined for developing successful

MW processes. The heating pattern and cold spot location in food pouches were determined by a chemical–marker based computer–vision method, and predicted by computer simulation to make sure the results obtained from the two methods agree with each other (Pandit et al., 2007). In our previous study (Chen et al. 2007), a commercial electromagnetic software combined with a customer-built heat transfer model was successfully developed to simulate the 915 MHz pilot- scale MW system which coupled electromagnetic heating and conventional heat transfer. In the current study, the same computer simulation models were used for predicting the heating pattern and cold spot location in sample pouches.

The simulation results of tomato pouches obtained above were compared with heating patterns experimentally determined using five different whey protein gel (model gel) formulations (Wang et al., 2009). The five model foods were made of whey protein gels containing salt and D-ribose at different concentrations. Heating patterns for five model foods in 8-oz pouches determined by chemical-mark based computer-vision method were in agreement with the results predicted by computer simulation method, so were the cold spot locations in the five model food pouches obtained by both methods. Meanwhile, the heating pattern and cold spot location predicted by computer simulation did not change within a ±20% deviation of dielectric properties of samples.

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It was our hypothesis that if the dielectric properties of tomato samples fell within the range of ±

20% deviation of the model foods’ dielectric properties, then the heating pattern and cold spot location inside the model foods could be applied to the tomato pouches (Fig. 2).

For the carrot products, the heating pattern and cold spots in the sample pouches were determined directly using the chemical marker method. Different from MATS processes using whey protein gel containing D-ribose and salts as the model food, gellan gel was selected for

MAP processes to determine the heating pattern. Through trial and error, gellan gel model food

(1% gellan gum, 0.2% Ca2+) contained 1% D-ribose and 0.5% L-lysine as chemical marker M-2 precursors has similar dielectric properties as the carrot products, and was used for heating pattern determination (Data not published). Gellan gel model food was processed under the same condition as the carrot products, heating pattern and cold spot location in carrot pouches were determined by the chemical–marker based computer–vision method as we mentioned above.

2.5. Heat penetration test

Heat penetration tests were conducted to determine temperature profiles at the cold spot inside food packages, and the information was used to design the desired thermal process to achieve target microbial inactivation.

For diced tomatoes, the temperature of thermal processing was selected based on the heat resistance of the target bacteria, the spores of B. coagulans ATCC 8038 in tomatoes. According to the obtained results (Chapter 3), D values of those spores in tomato juice (pH 4.3) at 105°C was 1.20 min. This temperature was the most appropriate for MW processing among the selected

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temperatures (90-115°C) and therefore was chosen as the processing temperature for diced tomato products. The process of diced tomatoes was designed as a 5D process, meaning a 5-log reduction of the target B. coagulans spores. Since the D value of the spores of B. coagulans

ATCC 8038 strain in tomato juice is 1.20 min at the processing temperature (105°C), a 5D process for diced tomatoes was designed as a target F value of 6.0 minutes for B. coagulans spores.

For the processing of diced carrots, 90°C was selected as the pasteurization temperature based on the kinetic results of carrot texture degradation (Chapter 4). For pasteurization of low-acid foods which allows a shelf life up to 6 weeks at 5ºC, a common practice to aim for a 6 log reduction of target pathogen psychrotrophic Clostridium botulinum is suitable (Vervoort et al., 2012; ECEF).

This is called a “6D” process. A general recommendation of F90°C=10 min (or equivalents) is generally accepted for most foods, which represents at least 6-log reduction of the most heat resistant non-proteolytic strains of C. botulinum (Vervoort et al., 2012; ECEF). In our current study, the target microorganism for carrot products is non-proteolytic C. botulinum type E spores

(Vervoort et al., 2012). Gaze and Brown (1990) studied the thermal resistance of NP C. botulinum type E spores in carrot from 75–90ºC and reported their D value of 0.48 min at 90°C.

Based on this literature, a 6D process of NP C. botulinum type E spores is calculated to be a process of F90°C=2.88 min. Given the two conditions mentioned above, two thermal treatment levels were used in processing of carrot product: one was F90°C=3 min which allows a 6D process of target microorganism, the other one was F90°C=10 min which is generally considered an adequate process for most of the foods.

The calculation of the F value was based on the following equation:

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T T t ref F  10 z dt  o (1) where T (ºC) is the temperature measured at the cold spot at time t during process, Tref is the reference temperature, and z is the z-value of the target bacteria in the products. In the current study, Tref was 105º C for tomato products and 90°C for carrots; z value was 8.31ºC for B. coagulans ATCC 8038 spores in tomatoes, and 9.84°C for NP C. botulinum type E spores in carrots (Gaze and Brown, 1990; Peng et al., 2012).

For the heat penetration tests, Ellab sensors (Ellab Inc., Centennial, CO) were used to trace temperatures inside the sample pouches during thermal processing. The tomatoes were used as an example to demonstrate this procedure for process development here (Fig. 3). For better fixation of the Ellab sensor into the tomato dice, a bigger tomato piece (25.4 × 57× 16 mm) sliced from the middle layer of tomato rather than the real sample size (12.7× 12.7× (6-8) mm) was used. Computer simulation was performed to see if the change in sample size would cause any change in the heating pattern or cold spot location in the sample pouch (Fig. 4). A MW transparent frame was used to fix the position of the Ellab sensor tip at the cold spot of sample pouch. A total of 100.0 ± 0.5g diced tomato, and 89.5 ± 0.5g puree added with 0.2% NaCl,

0.055% CaCl2 (w/w, total sample in each pouch) were carefully filled into the pouch. The prepared sample pouches were loaded to the MATS system.

In the heat penetration tests for diced tomatoes, the system pressure for MATS was maintained at

33 psig, and the power for four MW heating cavities were set at 7.0/6.2/2.6/2.5 kW. The supply water temperatures were set to 56/108/107/15°C for preheating, MW-heating, holding and

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cooling sections, respectively. The processing time was adjusted by changing the moving speed of food pouches on the conveyor belt to achieve the target 5D process. For the purpose of comparison, conventional HW heating was also conducted on the tomato pouches with the MW system. During the HW processes, the system pressure and circulating water temperature setting were the same as for the MW processing, the MW power was turned off, and the holding time of food pouches in the holding section was adjusted to achieve the same 5D process.

The heat penetration tests for carrot samples processed by the MAP system followed the same procedures as for tomato samples. The differences were that the system pressure was normal atmosphere for MAP processing; the total power for two MW heating cavities was 14 kW; the circulating water temperatures were set to 61/93/93/15°C for preheating, MW-heating, holding and cooling sections, respectively. The moving speed of the package carrier was adjusted to achieve the target processes of F90°C=3 min or F90°C= 10 min.

2.6. Incubation test for diced tomatoes

Incubation test for diced tomatoes was conducted to validate thermal processing and ascertain microbiological safety and stability. Processed sample pouches were incubated at 35 ± 1°C for

16 days, 3M Petrifilm plates were used for Aerobic (AC), E.coli/Coliform (EC), and Yeast and

Mold (YM) counts. In addition, microbial assay of the raw material (whole tomato and tomato products before processing) was also conducted in parallel. Before and after processing, two replicates were sampled for each process. Pouches were opened aseptically and 100 g of sample was placed into a Seward 400 circulator stomacher filter bag. The sample was stomached for 2 min at 200 rpm. One ml of sample was pipetted and tenfold-serially diluted in 9 ml of 0.1%

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peptone water. One ml diluents were pipetted onto AC, EC, and YM 3M Petrifilm count plates.

The AC and EC plates were incubated at 35 ± 1ºC for 2 days while the YM plates were incubated at room temperature for 5 days.

For whole fresh tomatoes, one tomato was sampled per case. A whole Roma tomato was put into a Seward 400 circulator stomacher bag with 50 ml Difco Buffered Peptone Water (BPW) and the opening was firmly twisted and closed. The surface of the tomato was gently rubbed and shaken in the solution by hand for 2 min to dislodge surface micro flora. One ml of BPW solution was tenfold-serially diluted in 0.1% peptone water. One ml diluents were pipetted onto AC, EC and

YM count plates and the incubation times were same as those described previously.

3. Results and discussions

3.1. Dielectric properties of tomato/carrot samples

The dielectric properties of tomato puree with 0.2% NaCl and 0.055% CaCl2obtained by blending tomato sample from a whole pouch are shown in Fig. 5A. The dielectric constant of tomato samples decreased with increasing temperature, about a 3-5 unit reduction for every 20°C temperature increase at the same frequency. The dielectric constant also decreased with increasing frequency. Unlike the dielectric constant, the effect of temperature on the loss factor behaves in the opposite manner; the loss factor increased with increasing temperature. It also decreased with increasing frequency, more sharply at lower frequencies (300-1500 MHz). The same trends of dielectric constant and loss factor with temperature and frequency were observed on carrot puree (Fig. 5B).

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The dielectric properties of tomato puree with and without added salts (NaCl and CaCl2) as a function of temperature at 915MHz are shown in Fig. 6A. The agreement of the dielectric constants at each temperature in both samples with and without salts is remarkable, the two lines are almost overlapping. This demonstrates that added salts had little effect on the dielectric constant of tomato puree. However, tomato samples with salts had higher loss factors than those without salts, and the increasing effect of salts on loss factor was more evident at higher temperatures.

Fig. 6B shows the dielectric properties of carrot puree and its two solutions (0.2% NaCl with

0.1% or 1.4% CaCl2) at 915 MHz. Overall, the dielectric constant of the carrot puree was very close to the two solutions at each temperature and ranged from 55-72, although at low temperatures (22-60°C) it showed slightly lower values than those of the two solutions. The dielectric loss factor of the carrot puree ranged from 17-40 when the temperature was increased from 22°C to 120°C, around 3-5 units higher than that of the solution with 0.2% NaCl and 0.1%

CaCl2 at each temperature. When the concentration of CaCl2 increased from 0.1% to 1.4%, the loss factor of the solution increased sharply, with values ranging from 50-148, around 3 times increase in its loss factor. This can be explained by the increased ionic conductivity resulting from the increased dissolved ions, which may lead to the increase in the dielectric loss factor.

More information about the effects of salt on the dielectric properties and their mechanisms can be found in Chapter 5.

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3.2. Heating pattern and cold spot in sample pouches

To trace the temperature of tomatoes in pouches, a tomato piece having a larger size than the real product was used to better fix the position of an Ellab sensor, as mentioned above. Therefore, computer simulation was conducted to check whether this change would lead to a change in the heating pattern or heating rate in the sample pouch. The simulation results showed that the same temperature profile was obtained at the centers of both the large and small tomato pieces (Fig. 7), and that the sample pouches with a large tomato piece and a small one had the same heating pattern and cold spot location (Fig. 8a). For comparison, the heating pattern experimentally obtained in the model foods by the chemical marker method is also shown in Fig. 8.

The previous simulation work showed that the heating pattern and cold spot location did not change within a ± 20% deviation of dielectric properties of samples. The loss factor of tomato puree with added salts at 105°C was in the range of those for five model foods, while the dielectric constant was higher but still in the 20% deviation range (Fig. 9). Therefore, the heating pattern predicated by computer simulation was the same as that obtained by chemical marker method (Fig. 8). As seen in Fig 8, the cold spot of the model foods was in the middle layer concerning depth, located 22.8 mm in the x-direction from the center point and -2.2 mm in the y- direction from the center point in that layer. Therefore, the cold spot location (22.8, -2.2) mm away from the center point in the middle layer of tomato pouches was applied to their heat penetration tests.

For the carrot products, the heating pattern obtained by the chemical-mark method using gellan gel as the model food is shown in Fig. 10. Our results showed that the cold spot in the model

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food was also located in the middle layer concerning depth. Fig. 10 shows the top view of heating pattern and cold spot location in the middle layer of model food. As can been seen, the cold spot in carrot sample pouch was located directly at the center point (0, 0) in the middle layer, and was used for their heat penetration tests.

3.3. Heat penetration results

Tomato samples were preheated in the preheating section at 55°C for 15 min, then they entered into the MW heating section, followed by the holding section (105°C), and finally came out from the cooling section (15°C). To achieve a 5D process (F105°C = 6.0 min), the speed of the conveyor belt was adjusted to 47 inch/min, with a total MW heating time of 2.7 min, and a total holding time of 2.8 min. Two Ellab sensors packaged in tomato pouches were used to record the temperature at the cold spot of the sample pouch in each test. Tests were conducted in triplicate.

The F value was calculated starting from the sample coming out of the last MW cavity and going into the holding section (Fig. 11). The F values obtained from the 3 tests varied from 7.7 to 16.0 min. Figure 11A shows a typical temperature profile of the tomato sample recorded by the Ellab sensor under MW processing.

For conventional HW processing of the tomatoes, the holding time at holding section (105°C) was adjusted to 20.1 min to achieve a target 5D process. Two Ellab sensors packaged in sample pouches were used to record temperatures at the cold spot in each test, and tests were done in duplicate. The F values obtained varied from 6.4 to 13.2 min. The typical temperature profile recorded by the Ellab sensor during hot water heating is shown in Figure 11B.

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For the MAP processing of diced carrots, samples were first preheated in the preheating section

(60°C) for 20 min, then went through the MW section, the holding section (90°C) and the cooling section (15°C), consecutively. The speed of the package carrier was adjusted to 42 and

39 inch/min to achieve a process of F90°C = 3 min and F90°C = 10 min respectively. The corresponding total processing time was 3.22 and 4.96 min for the two MW processes. For HW processing, the holding time in the holding section was adjusted to 7.80 and 13.94 min to achieve an equivalent process with an F value at 90°C of 3 min and 10 min, respectively. Fig. 12 shows typical temperature-time profiles at the cold spot in the diced carrot pouch under MW and HW processing, for a target process of F90°C= 3 min. The processing parameters to achieve a target process for each product were determined and summarized in Table 1.

3.4. Incubation results for diced tomatoes

After incubation of the MATS processed tomato pouches at 35 ± 1°C for 16 days, no swollen pouches were observed. Microbial test results of raw and processed samples are given in Table

2. No colonies were observed in the processed samples under the detection limit (1CFU/g). The microbiological results validated the success of the MATS processing for the tomato products.

4. Conclusions

Dielectric properties of tomato and carrot products for processing were determined and used for computer simulation of heating patterns and cold spot locations in the sample pouch. The heating pattern determined by the computer simulation method was confirmed by that obtained with the chemical-marker based computer-vision method. The cold spots of the tomato and carrot pouches were in the middle layer concerning depth. The tomato pouch’s cold spot was located

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22.8 mm in the x-direction from the center point and -2.2 mm in the y-direction from the center point, while the carrot pouch’s cold spot was located directly at the center point (0, 0) in the middle layer of the sample pouch. A MATS process achieving a target F value of no less than 6 min was developed for processing of diced tomatoes packaged in 8-oz pouches, which can deliver a 5D thermal treatment to B.coagualans ATCC 8038 spores. For diced carrots, MAP processes with F90°C= 3 min and F90°C=10 min were developed to achieve at least a 6 D reduction of NP C. botulinum type E spores. Incubation tests and microbial analyses of the processed tomato pouches verified the safety of the products produced from the developed MATS process.

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References

Chen, H., Tang, J., and Liu, F. 2007. Coupled simulation of an electromagnetic heating process

using the finite difference time domain method. J Microw Power Electromagn Energy. 41(3),

50–68.

Chen, H., Tang, J. and Liu, F. 2008. Simulation model for moving food packages in microwave

hating processes using conformal FDTD method. J Food Eng. 88, 294–305.

Dauthy, M.E. 1995. Fruit and vegetable processing. Food and Agriculture Organization of the

United Nations (ISBN 92-5-103657-8). Rome. Available at

http://www.fao.org/docrep/V5030E/V5030E00.htm#Contents.

Gaze, J.E., and Brown, G.D. 1990. Determination of the heat resistance of a strain of non-

proteolytic Clostridium botulinum Type B and a strain of Type E, heated in cod and carrot

homogenate over the temperature range 70 to 92°C. Campden Food and Drink Research

Association Technical Memorandum N. 592. Chipping Campden, UK.

Kim, H.J., and Taub, I.A. 1993. Intrinsic chemical markers for aseptic processing of particulate

foods. Food Tech. 47, 91–99.

Lau, M.H., Tang, J., Taub, I.A., Yang, T.C.S., Edwards, C.G., and Mao, R. 2003. Kinetics of

chemical marker formation in whey protein gels for studying microwave sterilization. J Food

Eng. 60, 397–405.

Luan, D., Tang, J., Pedrow, P.D., Liu, F., and Tang, Z. 2013. Using mobile metallic temperature

sensors in continuous microwave assisted sterilization (MATS) systems. J Food Eng. 119,

552–560.

Lucier, G., and Glaser, L. 2009. Vegetables and melons: tomatoes. USDA Economic Research

Service. Available at http://www.ers.usda.gov/briefing/vegetables/tomatoes.htm.

161

Lucier, G., and Lin, B.H. 2007. Factors affecting carrot consumption in the United States.

Outlook Report from the Economic Research Service/USDA. No. (VGS-31901): 1–21.

Pandit, R.B, Tang, J., Liu, F., and Pitts, M. 2007. Development of a novel approach to determine

heating pattern using computer vision and chemical marker (M-2) yield. J Food Eng. 78,

522–528.

Peng, J., Mah, J.H., Somavat, R., Mohamed, H., Sastry, S., and Tang, J. 2012. Thermal

inactivation kinetics of Bacillus coagulans spores in tomato juice. J Food Prot. 75(7), 1236–

1242.

Resurreccion, F.P., Tang, J., Pedrow, P., Cavalieri, R., Liu, F., and Tang, Z. 2013. Development

of a computer simulation model for processing food in a microwave assisted thermal

sterilization (MATS) system. J Food Eng. 118, 406–416.

Tang, J., Liu, F., Pathak, S., and Eves, G. 2006. Apparatus and Method for Heating Objects with

Microwaves, US Patent No. 7, 119, 313.

Tang, Z., Mikhaylenko, G., Liu, F., Mah, J.H., Pandit, R., Younce, F., and Tang, J. 2008.

Microwave sterilization of sliced beef in gravy in 7 oz trays. J Food Eng. 89, 375–383.

Wang, Y., Tang, J., Rasco, B., Wang, S., Alshami, A.A. and Kong, F. 2009. Using whey protein

gel as a model food to study dielectric heating properties of salmon (Oncorhynchus

gorbuscha) fillets. LWT-Food Sci & Tech. 42, 1174–1178.

Zhang, W., Liu, F., Nindo, C., and Tang, J., 2013. Physical properties of egg whites and whole

eggs relevant to microwave pasteurization. J Food Eng. 118, 62-69.

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Table 1. Processing conditions for carrot and tomato products for MW and HW processes with equivalent process severity regarding microbial safety.

Processing Target F90°C = 3min F90°C = 10 min

MW Water temperature 61/93/93/15°C processing setting (preheating, heating, holding and cooling sections) MW power setting 14 kW 14 kW Preheating time 20 min 20 min MW heating time 1.36 min 1.46 min Holding time 1.86 min 3.50 min Carrot Total processing time 3.22 min 4.96 min (pasteurization) Real F value 4.9 min 8.9 min

Preheating time 20 min 20 min HW Total processing time 7.80 min 13.96 min processing Real F value 3.9 min 13.4 min Processing Target 5D process (F105°C = 6.05 min) Water temperature 56/108/107/15°C setting (preheating, MW heating, holding and processing cooling sections) MW power setting 7.0/6.2/2.6/2.5 kW Preheating time 15 min Tomato MW heating time 2.71 min (sterilization) Holding time 2.79 min Total processing time 5.50 min Belt speed 47 inch/min

Water temperature 56/108/107/15°C setting (preheating, HW heating, holding and processing cooling sections) Preheating time 15 min Total processing time 20.1 min

The pressure of the sterilization system was 33 psig, and the pasteurization system was at atmospheric pressure.

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Table 2. Microbial assay of raw whole tomatoes, and diced tomatoes before and after processing.

Whole tomatoes Sample before 3M petrifilm count Sample after process (CFU/tomato) process (CFU/g) Aerobic Plate No colonies were (1.21 ± 0.62) ×106 (5.40 ± 0.42) ×104 Count observed Detection limit: 1 Coliforms (1.13 ± 0.40) ×103 (4.35 ± 0.78)×102 CFU/g Yeasts (1.11 ± 0.34) ×104 (6.05 ± 0.92)×102 Molds 63 ± 95 5 ± 7

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Figure 1a. Front view diagram of four sections in the MATS system at WSU.

Figure 1b. Front view of MAP system at WSU.

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Figure 2. Flowchart of determination of heating pattern in diced tomato pouches.

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Figure 3. Preparation of sample pouch with Ellab sensor

Figure 4. Illustration of computer simulation (a): sample pouch with Ellab sensor (b): sample pouch without Ellab sensor.

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Figure 5. Dielectric constants and loss factors of tomato puree (A) and carrot puree (B) to which salts were added, as a function of temperature and frequency.

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Figure 6. Dielectric properties of processed products with temperatures at 915 MHz (A tomato puree with and w/o salts; B carrot puree and its solutions).

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Figure 7. Simulation results of temperature profiles at the cold spot in sample pouches with a small tomato piece or a large piece for temperature measurement.

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Figure 8. Heating pattern and cold spot location in tomato sample pouch (top view of the middle layer). (a): Heating pattern obtained by computer simulation using dielectric properties of tomato puree. (b): Heating pattern obtained by chemical marker method.

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Figure 9. Dielectric properties of tomato sample compared with those of five whey protein model foods.

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Figure 10. Heating pattern and cold spot in carrot sample pouch (top view of the middle layer, obtained by chemical mark method).

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Figure 11. Example of temperature-time profile at the cold spot in the diced tomato pouch under

MW (A) and HW (B) processing. Samples were preheated at 55°C for 15 min.

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Figure 12. Example of temperature-time profiles at the cold spot in the diced carrot pouch under

MW (A) and HW (B) processing. Preheating was at 60°C for 20 min.

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Chapter 7. Quality Evaluation of Vegetable Products Thermally Processed

with Microwave and Conventional Methods

Abstract: This chapter presents the results of quality evaluation of diced carrots after microwave

(MW) pasteurization and diced tomatoes after MW sterilization, compared to conventional thermal processing. For diced carrots, non-proteolytic Clostridium botulinum type E spores were the target bacteria and the thermal process was designed to be either a 6D process for the target bacteria (F90°C=3min) or F90°C=10 min. For diced tomatoes, the target bacterium was Bacillus coagulans spores and the thermal process was designed to achieve a 5D reduction of the target bacterium. Pre-packaged vegetables (diced carrots or tomatoes) with added salts (NaCl and

CaCl2) were pasteurized or sterilized by the 915 MHz pilot-scale MW systems in a batch process. Carrot and tomato products were also processed by conventional hot water (HW) processing to achieve the same level of inactivation for the target microorganisms. Quality attributes of the products processed by MW and HW heating were compared. Results showed that compared to raw carrots, carrots heated by MW had a lower total color difference (∆E values) than those heated by HW processing on equivalent processing conditions, denoting better color retention. There was no significant difference in texture retention of carrots heated by MW and HW with the same process severity. No pectin methylesterase activity was detected in any of the processed carrots. For carotenoids, no significant differences were found in carrot samples processed by either methods, except in high CaCl2 solutions (1.4%) with intense processing

(F90°C=10 min), where the total carotenoids and β-carotene in samples treated by HW processing were higher than in those by treated by MW processing. For tomatoes, no significant differences were found in the color attributes (L*a*b* values), texture, ascorbic acid, or lycopene content of samples processed by either method using equivalent processing conditions. Adding CaCl2 to

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tomatoes significantly increased the texture retention and lycopene content in the processed products. Results showed that the impact of MW processing on the quality of vegetables depended on the characteristics of the vegetables and the specific quality parameters tested.

Keywords: Microwave processing; carrot; tomato; quality; equivalent

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1. Introduction

One of the recent trends in fruit and vegetable processing is using advanced preservation techniques (e.g., high pressure, microwave, ohmic heating and pulsed electric fields) combined with new packaging materials and technologies, which help provide consumers with more food choices (Barrett & Lloyd, 2011; Dauthy, 1995). Microwave (MW) heating can rapidly raise the temperature inside foods to the desired sterilization or pasteurization temperature (Sun et al.,

2005; Tang et al., 2002). Compared to conventional retorting, MW heating requires a relatively short heating time (e.g. several minutes) due to its ability to generate volumetric heating within food materials, and thus has the potential to produce high quality shelf-stable products for food pasteurization and sterilization processes.

The application of MW heating in food has drawn increased attention over the past few decades.

Many researchers have reported the application of MW heating in vegetable processing and its effect on a nutrients and quality, such as in carrot juice (Rayman and Baysal, 2011), carrot pieces

(Lemmens et al., 2009), Brussels sprouts (Olivera et al., 2008; Vian et al., 2007), potatoes

(Alvarez and Canet, 2001; Barba at el., 2008), peas and spinach (Hunter and Fletch, 2002), tomatoes (Begum and Brewer, 2001), Swiss chard and green beans (Villnaueva et al., 2000), asparagus (Sun et al., 2007), and sweet potato purees (Steed et al., 2008). In most of these publications, research was carried out in a 2450 MHz domestic MW oven or a modified MW oven with specially installed temperature sensors. Different heat treatment levels were achieved by adjusting input power or heating time; normally several minutes are required for blanching or pasteurization for enzyme inactivation. Limited information is available on the quality of vegetables processed by MW sterilization and pasteurization. Sun et al. (2007) processed

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asparagus packaged in 8 oz. pouches by a MW-circulated water combination (MCWC) heating system and pressurized HW heating (both with F0=3 min), and steam-heating in a retort using the industrial standard method (121°C for 17 min). They evaluated the quality and antioxidant activity of the sterilized asparagus and found asparagus processed by MCMC had greater antioxidant activity and greener color than samples treated by the other two methods. Steed et al.

(2008) sterilized pumpable sweet-potato purees using a continuous flow MW-assisted processing and aseptic packaging. Compared to unprocessed purees, samples processed by continuous MW heating using a 60 kW systems howed an increase in total phenolic content and gel strength, a decrease in anthocyanins, and good retention of the overall color change and antioxidant activity(Steed et al. 2008). Koskiniemi et al. (2013) evaluated the quality of acidified vegetables

(broccoli, red bell pepper, and sweet-potato) pasteurized by continuous MW processing with a rotation apparatus and observed good retention of color and after MW pasteurization. The US

Army Natick Soldier Center conducted accelerated shelf-life/sensory studies on three products processed at Washington State University: chicken breast in 10-oz. trays in 2004; chicken and dumplings in 8-oz. pouches, and chicken and pasta in 10-oz. trays in 2012. All three MW processed chicken and chicken products had significantly higher hedonic scores (a sensory assessment) than those processed by conventional retort processing with an equivalent process severity (F0=6 min) over a 6-month storage at 100°F (equivalent to three-year storage at 80°F).

These results suggested that MW processed chicken products had a better overall acceptance by sensory evaluation (data not published). However, there are no systematic studies on the influence of MW sterilization and pasteurization on the quality of tomatoes and carrots.

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Tomatoes and carrots are two of the most commonly consumed vegetables in the US. Three- fourths of the tomatoes consumed by Americans are in processed form, most of which are thermally processed; and one-fourth of carrots are consumed in processed form, largely canned and frozen (Lucier and Glaser, 2009; Lucier and Lin, 2007). In addition to the popularity of the two vegetables, another reason for selecting carrots and tomatoes was the differences in their pH and therefore the extent of β-elimination reaction proceeding in the vegetables under high temperatures. β-elimination is a non-enzymatic reaction that takes place at high pHs (>4.5) and high temperatures (>80ºC). Studies shows that the early, rapid phase of texture loss in both tomatoes and carrots under high temperatures is due to the turgor loss resulted from the loss of membrane integrity, while a 2nd slower, prolonged phase of softening occurred was observed in carrots, and mainly contributed to the breakdown of pectins through β-elimination reactions

(Grant et al., 1973; Gonzalez et al., 2010; Greve et al., 1994ab; Anthon and Barrett, 2005).

Because the pH of the carrots (5.2-5.8) is much higher than that of the tomatoes (3.9-4.4), β- elimination in carrots under thermal processing is much more noticeable than in tomatoes considering the high pH (pH 4.5) required for this reaction to take place. However, no information is available on quality changes in tomatoes or carrots processed by microwave sterilization or pasteurization.

In the current study, diced tomatoes and carrots were pre-packaged in 8-oz pouches with added salts (NaCl and CaCl2), in accordance with common commercial practices. Pouches were pasteurized or sterilized by a 915 MHz pilot-scale MW system in a batch process. Tomato or carrot products heated by conventional HW processing were conducted on an equivalent basis, achieving the same level of target microbial inactivation. Quality attributes of the products

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processed with MW and HW heating were compared. This study illustrates the application of

MW pasteurization and sterilization to pre-packaged tomato and carrot products and compares the impact of MW and HW processing on the products’ quality.

2. Materials and Methods

2.1. Sample preparation

Fresh carrots with their peel (Cultivar Imperator, from Bolthouse Farms, Inc., Bakersfield, CA) or Roma tomatoes were purchased from a local grocery store (Safeway, Pullman, WA) and stored at 4ºC. Carrots were cut into dices using a Hallde Flexi RG-7 dicer (Hicksville, NY) and processed immediately after. A total of 136.5 ± 0.5 g diced carrots and 90.5 ± 0.5 g CaCl2 solution (0.1% or 1.4%, w/w, total samples in each pouch) were filled into each 8-oz laminate pouch (18.5 × 13.2 ×1.6 cm, Printpack Inc., Atlanta, GA).

Only tomatoes with dark red color (‘a’ value between 22 and 25) were selected, washed, and cut into quarters. The pericarps were diced into 12.7× 12.7× (6-8) mm pieces with a food dicer

(Fengxing Trading Co., Ltd, Ningbo, China), and the remainder was blended into puree. Salts were added to the puree during the blending. Two types of samples were prepared, one with and one without added CaCl2. For the former, 0.2% NaCl (w/w, total samples in each pouch) and

0.055% CaCl2 (w/w, equivalent to containing 200 ppm calcium) were added. For the latter, only

0.2% NaCl was added to improve the taste. A total of 137.5 ± 0.5 g diced tomatoes and 89.5 ±

0.5 tomato puree with ingredients were filled into each 8-oz laminate pouch.

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All sample pouches were vacuum sealed (Koch Supplies Inc., Kansas city, MO, USA). Pre- packaged carrot/tomato pouches were loaded into the MW or HW heating system to process immediately.

For the processed samples, pH, soluble solids, drain weight, color and texture were measured on the same day of processing. Sample pouches used to assess the enzymes, carotenoids, ascorbic acid and lycopene were stored at -30°C until the day of analysis. Frozen samples were thawed at room temperature (22°C) before analysis. For color and texture assay of the carrot and tomato dices, samples were drained before analysis, using the procedures described below. For pH, soluble solids, enzymes, carotenoids, ascorbic acid and lycopene assays, the whole pouch of carrot or tomato samples was blended until homogenous and used for each measurement.

2.2. Thermal treatments

Based on the kinetic study of carrot texture degradation, 90°C was selected as the pasteurization temperature for the diced carrot products (Chapter 4). For pasteurization of low-acid foods which allows a shelf life of up to 6 weeks at 5ºC, a common practice to aim for a 6 log reduction of the target pathogen psychrotrophic C. Botulinumis suitable (Vervoort et al., 2012; ECEF). This is called a “6D” process. A general recommendation of F90°C=10 min (or equivalent) is universally accepted for the pasteurization of most foods, which represents at least 6-log reduction of the most heat resistant non-proteolytic strains of C. botulinum (Vervoort et al., 2012; ECEF). In the current study, the target microorganism for carrot products is non-proteolytic C. botulinum type

E spores (Vervoort et al., 2012). Gaze and Brown (1990) studied the thermal resistance of NP C. botulinum type E spores in carrot from 75–90ºC and reported a D value of 0.48 min at 90°C.

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Based on these data, the required F90°C was 2.88 min to achieve a 6D process for NP C. botulinum type E spores. Given the two conditions mentioned above, two thermal treatment levels were used for processing the carrot products: one was F90°C=3 min, which yields a 6-log reduction of target NP C. botulinum type E spores, and the other process was F90°C=10 min, which is generally considered an adequate process to pasteurize most foods.

For the diced tomato products, a MW sterilization process was designed to achieve a 5D reduction in the target bacteria, B. coagulans ATCC 8038 spores. This equated to an F105°C of 6.0 min, which is based on the thermal resistance data of this microorganism obtained in Chapter 3.

A MW assisted thermal pasteurization (MAP) process was developed for diced carrots packaged in 8-oz pouches using a 14-kW single-mode MW system, while a MW assisted thermal sterilization (MATS) process was developed for diced tomatoes using a semi-continuous,

915MHz single-mode MW system. Conventional HW processing was also conducted in order to achieve equivalent process severity with regard to microbial inactivation. Details on the systems and operation procedures are described in Chapter 6. The processing conditions of the two products for equivalent MW and HW processes are listed in Table 1.

2.3. Color of carrot and tomato dices

The CIE L*(lightness), a*(redness), and b*(yellowness) color attributes of processed carrot and tomato dices were determined using a computer vision system following the procedures of Kong et al. (2007). This system included photography lights, a camera, and a computer with Adobe

PhotoShop to analyze the surface color of food products. A Benchers Copymate copy stand was

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used to fix the camera perpendicular to the food samples. The camera was a Canon EOS 60D fitted with a Canon EF 100 mm f/2.8 USM macro lens. The Alzo 300 Table Top lighting System consisted of two parabolic reflectors covered with a diffuser cloth. Each reflector used three Alzo

27 Watt Fluorescent Daylight Balanced light Bulbs with a color temperature of 5500K. A light meter (Sekonic Corp., Tokyo, Japan) was used to evenly distribute light on the food surface

(12.2 EV). The Canon supplied EOS Utility software was used to remotely control the camera and to download the color images (.JPG) of the food samples into the computer. The color images were analyzed using Adobe PhotoShop. In CIE LAB color scale, L* varies from 0-100, a* and b* between -127 and +127; while these ‘L’, ‘a’ and ‘b’ values under “Lab color model” from

PhotoShop are encoded between 0-255. The color values obtained from Photoshop were converted to standard CIE L*, a*, and b* values using the following equations (Yam and

Papadakis, 2004):

(1)

(2)

(3)

The hue angle (H) and total color differences (∆E) were calculated by the following equations:

H=tan-1 ( ) (4)

∆E=√ (5) where the raw samples were used as the control in the calculation of ∆E.

2.4. Texture of carrot and tomato dices

The firmness of the treated, diced carrot pieces was determined using a TA.XT2 Texture analyzer (Stable Micro Systems Ltd., Godalming, UK) fitted with a 25 mm diameter aluminum

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cylinder probe using the methods described by Lemmens et al. (2009). The samples were compressed to 70% strain at a cross head speed of 1 mm/s. For each test, one piece of diced carrot sample was placed under the probe. The peak force of the first compression cycle of the sample was marked as the maximum force and was the quantitative indicator of sample firmness.

The measurements were made using six replicates for each treatment condition.

The firmness of the diced tomatoes was measured by a TA.XT2 texture analyzer (Stable Micro

Systems Ltd., Godalming, UK) fitted with a mini-Kramer shear cell. A total of 12.30 g drained samples after processing and 11.30 g fresh diced tomatoes before processing were used. Each measurement was replicated 6 times according to Anthon et al. (2005) and the maximum compression force of the first peak was recorded.

2.5. Pectin methylesterase (PME) activity of carrots

PME was extracted from diced carrot samples using a modified method from Vervoort et al.

(2012). Ten g of homogenized sample was mixed with 0.2 M Tris-HCl buffer containing 1M

NaCl (pH 8.0, 1:1.3 w/v) and stirred at room temperature for 30 min.

The PME activity of the sample was quantified as the production of H+ during pectin hydrolysis as a function of time at pH 7.0 and 30ºC, following the methods by Anthon et al. (2002ab).

Briefly, 30 ml solution containing 0.25 M NaCl and 0.25% citrus pectin was equilibrated to 30ºC and adjusted to pH 7.0. One ml of homogenized sample was added to the solution and the pH was readjusted to 7.0 and maintained at this pH for 10 minute by the addition of 0.01 N NaOH.

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The rate was calculated as μmol of NaOH consumed over the 10minute time period. The results were reported as percentages compared to the raw (unprocessed) control samples.

2.6. Carotene analysis of carrots

The extraction of carotene from the carrot sample followed the methods described by Sadler et al. (1990) with modifications. Briefly, 5 g of homogenized carrot sample was mixed with 50 ml extraction solvent hexane with acetone and ethanol (50:25:25) containing 0.1% BHT and mixed for 20 min. Fifteen ml Milli-Q water was added to the mixture and mixed for an additional 10 min and the mixture was centrifuged at 600 × g for 8 min to separate the organic layer from the water layer. The organic layer was collected and filtered through a 0.45 μm syringe filter as the final extract for assay.

The total carotenoid content of the extract was measured by a spectrophotometer (Pharmacia

Biotech Ltd., Cambridge, England) at 450 nm, the maximum absorbance wavelength of β- carotene. Hexane with 0.1% BHT was used as a blank. The total carotenoid concentration of the extract was calculated by Beer’s law, with the extinction coefficient of β-carotene in hexane

1% E 1cm=2560.

The α- and β-carotene contents of the carrot samples were determined by the method described by Vervoort et al. (2012), using an Agilent RP-HPLC system with a UV-DAD detector. A YMC

Carotenoid column (150×4.6 mm, 5 μm) was used to separate the carotenoids through linear gradient elution from 100% solution A (81% methanol, 15% methyl-t-butyl ether; 4% milli-Q water) to 100% solution B (41% methanol, 55% methyl-t-butyl ether; 4% milli-Q water) in 28

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min, held for 5 min, then returned to 100% solution A and equilibrated for 8 min. The flow rate was 1 ml/min and the detection wavelength was 450 nm. The standards of α- and β-carotenes were dissolved in hexane and used for standard curves.

The moisture content of the homogenized carrot sample was determined, and the concentrations of total carotenoids, α- and β-carotenes in carrot samples were calculated on a dry weight basis.

2.7. Determination of pH, soluble solids, drained weight of tomatoes

Analysis of the pH, soluble solids, drained weight and color of the tomato samples were performed immediately after processing. Fresh tomato samples taken before processing were used as a control. Total soluble solids were assessed by optical refractometer (Atago Co. LTD,

Japan) expressed as °Brix. Drained weight was determined according to 21 CFR 155.190-

Canned tomatoes (FDA 2005). The whole sample pouch was emptied onto a tilted U.S. #8 sieve, and samples were distributed as uniform as possible over the screen. The screen was tilted with a height of 2 inch. The weight of drained samples after 2 min draining on the screen was drained weight. The ratio of drained weight to the net weight of samples (weight before draining) was recorded as the percentage of sample drained weight (%).

2.8. Ascorbic acid content of tomatoes

Ascorbic acid content of the tomato samples was determined following the procedure described by Nisperos-Carriedo et al. (1992). Briefly, 50 g thawed sample was blended with 50 ml 0.05 N

H3PO4 for 3 min, and centrifuged at 10,000 × g for 10 min. The supernatant was collected and diluted to 100 ml. Three ml extract was purified by passing a 3cc C18 Sep-Pak cartridge which was preconditioned by flushing with 2 ml acetonitrile followed by 5 ml Milli Q water. The

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extract was filtered through 0.45 µm syringe filter before injection. Agilent 1100 HPLC system

(Santa Clara, CA) equipped with a XTerra RP18 column (4.6 × 250 mm; 5 µm) from Waters

(Milford, MA) was used for the separation and maintained at 30°C during analysis. Separation was performed using a 2% KH2PO4 as the mobile phase in the isocratic mode. The flow rate was

0.4 ml/min, and the detection was performed at 260 nm using a diode array detector.

2.9. Lycopene content of tomatoes

Lycopene in the processed tomato samples was extracted and quantified using a modified method from Halim and Schwartz (2006) and Gupta et. al (2010). Briefly, one gram thawed sample was mixed with 0.2 g CaCO3 and homogenized with 10ml methanol at 20,000 rpm for 1 min. The sample was centrifuged at 10,000 × g for 10 min, and the supernatant was discarded.

The pellet was re-suspended in 4 ml hexane/acetone (1:1 v/v) solution, 4 ml Milli Q water was added to induce phase separation. The nonpolar layer was collected and successive extraction was repeated 3 times. The collected fraction was made up to 10 ml with the extracting solvent and 1 ml extract was dried under nitrogen. Next, the sample was re-dissolved in 6 ml methanol/MTBE (1:1 v/v), and filtered through 0.45 µm syringe filter before injection. A YMC

Carotenoid column (4.6 × 250mm; 5 µm) from Waters (Milford, MA) was used for the separation. The mobile phase was made of A solvent (methanol/MTBE/2% aq. ammonium acetate, 88:5:7, v/v/v) and B solvent (methanol/MTBE/2% aq. ammonium acetate, 20:78:2, v/v/v). Separation was achieved by gradient elution of 0-85% B in 20 min, followed by a 10 min linear gradient to 100% and hold for 5 min, then returning to 0% B and holding for 15 min. The detection was performed at 471 nm using a UV-Vis detector. All analysis was performed under dim light to avoid sample degradation.

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2.10. Statistical analysis

Statistical analysis was conducted using SAS 9.2 (SAS Institute Inc., 2008). One-way analysis of variance (ANOVA) was used for all data. Differences among treatments by means were determined by least significant difference (LSD) multiple comparison test with significance defined as P < 0.05.

3. Results and Discussion

3.1. Color

The CIE LAB color values of raw and processed carrot and tomato dices are given in Table 2.

For processed carrots, color values varied depending on the treatment type. Processing

* significantly decreased a values of all carrot samples, and the carrot samples in 1.4% CaCl2

* * * solution after the F90°C=10 min HW processing had the lowest a value.TheL and b values of carrot samples were relatively less affected by thermal processing. No significant difference was found in b* values between the samples before and after processing, except for the samples in

0.1% CaCl2 solution after the F90°C=3 min MW processing. Samples in 1.4% CaCl2 solution with an F value of 3 min by MW processing had a significantly higher L* value compared to the raw material, while no significant difference was found in samples from other treatments. The changes in the a* and b* values indicate the a deterioration of the initial intense orange color of the untreated carrots (Vervoort et al., 2012). The b*/a* value, which is used to represent the yellowness of carrots, is shown in Fig. 1A. A significant increase in the b*/a* values was found for processed samples as compared to the untreated samples. No significant differences were found between samples heated by MW and HW under the same conditions, except for samples in

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1.4% CaCl2 solution with F90°C=10 min. For the hue angle of carrots, raw samples had the lowest value, which was significantly different from all processed samples, except those in 0.1% CaCl2 solution with F90°C=3 min by microwave processing. Changes in color were likely related to the degradation and isomerization of carotenoids during thermal processing, since carotenoids in carrots are responsible for their orange color. Another possible reason can be due to the expulsion of air from the tissue matrix during thermal process. This results in the cooking solution flooding the intercellular spaces where the air was, and makes the cells more translucent and therefore may affect their L* and a* values.

The color of tomatoes was stable over the course of processing. Neither MW nor HW processing significantly affected the L*a*b* values, although a decrease in all of these values was observed compared to the raw samples. The a*/b* values (red-yellow ratio), commonly used to represent the redness of tomatoes and tomato products, are shown in Figure 1B. There was a slight decrease in the a*/b* value after processing, but no significant differences existed between samples before and after processing. The processed samples had an increased hue angle, but again, no significant differences were found among samples with different treatments. The stability of the tomato color during thermal processing can be attributed to the heat stability of lycopene, which is the main pigment responsible for the red color of tomatoes. Shi (2002) reported that lycopene was relatively stable if heated at temperatures below 100°C, but the duration of heating must be taken into consideration. These results indicated that heating at

105°C for up to 20 min did not significantly affect the color of diced tomatoes.

Total color differences between the raw and processed carrot and tomato samples are summarized in Table 3. Theoretically speaking, a ∆E of 1 represents a barely-noticeable color

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difference to the human eyes under ideal viewing conditions; while ∆E values between 2 and 3 are considered equivalent by some viewers in less than ideal lighting (Vervoort, 2012). From

Table 3 it is clear that all the ∆E values were higher than 3, suggesting that the color differences between the raw and processed carrot or tomato samples are perceptible to the human eye under normal lighting conditions. Diced carrot samples in 1.4% CaCl2 solution processed by HW with

F90°C=10 min had the largest ∆E value, denoting the least color retention of the carrots. The ∆E values of processed carrots varied from 5.84 to 10.32, and all carrot samples processed by microwave heating had lower ∆E values than those processed by hot water under same conditions, denoting a better color retention. This result could be due to the shorter heating time of microwave processing compared to hot water processing.

3.2. Texture

Texture changes of diced carrot and tomato samples before and after processing are shown in

Fig. 2. Significant decreases in the texture before and after processing were found in both carrot and tomato products under all processing conditions. For diced carrots, the texture loss was 27-

30% for samples in 0.1% CaCl2 with F90°C=3 min, 16-21% for those in 1.4% CaCl2 with F90°C=3 min, 45-50% for those in 0.1% CaCl2 with F90°C=10 min and 21-25% for those in 1.4% CaCl2 with F90°C=10 min. The firming effort of CaCl2on texture was apparent based on these results; the higher the concentration, the better retention of carrot texture. However, no significant difference was found between carrot samples heated by MW and HW processing with the same process severity.

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For diced tomatoes, samples processed by MW and HW heating with added CaCl2 resulted in a

60% and 64% decrease in texture, respectively. For samples processed by MW heating without added CaCl2, the texture was almost completely lost (90% reduction), which demonstrates the important role of calcium in texture retention of tomato products. However, no significant difference was found for diced tomato processed by MW or HW with the same processing severity. The explanation for this result could be that the degradation of tomato texture occurs at the very beginning of high temperature processing (105°C). To achieve the 5D process, even a total processing time of 5.30 min with MW heating could only retain around 2/3 of the initial texture. Ma and Barrett (2001) found that when holding diced tomatoes at 88°C, a large reduction in firmness occurred in the 1st min of heating and then remained almost unchanged over the next 10 min. Greve et al. (1994ab) found that a fast phase of texture loss in vegetables occurs in the first minute at temperatures near 100°C and this can be attributed to a loss of turgor resulting from the breakdown of the cellular membranes.

3.3. PME activity of carrots

Pectin methylesterase (PME) is one endogenous enzyme that plays an important role in stabilizing the cell wall structure of carrots. PME can catalyze the de-esterification of pectins, creating binding sites for divalent cations, primarily Ca2+ (naturally present in the tissue or added during processing). The binding sites are on the polygalacturonic acid backbone of the pectin and the addition of calcium allows the formation of cross-linkages between pectin chains, which improves the texture. In this study, no PME activity was detected in the processed products.

Results indicated that after a preheating treatment of 60°C for 20 min, heating at 90°C for 3.22 min resulted in a complete loss of PME activity in the processed carrots. This finding is in

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accordance with published papers that indicate PMEs are heat sensitive. Anthon and Barrett

(2002) studied the kinetics of thermal inactivation of PMEs in carrot juice and reported D65.7°C=

5min for the labile form and D70.5°C=5min for the resistant form of PME. Lemmens et al. (2009) studied the thermal pretreatments of carrot pieces using different heating techniques and reported no PME activity detected after blanching at 90°C for 4 min.

3.4. Carotenoids of carrots

Carotenoids are responsible for the orange color of carrots. They are also one of the important bioactive compounds in carrots, known as vitamin A precursors, and can act as antioxidants to reduce the risk of developing degenerative diseases. α- and β-carotene are the two major carotenoids in carrots. The total carotenoids, α-carotene, and β-carotene contents in raw and processed carrot products are shown in Fig. 4. In raw carrots, the dry weight of the total carotenoids, α-carotene, and β-carotene contents were 130.13 ± 3.35, 34.15 ± 1.04 and 93.64 ±

3.12 mg/100 g, respectively. Both MW and HW processing significantly decreased the contents of the total carotenoids, α-carotene, and β-carotene in the carrot samples; the decrease depended on the intensity of heating. Applied heat treatments with F90°C=3 min caused a loss of 15-23% in total carotenoids compared to the initial value, while the loss increased to 19-35% for those with

F90°C=10 min. For β-carotene, the loss for processes with F90°C=3 min was 11-20%, and the loss increased to 17-37% when the processing intensity was increased to F90°C=10 min. The α- carotene seemed to be more heat sensitive than β-carotene at the beginning of a thermal process.

The loss in α-carotene for processes with F90°C=3 min was 22-31% and stayed relatively stable with increased processing intensity, as the loss value for F90°C=10 min was 30-33%. In most cases, no significant differences were found in the carotenoids for samples processed by MW

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and HW heating. However, for carrots dices in 1.4% CaCl2 solution by processing with F90°C=10 min, the total carotenoid and β-carotene loss by MW heating was larger than HW processing.

One possible explanation of this result could be that the longer heating time of HW processing caused more cell disruption of carrot tissue, which resulted in an improved extractability of the carotenoids. Veervoort et al. (2012) also observed decreased α- and β-carotene content in carrot products going from mild pasteurization at 70°C to sterilization. However, Knochaert et al.

(2011) reported an increased β-carotene content in sterilized carrots (F0=3min).

3.5. pH, soluble solids, drained weight of diced tomatoes

Results of pH, soluble solids, drained weight for MW and HW processed diced tomatoes are given in Table 3. Three types of samples were compared, MW processed with added CaCl2, MW without added CaCl2, and HW processed with added CaCl2. Samples before processing were used as controls. The pH of each processed sample was well controlled between 4.30-4.40, without significant differences. The soluble solids content of the three processed samples were around 4.6-4.7°Brix and no significant difference was found among samples. For drained weight, no significant differences were observed among raw and processed samples with CaCl2, yielding relatively higher values around 76-79%. However, HW processed samples without CaCl2 showed a much lower drained weight of 70.31 ± 2.05% compared to the other samples. The higher drained weight of samples processed with CaCl2 could be due to the interaction of calcium with the pectins in the cell wall, which helped maintain the cell structure during processing so the samples didn’t lose as much cell contents. This agrees with findings from Bradley (1966), which showed cell-wall rigidity had an important effect on the final drained weight.

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3.6. Ascorbic acid content of tomatoes

Ascorbic acid is the one of the most important micronutrients in tomato fruits and tomato products and has numerous physiological benefits with antioxidant values. The ascorbic acid content in sterilized tomato samples (with and without CaCl2) processed by MW or HW heating is shown in Fig. 5. The initial concentration of ascorbic acid in raw tomato samples was 7.31 ±

1.22 mg/100g. The value was a little lower than the concentration range reported in other varieties, which was 10-22 mg/100g fresh matter (Abushita et al., 2000; Marfil et al., 2008; Van den Broeck et al., 1998). Possible reasons for the relatively low concentration in the raw samples might be they were less mature tomatoes or grown in cold weather and low light. A decrease of around 1 mg/100g of ascorbic acid was found in samples processed by either MW or HW with the addition of calcium, around 14% loss compared to the initial value. Although the amount of ascorbic acid retained in samples without calcium added was slightly lower than the other two processing treatments, no significant difference was found between samples processed with and without calcium. Van den Broeck et al. (1998) studied the thermal degradation kinetics of L- ascorbic acid in squeezed tomatoes by heat (120-150°C), and reported D120°C=325-469 min depending on the variety, and a z value of 30.15°C. Based on these data, D105°C of ascorbic acid in squeezed tomatoes is calculated to be 1018-1469 min, higher than our results if same degradation tendency was applied.

3.7. Lycopene content of tomatoes

Lycopene contents of unprocessed and thermally processed tomato samples using MW and HW are shown in Fig. 6. Total lycopene content in tomatoes decreased in processed samples; the initial content was 17.45 mg/100g, which decreased to 15.25 (MW processed with CaCl2) and

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15.87 (HW processed with CaCl2). Processed samples without CaCl2 showed a much lower value (9.08 mg/100g), which was almost a 48% loss. The lycopene lost in processed tomato samples with added CaCl2 was relatively small and did not differ significantly from the raw samples, but did have significantly lower values than those without CaCl2 added. One possible explanation of this result was that the protective effect of calcium on the structural integrity strengthened the cell walls and better maintained the integrity of carrot cells, therefore reduced the exposure of lycopene to oxygen in the tissue during the heating process, and resulted in less lycopene loss. Shi et al. (2003) reported a greater stability of lycopene in tomato puree heated at

100°C or below for within 60 min, with a loss of 3.47% or less.

4. Conclusions

For carrots, the total color differences between all processed and raw products were perceptible by the human eye under normal viewing conditions. However, samples processed by MW heating had lower ∆E values than HW heated samples with the same process severity, indicating the MW heated samples had better color retention. The impact of processing on carrot texture was significant, but no significant difference in texture retention was found between MW and

HW heated samples treated with the same process severity. Both processing methods completely inactivated the PMEs in carrots. All processes lowered the total carotenoid, α-carotene, and β- carotene content in carrots. No significant differences were found in the carotenoid content in samples processed by either method in most cases. However, for diced carrots in high CaCl2 solutions (1.4%) subject to intense processing (F90°C = 10 min), the total carotenoids and β- carotene in samples heated with hot water processing had higher values than those heated with microwave processing. One possible explanation for this result could be that the longer heating

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time of hot water processing caused more cell disruption of carrot tissue, which resulted in an improved extractability of the carotenoids.

For tomatoes, no significant differences were found in the color attributes (L*a*b* values), ascorbic acid, or lycopene content of samples processed with MW and HW with equivalent processing severity. Similar to carrots, the impact of processing on the texture loss of tomato samples was significant. However, the texture loss in samples treated with both processing methods with the same severity did not show significant differences from each other. Addition of

CaCl2 to tomatoes significantly increased texture retention and increased the lycopene content in the processed products.

These results indicate that the impact of MW heating on quality attributes of vegetables depends on the characteristics of the vegetables and the specific quality parameter tested. Some quality parameters, like texture and enzymes, are quickly reduced, degraded, or inactivated during a very short time at the beginning of the process (temperature at 105ºC for tomatoes and 90°C for carrots). In this case, even the MW processing time was not short enough to retain these characteristics. There are some relatively heat-stable quality parameters, such as lycopene in tomatoes; results showed that a 20 min holding time at 105ºC was not long enough to lead to a significant reduction in its total concentration. Other quality parameters have intermediate heat- stability, such as color in carrots. Carrots heated with MW processing showed better color retention than HW processing. These results can be used in optimizing thermal processing for carrot or tomato products.

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References

Abushita, A.A., Daood, H.G., and Biacs, P.A. 2000. Change in carotenoids and antioxidant

vitamins in tomato as a function of varietal and technological factors. J Agric Food Chem. 48,

2075–2081.

Alvarez, M.D., and Canet, W. 2001. Kinetics of thermal softening of potato tissue heated by

different methods. Eur Food Res Technol.212, 454–464.

Anthon, G.E., and Barrett, D.M. 2002a. Kinetic parameters for the thermal inactivation of

quality-related enzymes in carrots and potatoes. J Agric Food Chem. 50, 4119–4125.

Anthon, G.E., Sekine, Y., Watanabe, N., and Barrett, D.M. 2002b. Thermal inactivation of pectin

methylesterase, polygalacturonase, and peroxidase in tomato juice. J Agric Food Chem. 50,

6153–6159.

Anthon, G.E., Blot, L., and Barrett, D.M. 2005. Improved firmness in calcified diced tomatoes

by temperature activation of pectin methylesterase. J Food Sci. 70(5), C342–C347.

Barba A.A., Calabretti, A., Amore, M., Piccinelli, A.L., and Rastrelli, L. 2008. Phenolic

constituents levels in cv. Agria potato under microwave processing. LWT-Food Sci & Tech.

41, 1919–1926.

Barrett, D.M., and Lloyd, B. 2011. Advanced preservation methods and nutrient retention in

fruits and vegetables. J Sci Food Agric. 92, 7–22.

Begum, S., and Brewer, M.S. 2001. Chemical, nutritive and sensory characteristics of tomatoes

before and after conventional and microwave blanching and during frozen storage. J Food

Quality. 24, 1–5

Bradley, B.F. 1966. Factors influencing the drained weight, texture and other processing

characteristics of canned strawberries. J Sci Food Agric. 17(5), 226–232.

198

Bourne, M.C. 1989. Applications of chemical kinetic theory to the rate of thermal softening of

vegetable tissue. In Quality Factors of Fruits and Vegetables, Vol.ACSSymp. Ser. 405 (J.J.

Jen, ed.). American Chemical Society, Washington, 98–110.

Dauthy, M.E. 1995. Fruit and vegetable processing. Food and Agriculture Organization of the

United Nations (ISBN 92-5-103657-8). Rome. Available at

http://www.fao.org/docrep/V5030E/V5030E00.htm#Contents.

ECFF (European Chilled Food Federation). 2010. Recommendations for the production of

prepackaged chilled food. Retrieved on Feb 6, 2013, from

http://www.chilledfood.org/Resources/Chilled%20Food%20Association/Public%20Resources

/ECFF_Recommendations_2nd_ed_18_12_06.pdf

Gaze, J.E., and Brown, G.D. 1990. Determination of the heat resistance of a strain of non-

proteolytic Clostridium botulinum Type B and a strain of type E, heated in cod and carrot

homogenate over the temperature range 70 to 92°C. Campden Food and Drink Research

Association Technical Memorandum N. 592. Chipping Campden, UK.

Gonzalez, M.E., Jernstedt, J.A., Slaughter, D.C., Barrett, D.M. 2010. Influence of cell integrity

on textural properties of raw, high pressure, and thermally processed onions. J Food Sci. 95,

E409–E416.

Grant G.T., Morris E.R., Rees D.A., Smith P.J.C., and Thom D. 1973. Biological

interactionsbetween polysaccharides and divalent cations: the egg-box model. FEBS Lett. 32,

195–198.

Greve LC, McArdle RN, Gohlke JR, and Labavitch JM. 1994a. Impact of heating on

carrotfirmness. Changes in cell wall components. J Agric Food Chem.42, 2900–2906.

199

Greve LC, Shackel KA, Ahmadi H, McArdle RN, Gohlke JR, and Labavitch JM. 1994b. Impact

of heating on carrot firmness: contribution of cellular turgor. J Agric Food Chem. 42, 2896–

2899.

Gupta, R., Balasubramaniam, V.M., Schwartz, S.J., and Francis, D.M. 2010. Storage stability of

lycopene in tomato juice subjected to combined pressure-heat treatments. J Agri Food Chem.

58, 8305–8313.

Halim, Y., and Schwartz, S.J. 2006. Direct determination of lycopene content in tomatoes

(lycopersicon esculentum) by attenuated total reflectance infrared spectroscopy and

multivariate analysis. J AOAC Intl.89, 1257–1262.

Knockaert, C., De Roeck, A., Lemmens, L., Van Buggenhout, S., Hendrickx, M., and Van Loey,

A. 2011. Effect of thermal and high pressure processes on structural and health-related

properties of carrots (Daucus carota). Food Chem. 125, 903–912.

Kong, F., Tang, J., Rasco, B., Crapo, C. and Smiley, S. 2007. Quality changes of Salmon

(Oncorhynchus gorbuscha) muscle during thermal processing. J Food Sci. 72, 103–111.

Koskiniemi, C.B., Truong, V.D., Mcfeeters, R.F., and Simunovic, J. 2013. Quality evaluation of

packaged acidified vegetables subjected to continuous microwave pasteurization. LWT-Food

Sci & Tech. 54, 157–164.

Lucier, G., and Glaser, L. 2009. Vegetables and melons: tomatoes. USDA Economic Research

Service. Available at http://www.ers.usda.gov/briefing/vegetables/tomatoes.htm

Lucier, G., and Lin, B.H. 2007. Factors affecting carrot consumption in the United States.

Outlook Report from the Economic Research Service/USDA. No. (VGS-31901): 1–21.

Hunter, K.J., and Fletch, J.M. 2002. The antioxidant activity and composition of fresh, frozen,

jarred and canned vegetables. Innov Food Sci & Emerg Tech. 3, 399-406.

200

Lemmens, L., Tiback, E., Svelander, C., Smout, C., Ahrne, L., Langton, M., Alminger, M., Loey,

A.V., and Hendrickx, M. 2009. Thermal pretreatments of carrot pieces using different heating

techniques: effect on quality related aspects. Innov Food Sci & Emerg Tech.10, 522–529.

Ma, W.H., and Barrett, D.M. 2001. Effects of raw materials and process variables on the heat

penetration times, firmness and pectic enzyme activities diced tomatoes (Halley Bos 3155 cv).

J Food Proc Pres. 25, 123–136.

Marfil, P.H.M., Santos, E.M., and Telis, V.R.N. 2008. Ascorbic acid degradation kinetics in

tomatoes at different drying conditions. LWT-J Food Sci & Tech. 41, 1642–1647.

Nisperos-Carriedo, M.O., Buslig, B.S., & Shaw, P.E. 1992. Simultaneous detection of

dehydroascorbic, ascorbic, and some organic acids in fruits and vegetables of HPLC. J Agri

Food Chem. 40, 1127–1130.

Olivera, D.F., Vina, S.Z., Marani, C.M, Ferreyra, R.M., Mugridge, A., Chaves, A.R., and

Mascheroni, R.H. 2008. Effect of blanching on the quality of Brussels sprouts (Brassica

oleracea L. gemmifera DC) after frozen storage. J Food Eng. 84, 148–155.

Rayman, A., and Baysal, T. 2011. Yield and quality effects of electroplasmolysis and microwave

applications on carrot juice production and storage. J Food Sci. 76(4), C598–C605.

Sadler, G., Davis, J., and Dezman, D. 1990. Rapid extraction of lycopene and β-carotene from

reconstituted tomato paste and pink grapefruit homogenates. J Food Sci. 55, 1460–1461.

Shi, J., Maguer, M.L., Bryan, M., and Kakuda, Y. 2003. Kinetics of lycopene degradation in

tomato puree by heat and light irradiation. J Food Proc Engr. 25, 485–498.

Steed, L.E., Truong, V.D., Simunovic, J., Sandeep, K.P., Kumar, P., Cartwright, G.D., and

Swartzel, K.R. 2008. Continuous flow microwave-assisted processing and aseptic packaging

of purple-fleshed sweetpotato purees. J Food Sci. 73(9), E455–E462.

201

Sun, T., Tang, J., and Powers, J.R. 2007. Antioxidant activity and quality of asparagus affected

by microwave-circulated water combination and conventional sterilization. Food Chem. 100,

813–819.

Tang, J., Feng, H., and Lau M. 2002. Microwave heating in Food Processing. Advances in

Bioprocessing Engineering. 1–44. River Edge, NJ: World Scientific.

U.S. Food and Drug Administration. 2005. Code of Federal Regulations-Title 21: Food and

Drugs (21 CFR 155.190).

Van den Broeck, I., Ludikhuyze, L., Weemaes, C., Van Loey, A., and Hendrickx, M. 1998.

Kinetics for Isobaric-Isothermal degradation of L-ascorbic acid. J Agric Food Chem. 46,

2001–2006.

Vervoort, L., Van der Plancken, I., Grauwet, T., Verlinde, T., Matser, A., Hendrickx, M., and

Van Loey, A. 2012. Thermal versus high pressure processing of carrots: a comparative pilot-

scale study on equivalent basis. Innov Food Sci & Emerg Tech. 15, 1–13.

Vian, S.Z., Olivera, D.F., Mariani, C.M., Ferreyra, R.M., Mugridge, A., Chaves, A.R., and

Mascheroni, R.H. 2007. Quality of Brussels sprouts (Brassica oleracea L. gemmifera DC) as

affected by blanching method. J Food Eng. 80, 218–225.

Villanueva M.O., Marquina, A.D., Vargas, E.F., and Abellan, G.B.2000. Modification of

vitamins B1 and B2 by culinary processes: traditional systems and microwaves. Food Chem.

71, 417–421.

Yam, K.L., and Papadakis, S.E. 2004. A simple digital imaging method for measuring and

analyzing color of food surfaces. J Food Eng. 61, 137–142.

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Table 1. Processing conditions for carrot and tomato products for MW and HW processes with equivalent process severity.

Processing Target F90°C = 3 min F90°C = 10 min

Water temperature 61/93/93/15°C setting (preheating, MW heating, holding and processing cooling sections) MW power setting 14 kW 14 kW Preheating time 20 min 20 min MW heating time 1.36 min 1.46 min Holding time 1.86 min 3.50 min Carrot Total processing time 3.22 min 4.96 min (pasteurization) Real F value 4.9 min 8.9 min

Preheating time 20 min 20 min HW Total processing time 7.80 min 13.96 min Processing Real F value 3.9 min 13.4 min Processing Target 5D process (F105°C = 6.05 min) Water temperature 56/108/107/15°C setting (preheating, MW heating, holding and processing cooling sections) MW power setting 7.0/6.2/2.6/2.5 kW Preheating time 15 min Tomato MW heating time 2.71 min (sterilization) Holding time 2.79 min Total processing time 5.50 min Belt speed 47 inch/min

Water temperature 56/108/107/15°C setting (preheating, HW heating, holding and processing cooling sections) Preheating time 15 min Total processing time 20.1 min

The pressure of the sterilization system was 33 psig, and the pasteurization system was atmospheric pressure.

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Table 2. CIE L*, a*, b* values, total color differences (∆E) and hue angles of carrot and tomato dices following different treatments. The color attributes of fresh carrots were used as the control.

Sample L* a* b* Hue angle Total color difference

(H) (∆E)

Raw 61.68 ± 2.48a 48.32 ± 3.16a 59.51 ± 2.62abc 0.89 ± 0.01a Ref

a b d ae F3min, MW, 0.1% CaCl2 61.15 ± 3.85 42.60 ± 4.65 57.05 ± 4.45 0.92 ± 0.02 6.25

a b ab bcd F3min, HW,0.1% CaCl2 61.17 ± 2.35 42.04 ± 4.86 61.14 ± 2.16 0.97 ± 0.04 6.51

c bc c bce F3min, MW, 1.4% CaCl2 63.07 ± 2.42 41.84 ± 4.08 58.64 ± 3.31 0.95 ± 0.03 6.69

ab bc ab bd carrot F3min, HW, 1.4% CaCl2 62.24 ± 2.86 39.79 ± 4.24 60.83 ± 2.35 0.99 ± 0.05 8.65

ab b bc bcd F10min, MW, 0.1% CaCl2 62.92 ± 2.78 42.62 ± 4.22 59.23 ± 3.17 0.99 ± 0.02 5.84

a bc bc bd F10min, HW, 0.1% CaCl2 61.19 ± 2.78 40.16 ± 3.45 59.17 ± 2.50 0.97 ± 0.03 8.18

ab bc cd ce F10min, MW, 1.4% CaCl2 62.16 ± 2.61 42.26 ± 5.83 57.88 ± 3.21 0.94 ± 0.05 6.30

ab c a d F10min, HW, 1.4% CaCl2 62.57 ± 3.04 38.40 ± 5.41 61.62 ± 2.73 1.01 ± 0.06 10.32

a a a a Unprocessed w/ CaCl2 48.50 ± 6.44 48.45 ± 7.06 53.34 ± 3.21 0.83 ± 0.08 Ref

a a a a tomato MW w/ CaCl2 44.95 ± 9.00 40.61 ± 6.51 50.44 ± 5.37 0.89 ± 0.09 9.08

a a a a MW w/o CaCl2 47.51 ± 6.63 41.00 ± 6.78 51.52 ± 3.90 0.90 ± 0.09 7.74

a a a a HW w/ CaCl2 45.40 ± 8.33 40.57 ± 6.77 50.83 ± 4.80 0.90 ± 0.09 8.83

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Table 3. pH, drained weight, color, soluble solids of tomato samples before and after processing.

pH (after processing) °Brix Drained weight, % Unprocessed w/ Ca 4.6 ± 0.0a 76.16 ± 1.17a MW w/ Ca 4.32 ± 0.02a 4.7 ± 0.1a 78.04 ± 1.51a MW w/o Ca 4.36 ± 0.01a 4.7 ± 0.0a 70.31 ± 2.05b HW w/ Ca 4.30 ± 0.01a 4.7 ± 0.1a 79.13 ± 2.13a

Note: The pH of diced samples before processing was 4.27±0.01, and the corresponding electrical conductivity was 8.42 ± 0.17 mS/cm. Calcium concentration was 0.02% (w/w) for both

HW and MW processed samples.

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Figure 1. Color parameter b*/a* of diced carrots (A) and a*/b* of tomatoes (B) following different treatments. Columns labeled with the same letters are not significant different (p<0.05).

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Figure 2. Texture of diced carrots (A) and diced tomatoes (B) treated by MW and HW processing under different conditions.

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Figure 3. Residual PME activity of carrot dices by MW and HW processing under different conditions.

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Figure 4. Total carotenoid content, α-carotene, and β-carotene contents of diced carrots processed under different treatments.

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Figure 5. Ascorbic acid content of tomato samples processed under different conditions.

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Figure 6. Lycopene content of tomato samples under different conditions.

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Chapter 8. Conclusions and Recommendations

1. Major conclusions

1.1 B. coagulans ATCC 8038 strain can produce consistent heat-stable spores during refrigerated storage. The spores of this strain had higher thermal resistance compared to strain

185A, and ATCC 8038 was therefore chosen as the target bacterium for developing and validating thermal processes of tomato products.

1.2 Thermal degradation of carrot texture with pretreatments (preheating and calcium addition) under temperatures ranging from 80 to 110ºC followed a 2nd order reaction.

1.3 The dielectric loss factors were significantly different among the three tomato tissues studied (pericarp tissue with the skin, locular tissue with the seeds, and placental tissue), and among samples with and without salt. However, no significant differences were found in their corresponding dielectric constants.

1.4 Salt addition at the typical commercial canned tomato product level (0.2gNaCl/100gor

0.055 gCaCl2/100g) sharply increased the loss factor of the three tomato tissues, but didn’t affect their dielectric constants at MW frequencies of 915 and 2450 MHz.

1.5 Similar trends for changes in dielectric loss factor were observed for the three tomato tissues, that is, it decreased with increasing frequency, and increased with salt addition. For the effects of temperature, increasing temperature resulted in an increase in dielectric loss factor at

915 MHz; while at 2450 MH, temperature increase initially caused an increase, followed by a decrease of dielectric loss factor, resulting from different dominant loss mechanisms at the two frequencies.

1.6 The cold spot of tomato pouches was located at (22.8, -2.2) mm from the central point in the middle layer; while the cold spot was located in the center point (0, 0) mm in carrot pouches.

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1.7 A MATS process achieving a target F value of no less than 6 min was developed for processing diced tomatoes packaged in 8-oz pouches, which can deliver a 5D thermal treatment to B. coagualans ATCC 8038 spores.

1.8 For diced carrots, MAP processes with F90°C= 3 min and F90°C=10 min were developed to achieve at least a 6 D reduction of NP C. botulinum type E spores.

1.9 Incubation tests and microbial analyses of the processed tomato pouches verified the safety of products produced by the developed MATS processes.

1.10 Compared to raw carrots, carrots processed by MW heating had lower total color difference (∆E values) than those produced by HW processing under equivalent processing conditions, denoting better color retention.

1.11 The impact of processing on carrot texture was significant, but no significant difference was found in texture retention after MW and HW heating under the same conditions.

1.12 For carotenoids in carrots, all processes lowered levels of total carotenoids, α- and β- carotene contents in carrots. No significant differences were found in carotenoid content between samples subjected to MW and HW processing in most cases.

1.13 For tomatoes, no significant differences were found in the color attributes (L*a*b* values), ascorbic acid, and lycopene content of samples processed by MW and HW on an equivalent basis.

1.14 The impacts of MW heating on quality attributes of vegetables depend on the characteristics of the vegetables and the specific quality parameter selected. For some quality parameters like texture or enzymes, which would be quickly reduced, degraded, or deactivated during a very short time at the beginning of the process (temperature at 105ºC for tomatoes and

90°C for carrots), even the MW processing time was not short enough to retain these

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characteristics. For some relatively heat-stable quality parameters like lycopene in tomatoes, our results show that a 20 min holding time at 105ºC was not long enough to lead to a significant change in its total concentration. For some parameters, like color in carrots, MW processing showed a better color retention than HW processing.

2. Contributions to knowledge

2.1 The thermal resistance of a contaminant (B. coagulans spores) in tomato juice at different acidic pH levels between 4.0 and 4.5 (the commonly controlled pH range for canned tomato processes) under high temperatures were characterized. It included cold storage influence on thermal resistance of the spores, which has not been reported before. This information is useful for development of thermal processing of tomato products (Chapter 3).

2.2 The obtained kinetic model for carrot texture degradation was used to draw the texture retention/microbial or enzyme inactivation charts for carrot processing over a relatively large temperature range (80-110°C), which provide information for recommending processing conditions for carrot products that could control food pathogens and inactivate enzymes (Chapter

4).

2.3 Obtained results of dielectric properties of different tomato tissues may be used for developing MW pasteurization and sterilization processes for different tomato products, and also add new information to the database for computer simulation (Chapter 5).

2.4 This study provides systematic studies on developing pilot-scale MW sterilization processes for pre-packaged tomato dices and MW pasteurization processes for pre-packaged carrot dices, and for evaluating the influences of MW sterilization/pasteurization on their quality attributes (Chapters 6 &7).

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2.5 Quality evaluation results of the MW/HW processed products suggest that the impacts of

MW processing on the quality of vegetables depend on the characteristics of the vegetables and their specific quality parameters. This provides information for choosing suitable vegetables and vegetable products for MW processing in later studies (Chapter 7).

3. Recommendations for future research

3.1 A storage/shelf-life study should be carried out after the pasteurization of carrot products, and the effects of storage conditions (temperature/time) on the quality of pasteurized products should be investigated.

3.2 Microbial safety and stability of the pasteurized products should be assessed, along with the quality/sensory changes with storage to determine the shelf-life of pasteurized products. This is required for a developed pasteurized product to become a potential commercial product.

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