HYPERBARIC TREATMENT TO ENHANCE QUALITY ATTRIBUTES OF FRESH HORTICULTURAL PRODUCE

By Bernard Goyette

Department of Bioresource Engineering McGill University, Montreal Quebec, Canada

February, 2010

A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy

Bernard Goyette 2010

ABSTRACT
Bernard Goyette Ph.D. (Bioresource Engineering)

HYPERBARIC TREATMENT TO ENHANCE QUALITY ATTRIBUTES OF FRESH HORTICULTURAL PRODUCE

An experimental hyperbaric system was conceptualized, designed and built to explore the effect of hyperbaric treatment on the respiration rate (RR), respiratory quotient (RQ) and quality attributes of tomato. Housing five containers that could be individually pressurized from 1 to 9 atmabs, the respirometer was equipped with a flow meter, control valve, pressure transducer; CO2 and O2 gas analyzer and type T thermocouples, all connected to a data acquisition and control card. A software interface was programmed to allow control of the air flow rate through the proportional valve of the flow meter, based on a PID (Proportional, Integral, and Derivative) algorithm. Hyperbaric treatments on tomato fruit showed RR to be inversely proportional to the pressure applied: RR was reduced by 20% at 9 atmabs compared to the control (1 atmabs). At the onset of pressure application the RQ was low and increased to reach a value of approximately 1 within 120 hours. Low RQ values were caused by solubilization of CO2 in the tomato cells at the beginning of the process. Early breaker stage tomatoes were subjected to hyperbaric pressures of 1, 3, 5, 7 or 9 atmabs for different durations (5, 10 or 15 days) at 13C, followed by a storage period of 12 days at 20oC. The effect of hyperbaric treatment on postharvest quality of tomato fruits was evaluated with an emphasis on weight loss, firmness, color, lycopene content, titratable acidity (TA) and total soluble solids (TSS). Based on firmness values, control tomatoes were no longer I

acceptable for consumption after 12 days of post-treatment storage. Being subjected to hyperbaric pressures of 7 and 9 atmabs for 15 days caused irreversible physiological damage to the tomatoes. Treatments of 3, 5 or 7 atmabs applied over 10 days, or 5 atmabs applied over 5 days maintained marketable firmness. The lowest weight loss occurred with treatments of 3 or 5 atmabs for 5 days, or 5 atmabs for 10 days. Lycopene content of the tomatoes was improved by hyperbaric pressure followed by 12 days of maturation. The greatest lycopene content 28% more than in the control was obtained for tomatoes subjected to 5 atmabs over 10 or 15 days.

II

RSUM
Bernard Goyette Ph.D (Gnie des bioressources)

TRAITEMENT HYPERBARE POUR AMLIORER LA QUALIT DES PRODUITS HORTICOLES FRAIS

Un respiromtre hyperbare exprimental a t conu et construit pour tudier l'effet du traitement hyperbare sur le taux de respiration (RR), le quotient respiratoire (RQ) et les attributs de qualit de la tomate. Il tait compos de cinq contenants qui pouvaient tre individuellement pressuriss de 1 9 atmabs. Le respiromtre tait quip d'un dbitmtre, valve, capteur de pression, un analyseur de CO2 et O2 et de thermocouples de type T. Tous les capteurs taient relis une carte dacquisition de donnes et de contrle. Un logiciel servant dinterface a t programm pour permettre le contrle du dbit d'air travers la valve proportionnelle du dbitmtre bas sur un contrleur PID (proportionnel, intgral, drive). Les traitements hyperbares ont t effectus sur les tomates et il a t observ que RR tait inversement proportionnel la pression. Le RR a t rduit de 20% 9 atmabs compar au contrle (1 atmabs). Au dbut de l'application de pression le RQ tait faible et a augment graduellement durant 120 heures pour atteindre une valeur d'environ 1. Les faibles valeurs de RQ ont t vraisemblablement causes par la solubilisation CO2 dans la chair des tomates au dbut du processus. Leffet de la pression hyperbare a t test sur la qualit de la tomate. Les pressions utilises taient de 1, 3, 5, 7 ou 9 atmabs et ont t utilises avec trois diffrentes dures de traitement: 5, 10 ou 15 jours, 13C, et suivie d'une priode dentreposage de 12 jours 20C. L'effet du traitement hyperbare sur la qualit postrcolte de la tomate a t tudi en mettant l'accent sur la perte de poids, la fermet, la couleur, le lycopne, l'acidit titrable (TA), et les solides solubles totaux (TSS). Aprs 12 jours dentreposage, III

la fermet de toutes les tomates du groupe contrle (1 atmabs) tait un niveau non acceptable pour la consommation. Le maintient pendant 15 jours de pressions hyperbares leves (7 et 9 atmabs) ont caus des dgts physiologiques irrversibles chez les tomates. Les valeurs de fermet considres commercialisables ont t obtenues par des traitements de 3, 5 ou 7 atmabs maintenus pendant 10 jours ou 5 atmabs maintenus pendant 5 jours. Les plus faibles valeurs de perte de poids ont t observes avec des traitements de 3 ou 5 atmabs pendant 5 jours ou 5 atmabs pendant 10 jours. La teneur en lycopne des tomates sest amliore avec des pressions hyperbares suivis de 12 jours de maturation. La plus haute valeur de lycopne a t obtenue avec des tomates soumises 5 atmabs pendant 10 ou 15 jours. Le taux de lycopne tait significativement plus lev pour ces tomates soit 28% plus lev que celles du groupe contrle.

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ACKNOWLEDGMENTS
I would like to express my sincere gratitude to my academic supervisor, Professor Vijaya Raghavan, for his support throughout this study. My sincere thanks to my co-supervisor, Dr. Clment Vigneault, for his support and mentorship. Thank you for being patient with me and giving me the time to produce a quality work. Overall, thank you for giving me the opportunity to experience graduate studies. I sincerely thank Dr. Marie Thrse Charles, for her help in the study of the physiological aspects. I express my special thanks to Jrme Boutin and Dominique Roussel, my colleagues at Agriculture and Agri-food Canada, and to my daughter Amlie Goyette, for their great help. Thanks to my children, Amlie, Mathieu-Vincent, Nicolas and Thomas, who understood I needed to work so often. A special thanks to my friend Simona Nemes: you were my rayon de soleil on the campus. Finally, I would like to thank my wife, Marlne Pich, for her assistance and collaboration during these interminable working hours. I sincerely believe that this accomplishment would not have been possible without your support. I gratefully acknowledge the financial support of Agriculture and Agri-Food Canada.

TABLE OF CONTENTS
ABSTRACT............................................................................................................I RSUM..............................................................................................................III ACKNOWLEDGMENTS....................................................................................... V TABLE OF CONTENTS ...................................................................................... VI LIST OF FIGURES.............................................................................................. XI LIST OF TABLES ............................................................................................. XVI NOMENCLATURE .......................................................................................... XVII 1. GENERAL INTRODUCTION ........................................................................1

1.1. POSTHARVEST HISTORY CONSIDERING CONSUMER DEMAND IN NORTH AMERICA ............................................................................................1 1.2. FUNCTIONAL FOOD AND NUTRACEUTICAL INDUSTRY .........................4 1.3. TOMATO.......................................................................................................5 1.3.1. 1.3.2. 1.3.3. 2. LYCOPENE .........................................................................................6 TOMATO AND HUMAN HEALTH .......................................................6 TOMATO PRODUCTION ....................................................................9

1.4. PROBLEM STATEMENT..............................................................................9 OBJECTIVES.............................................................................................. 11 2.1. HYPOTHESIS............................................................................................. 11 2.2. MAIN OBJECTIVES.................................................................................... 11 3. LITERATURE REVIEW............................................................................... 12 3.1. INTRODUCTION ........................................................................................ 12 3.2. RESPIRATION RATE ................................................................................. 13 3.2.1. RESPIRATION DEFINITION ............................................................. 13 3.2.1.1. RESPIRATION DEFINITION.................................................................... 14 3.2.1.2. MONITORING METABOLIC ACTIVITY ................................................... 15 3.2.2. 3.2.3. RESPIRATION QUOTIENT DEFINITION.......................................... 15 RELATIONSHIP BETWEEN RESPIRATION RATE AND RESPIRATION QUOTIENT AND THEIR EFFECT ON PRODUCT METABOLISM ................................................................................... 16 ENVIRONMENTAL FACTORS AFFECTING RESPIRATION RATE AND RESPIRATION QUOTIENT ...................................................... 16 VI

3.2.4.

3.2.5. 3.2.6. 3.3.1.

RQ VALUES AND THEIR SIGNIFICANCE ....................................... 18 METHODS TO MEASURE RESPIRATION RATE AND RQ ............. 19 HEAT ................................................................................................. 20

3.3. PHYSICAL TREATMENT ........................................................................... 20 3.3.1.1. THERMOTOLERANCE............................................................................ 20 3.3.1.2. EFFECTS ON DISINFECTION AGAINST HUMAN PATHOGENS .......... 21 3.3.1.2.1. CANTALOUPE AND MELONS ......................................................... 21 3.3.1.2.2. LEAFY VEGETABLES ...................................................................... 21 3.3.1.2.3. TOMATOES ...................................................................................... 22 3.3.2. UV-C.................................................................................................. 22 3.3.2.1. EFFECTS ON DISEASE .......................................................................... 23 3.3.2.2. HORMESIS EFFECTS IMPROVEMENTS OF QUALITY ATTRIBUTES ........................................................................................... 23 3.3.2.2.1. POSITIVE CHANGES ....................................................................... 23 3.3.2.2.2. ADVERSE EFFECTS........................................................................ 24 3.3.2.3. EFFICACY OF UV TREATMENT............................................................. 24 3.3.3. PRESSURE....................................................................................... 25 3.3.3.1. HIGH PRESSURE PROCESSING (HPP) ................................................ 27 3.3.3.1.1. EFFECTS ON PATHOGEN .............................................................. 27 3.3.3.1.2. EFFECTS ON ENZYMES ................................................................. 28 3.3.3.1.3. EFFECTS ON PHYSICAL PROPERTIES AND QUALITY OF PROCESSED FRUITS AND VEGETABLES ........................................ 29 3.3.3.2. HPP COMBINED WITH MILD THERMAL TREATMENT ......................... 29 3.3.3.3. HPP COMBINED WITH LOW-TEMPERATURE TREATMENT ............... 30 3.3.3.3.1. EFFECTS ON RESPIRATION RATE OF FRUITS AND VEGETABLES UPON STORAGE ........................................................ 31 3.3.3.4. HYPOBARIC AND HYPERBARIC PRESSURE TREATMENT ................ 31 3.3.3.4.1. HYPOBARIC PRESSURE TREATMENT.......................................... 31 3.3.3.4.2. EFFECTS ON PATHOGENS AND DISEASES................................. 32 3.3.3.4.3. EFFECTS ON FRUITS AND VEGETABLES CONSERVATION....... 33 3.3.3.4.4. HYPERBARIC PRESSURE TREATMENT ....................................... 34 3.3.3.4.5. EFFECTS ON FRUITS AND VEGETABLES QUALITY .................... 34 3.3.3.5. BENEFICIAL SUBSTANCES AND FUNCTIONAL PROPERTIES OF FRUITS AND VEGETABLES INDUCED BY PRESSURE TREATMENT. 35 VII

3.4. TOMATO..................................................................................................... 36 3.4.1. PHYSIOLOGY OF TOMATO............................................................. 36 3.4.1.1. COMPOSITION........................................................................................ 36 3.4.1.2. CHARACTERISTICS ............................................................................... 36 3.4.2. QUALITY PARAMETERS.................................................................. 37 3.4.2.1. MATURITY ............................................................................................... 38 3.4.2.2. COLOR .................................................................................................... 38 3.4.2.2.1. COLOR MEASUREMENT................................................................. 40 3.4.2.3. TEXTURAL QUALITY .............................................................................. 40 3.4.2.4. TOMATO SOLIDS.................................................................................... 40 3.4.2.5. FLAVOUR QUALITY ................................................................................ 41 3.5. LYCOPENE ................................................................................................ 41 3.5.1. 3.5.2. 4. LYCOPENE IN TOMATO .................................................................. 41 LYCOPENE AND HUMAN HEALTH ................................................. 42

3.6. SUMMARY.................................................................................................. 43 CONCEPTUALIZATION, DESIGN AND EVALUATION OF A HYPERBARIC RESPIROMETER ............................................................... 45

4.1. INTRODUCTION ........................................................................................ 45 4.2. PRELIMINARY WORKBENCH ...................................................................48 4.2.1. 4.2.2. 4.2.3. RESPIROMETER DESIGN ............................................................... 48 CALIBRATION OF THE RESPIROMETER ....................................... 53 EVALUATION OF THE EFFICACY OF THE SYSTEM TO MEASURE THE RESPIRATION RATE OF LIVING PRODUCE ....... 53 RESPIROMETER DESIGN ............................................................... 55 CALIBRATION OF THE RESPIROMETER ....................................... 60 EVALUATION OF THE EFFICACY OF THE SYSTEM TO MEASURE THE RESPIRATION RATE OF LIVING PRODUCE ....... 62

4.3. RESULTS AND DISCUSSION.................................................................... 55 4.3.1. 4.3.2. 4.3.3.

4.4. CONCLUSION ............................................................................................ 65 4.5. REFERENCES ........................................................................................... 65 CONNECTING TEXT.......................................................................................... 67 5. EFFECT OF HYPERBARIC TREATMENT ON RESPIRATION RATE AND RESPIRATORY QUOTIENT OF TOMATO ........................................ 68

5.1. INTRODUCTION ........................................................................................ 68 VIII

5.2. MATERIAL AND METHOD ......................................................................... 70 5.2.1. 5.2.2. HYPERBARIC RESPIROMETER DESIGN ....................................... 70 BIOLOGICAL MATERIAL .................................................................. 73

5.3. EXPERIMENTAL DESIGN.......................................................................... 73 5.4. RESULTS AND DISCUSSION.................................................................... 74 5.4.1. 5.4.2. 5.4.3. 5.4.4. RESPIRATION RATE CALCULATION.............................................. 75 MODEL FOR PREDICTING THE RESPIRATION RATE .................. 81 RQ VALUES AND THEIR SIGNIFICANCE ....................................... 89 EFFECT OF HYPERBARIC TREATMENT ON RR AND RQ ............ 95

5.5. CONCLUSION ............................................................................................ 96 5.6. REFERENCES ........................................................................................... 97 CONNECTING TEXT.......................................................................................... 99 6. EFFECT OF HYPERBARIC TREATMENT ON QUALITY ATTRIBUTES OF TOMATO............................................................................................. 100

6.1. INTRODUCTION ...................................................................................... 100 6.2. MATERIALS AND METHODS .................................................................. 103 6.2.1. 6.2.2. 6.2.3. 6.2.4. HYPERBARIC SYSTEM.................................................................. 103 BIOLOGICAL MATERIAL ................................................................ 106 EXPERIMENTAL SET UP ............................................................... 106 EVALUATION OF QUALITY PARAMETERS .................................. 108

6.2.3.1. DECOMPRESSION ............................................................................... 107 6.2.4.1. WEIGHT LOSS ...................................................................................... 108 6.2.4.2. FIRMNESS............................................................................................. 108 6.2.4.3. COLOR AND LYCOPENE ..................................................................... 109 6.2.4.4. TOTAL SOLUBLE SOLIDS (TSS) AND TITRATABLE ACIDITY (TA).... 111 6.2.5. 6.3.1. STATISTICAL ANALYSIS ............................................................... 112 WEIGHT LOSS................................................................................ 113 6.3. RESULTS AND DISCUSSION.................................................................. 112 6.3.1.1. OPENING............................................................................................... 113 6.3.1.2. AFTER 12 DAYS OF STORAGE ........................................................... 115 6.3.2. FIRMNESS ...................................................................................... 115 6.3.2.1. OPENING............................................................................................... 117 6.3.2.2. AFTER 12 DAYS OF STORAGE ........................................................... 117 IX

6.3.3. 6.3.4.

COLOR AND LYCOPENE............................................................... 120 COLOR............................................................................................ 120

6.3.4.1. OPENING............................................................................................... 120 6.3.4.2. AFTER 12 DAYS OF STORAGE ........................................................... 122 6.3.5. LYCOPENE ..................................................................................... 122 6.3.5.1. OPENING............................................................................................... 122 6.3.5.2. AFTER 12 DAYS OF STORAGE ........................................................... 126 6.3.6. TITRATABLE ACIDITY.................................................................... 127 6.3.6.1. OPENING............................................................................................... 127 6.3.6.2. AFTER 12 DAYS OF STORAGE ........................................................... 129 6.3.7. TOTAL SOLUBLE SOLIDS (TSS) ...................................................129 6.3.7.1. OPENING............................................................................................... 129 6.3.7.2. AFTER 12 DAYS OF STORAGE ........................................................... 129 6.3.8. TSS TA RATIO ............................................................................. 133 6.4. GENERAL DISCUSSION.......................................................................... 133 6.5. CONCLUSION .......................................................................................... 136 6.6. REFERENCES ......................................................................................... 137 7. 8. 9. GENERAL SUMMARY AND CONCLUSIONS.......................................... 141 RECOMMENDATIONS FOR FUTURE STUDIES .................................... 144 CONTRIBUTIONS TO KNOWLEDGE ...................................................... 145

10. REFERENCES ......................................................................................... 146

LIST OF FIGURES
Figure 3.1: Representation of pressure range treatment and the type of produce on which the treatment can be applied. Figure Figure 3.2: Maturity and ripening stages of tomatoes. 4.1: Pictorial and schematic view of the hyperbaric respirometer. Dotted lines represent the gas flow pathway thru the dynamic respiration system. Figure 4.2: Chamber and equipment details of the dynamic respiration system developed. Figure 4.3: Theoretical % of flushing during the calibration as a function of time using the general dilution equation (Eq. 4.1). The volume used was 442 mL and an airflow rate of 50 mL min-1. Figure 4.4: Respiration rate pattern of 200 g tomato stored inside of the 442 mL airtight chamber pressurized at 2 atmabs and maintained at 13C with an airflowrate of 50 mL min-1. Figure 4.5: Respiration rate pattern using the outer chamber having a volume of 8863 mL with 1218 g of tomato at 13C and pressurized at 7 atmabs and an airflow rate of 110 mL min-1. Figure 4.6: Error of the respiration rate (RR) reading as a function of the RR. Figure 4.7: Air flow rate (mL min-1) required to maintain the CO2 concentration of the exhausting gas at 1478 ppm as a function of the commodity respiration rate (mL of CO2 min-1). 59 58 57 56 54 50 39 49 26

XI

Figure

4.8: Calibration curves obtained using the dynamic respiration system developed and a calibration gas containing 1478 ppm of CO2.

61

Figure

5.1: Schema of the automated respirometer developed to measure the respiration rate and the respiration coefficient using a continuous flow through system.

71

Figure

5.2: Detail of the closed container unit showing the air inlet for injecting Qin, the air outlet through which the airflow Qout exhausts from the system, and the inside volume (V) and the gas (G) produced or used by the produce.

72

Figure

5.3: Respiration rate (RRCO2) of tomato based on CO2 production as a function of time for various hyperbaric pressure and equivalent CO2 partial pressure.

76

Figure

5.4: Respiration rate (RRO2) of tomato based on O2 production as a function of time for various hyperbaric pressure and equivalent CO2 partial pressure.

77

Figure

5.5: Respiration quotient (RQ) of tomato as a function of time for various hyperbaric pressure and equivalent CO2 partial pressure.

78

Figure

5.6: Linear portion of respiration rate data based on CO2 used for linear regression analysis.

79

Figure

5.7: Linear portion of respiration rate data based on O2 used for linear regression analysis.

80

Figure

5.8: Respiration rate based on CO2 released as a function of CO2 partial pressures. The respiration rate presented in this figure is obtained from the intercept of the linear regression analysis presented in Table 1.

83

XII

Figure

5.9: Decrease of respiration rate in time based on CO2 released as a function of CO2 partial pressures. The respiration rate presented in this figure is obtained from the slope of the linear regression analysis presented in Table 1.

84

Figure 5.10: Respiration rate based on O2 released as a function of CO2 partial pressures. The respiration rate presented in this figure is obtained from the intercept of the linear regression analysis presented in Table 2. Figure 5.11: Decrease of respiration rate in time based on O2 released as a function of CO2 partial pressures. The respiration rate presented in this figure is obtained from the slope of the linear regression analysis presented in Table 2. Figure 5.12: Comparison between the theoretical RR calculated using Eq. 5.17 (continuous lines) and experimental RR data measured (data points). Figure 5.13: Apparent RQ results calculated for the first 120 hours after submitting tomato fruits to different absolute pressures ranging from 1 to 9 atmabs. Figure 5.14: Respiration coefficient (RQ) measured after CO2 gas reached equilibrium during pressure tomato fruit treatments at different absolute pressures ranging from 1 to 9 atmabs. Figure 6.1: Hyperbaric system used to test the effect of pressure on tomato. Figure 6.2: The percentage of tomato weight loss after 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. Vertical bars indicate standard deviation.

86

87

90

92

94

104

114

XIII

Figure

6.3: The percentage of tomato weight loss after 12 days maturation at 20C and 80% RH for 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. Vertical bars indicate standard deviation.

116

Figure

6.4: Initial tomato firmness and after 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. The firmness is expressed in N mm-1 required to penetrate the tomato by 3 mm using a flat punch 6 mm diameter. Vertical bars indicate standard deviation.

118

Figure

6.5: Tomato firmness after 12 days maturation at 20C and 80% RH for 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. The firmness is expressed in N mm-1 required to penetrate the tomato by 3 mm using a flat punch 6 mm diameter. Vertical bars indicate standard deviation. Vertical bars indicate standard deviation.

119

Figure

6.6: Initial Tomato color and after 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. Vertical bars indicate standard deviation.

121

Figure

6.7: Tomato color after 12 days maturation at 20C and 80% RH for 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. Vertical bars indicate standard deviation.

123

Figure

6.8: Lycopene concentration of tomato after 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. Vertical bars indicate standard deviation.

124

XIV

Figure

6.9: Lycopene concentration of tomato after 12 days maturation at 20C and 80% RH for 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. Vertical bars indicate standard deviation.

125

Figure 6.10: Initial TA of tomato and after 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. Vertical bars indicate standard deviation. Figure 6.11: TA of tomato after 12 days maturation at 20C and 80% RH for 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. Vertical bars indicate standard deviation. Figure 6.12: Initial TSS of tomato and after 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. Vertical bars indicate standard deviation. Figure 6.13: TSS of tomato after 12 days maturation at 20C and 80% RH for 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. Vertical bars indicate standard deviation. Figure 6.14: Initial TSS/TA ratio of tomato and after 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. Vertical bars indicate standard deviation. Figure 6.15: Initial TSS/TA ratio of tomato after 12 days maturation at 20C and 80% RH for 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. Vertical bars indicate standard deviation.

128

130

131

132

134

135

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LIST OF TABLES
Table 1.1 : Ripening index values for tomato fruits at different color stages. Adapted from Lopez Camelo and Gomez, 2004. Table 4.1 : Respiration rate (RR) of tomato fruits exposed to a pressure of 2 and 7 atmabs and a temperature of 13C. Table 5.1 : Parameter of the linear regression analysis of respiration rate based on CO2 production as a function of time for various CO2 partial pressures (Fig 5.6). Table 5.2 : Parameter of the linear regression analysis of respiration rate based on O2 production as a function of time for various CO2 partial pressures (Fig 5.7). Table 5.3 : Parameter of the linear regression analysis of RQ as a function of time for various CO2 partial pressures (Fig. 13). Table 6.1 : Ripening index values for tomato fruits at different color stages. Adapted from Lopez Camelo and Gomez, 2004. 110 93 85 82 64 8

XVI

NOMENCLATURE
a* = confidence interval used during statistical analysis = CIE standard nomination for one of the three colour components obtained from a chromameter b* CO2 = CIE standard nomination for one of the three colour components = difference in CO2 concentration between the inlet and outlet of the respiration chamber, ppm C C1 Cfinal CIE Cinitial Dt H L* = concentration of a gas inside the respiration chamber, ppm = constant = final concentration of diluted gas, ppm = Commission Internationale de lclairage = initial concentration of the gas to be diluted, ppm = dilution time, min = color parameter hue angle = CIE standard nomination for one of the three colour components obtained from a chromameter m N O2 = mass of produce, kg = number of samples required to produce a significant difference = difference in O2 concentration between the inlet and outlet of the respiration chamber, ppm p Q RQ = pressure inside of the respiration chamber, atmabs = flow rate, mL h-1 or mL min-1 = ratio between the amounts of CO2 released (Rx) over the amount of O2 consumed (Ry)

XVII

RR RRCO2 RRD

= respiration rate, mL gas kg-1 h-1 = respiration rate, mL CO2 kg-1 h-1 = respiration rate decrease in time, mL gas kg-1 h-1

RRmeasured = measured respiration rate, mL gas kg-1 h-1 RRO2 RRreal Rx Ry

2 2 Sx , Sy

= respiration rate, mL O2 kg-1 h-1 = real respiration rate, mL gas kg-1 h-1 = amounts of CO2 released, mL CO2 kg-1 h-1 = amounts of O2 released, mL O2 kg-1 h-1 = standard deviation of the population x and y, respectively, = time, h = time at the event 1, h = time at the event 2, h = two-tailed Student t-test value = time differential, h = void volume of gas to be diluted in the chamber, mL or L = CO2 volume difference inside the respiration chamber, mL or L = mean values of the population x and y, respectively

t t1 t2 t2 t V v

x, y

XVIII

CHAPTER I 1. GENERAL INTRODUCTION

1.1. POSTHARVEST HISTORY CONSIDERING CONSUMER DEMAND IN NORTH AMERICA Quality attributes of fruits and vegetables after harvest have been a worldwide concern for many years. Over the XXth century, innovations of all kinds have taken place to keep produce fit for consumption year round, ranging from natural cold, cooking and canning, salting and drying to refrigeration and transformation with the advent of new technologies such as fuel power, electricity and biotechnology. A 1921 article in the New York Times reported the studies of Dr. H.B Cox, who suggested that eating fresh fruits and vegetables was important to prevent malnutrition (Cox, 1921). The difficulty reported at that time was the unavailability of such commodities, as fresh vegetables delivered to households were often no longer fresh due to poor conservation methods. Given the necessity of making available fresh fruits and vegetables that would stay fresh for a certain time on the market counter without damage caused by diseases, transport or handling, conservation techniques were the subject of many studies. In 1950, the cooling of fruits and vegetables had increased the availability of a wide variety of produce, and to the establishment of systems for storage and long distance transportation. These changes brought new challenges to the field. It became obvious that not all commodities required the same cooling temperature to preserve food quality, and that improper conservation temperatures could enhance physiological damage. Several hours were needed to lower the produce temperature and since the temperature in the product was non-uniform, it allowed decay-organisms to grow (Bratley and Wiant, 1950).

2 Considering the non-uniformity problem of the cooling systems, the principle of pre-cooling emerged. Pre-cooling with ice and forced cold-air was investigated by Bratley and Wiant in 1950. Hydrocooling was introduced in the late 50s, but represented an important source of inoculums for pathogens as water was constantly re-circulated through the mass of produce. Contamination was reduced by the addition of chemicals to the cooling water (Smith, 1962). Diseases affecting fruits and vegetables after harvest were long known to be detrimental to conservation. Different antiseptic treatments were proposed over the years (Fulton and Bowman, 1925; Pryor and Baker, 1950), but it is only in the 50s that diseases affecting fresh fruits and vegetables became a main concern not only for producers but for handlers and consumers. Some diseases appeared to arise in the field but others resulted from poor operating conditions after harvest. Increasing attention was then given to treatments for decay prevention carried out after harvest and prior to shipment (Bratley and Wiant, 1950). Many methods were investigated to reduce postharvest decay, most of them of a chemical nature. Chemicals were applied through dipping or washing of the fresh produce, by fumigation of the storage facility or by coating the wrappers or liners of the shipping boxes. The chemicals used, fungicides, bactericides and antibiotics, were toxic to microorganisms (Smith, 1962). Since a cold chain was not in practice in the earlier years, it led to many designs of conservation devices over the last century. In 1917, a Patent was given for a sealed container subjected to the cooling effect caused by the expansion of a compressed gas. It was proposed to maintain organic material freshness (Darden, 1917). In 1918, the idea of vacuum followed by an addition of carbon dioxide to a sealed container to reduce the tendency to decay or ferment was proposed. The recommendation was to apply CO2 at 4 atm to maintain freshness from a few hours to a few weeks (Franks, 1918). In 1931, Edward Milani made a request to the US Patent office to protect the invention of the reduced oxygen and high carbon dioxide sealed container to maintain produce quality (Milani, 1931). He explained the principle of controlled atmosphere storage, how produce reacted and the importance of venting the container to maintain product quality. In the

3 1940s, some studies explored the use of low O2 or high CO2 atmosphere for the reduction of respiration rate and conservation at a larger scale. British studies found that the storage life of apples could be doubled by holding them at 14% CO2 and 8% O2 (Bratley and Wiant, 1950). These results led to the implementation of a number of such systems in England. As CA storage became widely used, it was observed that in certain cases it altered the quality of the produce. CA storage expanded worldwide and was widely evaluated and optimized to maintain an optimal quality of fresh produce (Murata and Minamide, 1970; Little et al., 1973; Gariepy et al., 1984; Reust et al., 1984; Goyette et al., 2002; Amodio et al., 2005; Lvesque et al., 2006). After the Second World War, mass marketing strategies for food production became the norm, resulting in the export of products worldwide. There were fewer varieties available (Cook, 2002). Efforts were made to export large quantities of non-perishable products, as well as canned or easily preserved commodities at relatively lower temperatures (12C). The canning industry was very important at that time (Bratley and Wiant, 1950). With the demographic and lifestyle trends of the 1970s, changes occurred and consumers demands diversified. Targeted marketing replaced mass marketing in the 80s and kept changing from there on (Cook, 2002). Between 1978 and 1988, fresh vegetable consumption per capita increased by 26%. In the 90s, 98% of American consumers stated that the quality of fresh fruits and vegetables had a determinant influence on where they shopped for food (Food Marketing Institute, 1990). A new trend towards organic foods also influenced the late 90s. Knowing the public health and environmental risks related to the use of pesticides, especially post-harvest fungicide treatments, favored the introduction of integrated pest management programs (IPM) and environmentally friendly technologies to improve the quality of fruits and vegetables from the field to the consumers table.

4 1.2. FUNCTIONAL FOOD AND NUTRACEUTICAL INDUSTRY Fruits and vegetables are a good source of vitamins, minerals and phytochemicals. Phytochemicals are non-nutritive bioactive plant substances considered to have beneficial effects on human health (Basu et al., 2007). People are more and more informed and aware of the importance of these phytochemicals, often presented to consumers as antioxidants. Antioxidants are protective agents that significantly delay or prevent oxidative damage in cells caused by reactive oxygen species and appeared to have a wide range of anticancer and antiatherogenic properties (Agarwal and Rao, 2000). The presence of natural plant compounds have been found to be further enhanced by inducing known quantities of physical stress. Heat treatment, controlled atmosphere storage, UV radiation and other chemical-free treatments have been studied. Many researches have reported that such stress or treatments induce positive physiological changes to the fruits and vegetables, such as natural disease resistance or improved quality attributes (Hodges and DeLong, 2007). The concept is called plant hormesis and is, by definition, the stimulation of a beneficial plant response by low or sub-lethal dosage of an elicitor such as a chemical, biological or physical stress (Luckey, 2003). The natural disease resistance of harvested horticultural crops induced by elicitors has been investigated (Terry and Joyce, 2004) and is very attractive considering the importance of the concept of reducing the use of pesticides and the enhancement of quality of fresh produce to serve as a functional food. Functional foods are products that are similar in appearance to conventional food, are consumed as part of a usual diet, and have demonstrated health benefits beyond basic nutrition such as the prevention, protection and treatment of chronic diseases (Anon., 2005; Basu et al., 2007). A nutraceutical is a product isolated or purified from foods, demonstrated to have physiological benefit or provide prevention, protection and treatment against diseases (Basu et al., 2007). In

5 recent years, functional food and nutraceutical industry have been constantly growing in the global marketplace. Agriculture and Agri-Food Canada has presented the Canadian Food Trends to 2020 - A long range consumer outlook report (Anon., 2005). The rapidly growing market for functional food and natural health products is benefiting Canadas functional foods and nutraceuticals (FFN) industry. According to Statistics Canada (Anon., 2008), in 2004 this industry generated $2.9 billions in total revenues, representing a 15% increase since 2002. But Canada has a long way to go to compete with other countries. In 2004, Canadas production represented only 3% of the global market. The United States of America is the World leader with 35% of the market, followed by Japan and the European Union. Other Eastern Countries such as China and India produce large quantities of traditional functional food products, but are limited in access to World markets by the necessity to properly label and assess the health effects of the products for export. As of 2006, Australia and New Zealand are emerging as international competitors. South, Central and Latin America are still developing and the principle of functional food lacks popularity. African markets are still not well organized although functional food and nutraceuticals are part of the African culture. In 2007, the functional foods and nutraceutical industry represented $75.5 billion US and is expected to grow to $167 billion by 2010 (Basu et al., 2007). 1.3. TOMATO Tomato is the third most important fresh vegetable consumed in Canada (Anon., 2008), the second most important vegetable crop in the world next to potato, and is the leading processed vegetable available (Gould, 1992). The origin of tomato is somewhat uncertain but it appears that it started out as a wild growing fruit in South America. The name tomato is derived from the Aztec word xitomate but the wild tribes of Mexico called it tomati. The fruit was taken to Europe from Mexico or Peru during the early 16th century and was grown extensively in Italy,

6 where it was called pomi doro or golden apple. As of 1800, six varieties of tomatoes were grown for market purposes in Europe. Its large production brought curiosity and interest in England and North America. Tomato was first brought to America in 1798 but the fruit was not sold on the market until 1829. It then rapidly gained popularity thus making it almost indispensable today, as it is used fresh, canned or processed as soup, sauce, or ketchups (Gould, 1992). Globally, the tonnage of tomato production and per capita consumption has kept increasing. In the U.S., per capita consumption of tomato has increased by almost 20% between 1985 and 2000 (FAOSTAT Database, 2004). Tomato consumption is anticipated to keep increasing since tomato fruits have been well identified as an important source of lycopene, a most potent antioxidant, and associated with high vitamine C and A content, which have considerable health benefits. 1.3.1. LYCOPENE Lycopene is a carotenoid, an acyclic isomer of -carotene. It is the most predominant carotenoid in human plasma and is found to concentrate in the adrenal gland, testes, liver and prostate gland. It is a natural pigment synthesized by plants and microorganisms but not by animals. As other antioxidants, like vitamin E, vitamin C and polyphenols, carotenoids are available from plant food. The lycopene present in natural plant sources is the most thermodynamically stable form (Agarwal and Rao, 2000). Antioxidant properties increase cellular defence against oxidative damage but lycopene may also have bioactivities capable of enhancing DNA repair (Astley and Elliott, 2005). 1.3.2. TOMATO AND HUMAN HEALTH Some genetic transformation, like genetically modified organisms (GMO), to improve the quality or resistance of eatable products is not well accepted by consumers. Hence alternative and natural sources should be favoured.

7 Tomatoes contain naturally-occuring beneficial ingredients and constitute the major dietary source of lycopene. They contain higher levels of lycopene than any other fruit or vegetable (Anon., 2005) and have been associated with decreased risk of certain chronic diseases, such as cancer and cardiovascular disease (Agarwal and Rao, 2000; Rivero et al., 2006). Since tomatoes undergo extensive processing and storage before being consumed, researchers have studied the stability of lycopene in tomato under processing and storage conditions. Results indicate that lycopene present in fresh tomatoes and tomato products, is stable, stays bioavailable and acts as an in vivo antioxidant providing protection against lipid, protein, and DNA damage (Agarwal et al., 2001). The presence of dietary lipids and heat during tomato processing also presented a positive effect resulting in the higher release of lycopene and easier absorption by human body (Porrini, 2003). To have a beneficial effect on the human body and to prevent certain diseases, the suggested daily dosage of lycopene is of 10 to 50 mg per day for adults. Since lycopene is valued at $100 per mg, supplements would be too expensive for most people to afford. As presented in Table 1.1, eating raw or processed tomatoes represent the easiest and cheapest way to get the recommended lycopene dosage in ones daily diet (Anon., 2005). Most importantly, one should know that it is impossible to separate with certainty the effect of vitamin C and lycopene in tomato consumption, since tomato is also a good source of vitamin C (Porrini, 2003). A single carotenoid or a single phytonutrient may have a small beneficial effect, but when they are combined, they often show a synergistic effect (Levy, 2003). The emphasis should then be on the consumption of the whole fruit rather than a single component, since food components work in concert.

Table 1.1 : Ripening index values for tomato fruits at different color stages. Adapted from Lopez Camelo and Gomez, 2004. Lycopene Product Tomato juice Tomato ketchup Tomato paste Tomato soup Tomato sauce Fresh tomatoes c
a

Lycopene Serving size b 240 mL 15 mL 30 g 245 g 60 g 148 g (1 medium) (mg/serving) 22.9 2.9 8.8 13.1 9.6 18.5

(mg/100g) a 9.3 17.0 29.3 10.9 15.9 12.5

USDA-NCC Carotenoid Database for U.S. Foods 1998 FDA Reference Amounts; Guidelines for Voluntary Nutrition Labeling of Raw

Fruit, Vegetables and Fish, Database Updated April 1, 2008.

c

Agarwal et al. 2001

9 1.3.3. TOMATO PRODUCTION Global tomato production (processing and fresh) has increased by 291% from 1961 to 2002 (FAS/USDA, 2003). World tomato production was about 100 million tons of fresh fruits in 2004, grown in 144 countries (FAOSTAT Database, 2004). In North America,
-1

California followed by

is

the Ontario

largest with

tomato a

producer of

with over

11.7 million tons yr-1,

production

0.5 million ton yr (Anon., 2005). Worldwide, China is becoming the largest fresh tomato producer with 25.9 million tons yr-1 which represents about 25% of the worlds tomato production. The United States is the second leading producer, with 95% of production occurring in California (Anon., 2004). Tomato is the second largest US fresh vegetable export. The top fresh tomato exporters are Spain, Mexico, Canada, United States, Italy, France and Turkey. Canada is the second most important supplier of fresh tomatoes to the United States after Mexico (Anon., 2004). On the other hand, Italy is the world leader in canned tomato exports, with approximately 80% of the world market. Chinas exports of tomato have grown over the last decade and China has become the second largest producer of tomato paste. Tomatoes are grown commercially across the world and represent one of the leading fruit productions. But tomatoes are also an important part of home-grown gardens. In the US, it is estimated that 35 million backyard gardens grow tomatoes (Cox, 2001). 1.4. PROBLEM STATEMENT Since tomatoes are consumed in many countries, it is obvious that they must be regarded as a part of a comprehensive strategy to prevent cancer through diet and contribute to better human health worldwide. There is epidemiological evidence that an increase in intake of tomatoes decreases the risk of certain

10 cancer and none of the studies reviewed showed adverse effects of high tomato intake or high lycopene levels (Giovannucci, 1999; Dwyer, 2003). Compared to 20 years ago, Canadians now eat 10.9% more fresh vegetables and 10.2% more fresh fruits (Anon., 2008). Considering that 50% of all cancer have been attributed to diet (Agarwal and Rao, 2000), the populations awareness of this fact incites people to seek fresh produce exempt of chemical preservatives. Preservation methods free of chemicals have been investigated to prevent rotting of fruits and vegetables through handling and storage. In this light, some physical treatments have been studied and it was observed that, along with preventing produce deterioration, these treatments can enhance beneficial nutrients and nutraceutical substances in the treated produce. More work has been done on tomatoes to improve their beneficial substances, such as lycopene. But physical treatments, like UV and heat treatments used as surface sterilisation treatments, are not easy to apply uniformly. The response to these treatments is also not uniform. Hyperbaric treatment is not a surface sterilising treatment but it has the advantage of being uniform. It can be used to create a hormic stress to the commodity being treated. The response reaction to a hormic stress would be a defence reaction that can enhance beneficial nutrients and nutraceutical substances in the treated produce. A hypothesis is being proposed that hyperbaric treatment can enhance quality attributes of fresh tomatoes. The overall purpose of the current study investigates some unique and innovative features for handling fresh produce.

11

CHAPTER 2 2. OBJECTIVES
2.1. HYPOTHESIS The hypothesis of this study is that hyperbaric pressure treatment can affect the physiological development of freshly harvested fruits and vegetables and hence modify their quality attributes. 2.2. MAIN OBJECTIVES The main objectives of this research are: To conceptualize, design and build a dynamic respirometer that can be used for hyperbaric treatments on fresh horticultural produce. The set up should resist pressures up to 9 atmabs, record environmental conditions such as temperature, O2 and CO2 concentrations, control gas flow rate, record, in real time, respiration rate and respiratory quotient. To measure the effect of hyperbaric treatments on respiration rate and respiratory quotient, of tomato fruit. To evaluate the effect of hyperbaric treatments on quality attributes of tomato fruit. To determine the optimal operational parameters of the system to effectively use hyperbaric treatments as a postharvest treatment on tomato fruit.

12

CHAPTER 3 3. LITERATURE REVIEW

3.1. INTRODUCTION Fruits and vegetables are an important part of healthy eating habits. Consumer demands for fresh fruits and vegetables of high quality, exempt of chemical preservatives, and with good or improved nutritional properties incited the industry to improve existing technologies and have lead to many research efforts on novel technologies (Garcia and Barrett, 2002). However, the development of new food processing technologies presents a variety of challenges related to consumers perception, acceptance and purchasing behaviour. In order to improve expected consumer appreciation and increase the chances of their eventual acceptance, novel technologies should improve the sensory quality of the food and be presented to consumer with factual information and clear statements about their safety and benefits (Cardello, 2003). Many studies reported include the occurrence of natural disease resistance in fruits and vegetables. Some natural disease resistance seems to be induced through external factors and others tend to be induced by the defence mechanism of the plant (Terry and Joyce, 2004). The enhanced protection of host plant tissue is an important factor in the development of new postharvest technologies. This concept was defined by Luckey (2003) as plant hormesis which involves the stimulation of a beneficial plant response by low or sub-lethal doses of an elicitor/agent, such as a physical stress. Physically-induced resistance by treatments such as heat, ionising radiation, UV irradiation and pressure has received increasing attention over the recent years. The primary mode of action of these treatments is disinfection of the commodity. In some cases, the physical stress also induced resistance against future

13 infection (Terry and Joyce, 2004) and enhanced the production of beneficial substances in the treated commodity (Luckey, 2003). One should not underestimate the public concern over biotechnology. Some new methods, such as genetically modified organisms or irradiation, are not well received by consumers as they are considered potentially risky technology when applied to human food. The lack of information available makes the public fearful about possible unknown and unforeseen side effects (Cardello, 2003). 3.2. RESPIRATION RATE Respiration is the process by which all fresh fruits and vegetables use their carbohydrates, proteins and fats to transform oxygen into carbon dioxide. Generally, as respiration occurs, the commoditys reserves are exhausted and it results in reduced food value. The rate of deterioration of the commodities is proportional to the respiration rate. Ethylene is a natural organic compound affecting plant metabolism. This hormone regulates growth, development and senescence. Generally, ethylene production increases with maturity at harvest and with various physical stresses, such as bruises, cuts, disease incidence, high temperatures and water (Kader, 2002). To preserve fresh fruits and vegetables for long periods, it is important to reduce the respiration rate and reduce the ethylene production. Many storage techniques induce a reduction in the respiration rate. Horticultural crops are classified according to their respiration rate (Kader, 2002). The classification proposed a range from very low to extremely high respiration rate, measured for different commodities under a 5C environment. Respiration rate is presented as mL CO2 kg-1 h-1 or mg CO2 kg-1 h-1. 3.2.1. RESPIRATION DEFINITION The respiration of fruits or vegetables is a biochemical reaction by which complex substrate molecules like carbohydrates, proteins and fats are broken down into

14 simpler molecules such as CO2 and H2O. Along with this reaction, energy and intermediate molecules are produced (Kader, 2002). Normally, when the respiratory quotient is equal to 1, the respiration process can be represented by the following Eq. 3.1. C 6 H 12 O6 + 6 O2 6 CO2 + 6 H 2 O + 686 kcal / mole ( 3.1 )

In this case, the respiratory process releases as many molecules of CO2 as O2 molecules were consumed. The rate at which the commodity uses oxygen to consume carbohydrates is called the respiration rate and is dependent on the metabolic activity. Respiration rate is presented in Eq. 3.2 (Lencki et al. 2004).
Respiration rate = volume of CO2 evolved or O2 consumed kg of commodity time

( 3.2 )

3.2.1.1. RESPIRATION DEFINITION Horticultural commodities are classified according to their respiration rate and pattern during maturation and ripening. There are two large classes of produce: climacteric and non-climacteric. Climacteric fruits show an increase of CO2 production during ripening and non-climacteric fruits show no change in their generally low CO2 production during ripening (Kader, 2002). Tomatoes are climacteric fruits. They are considered to have a moderate respiration rate with values varying between 10-20 mg of CO2 kg-1 h-1. During the climacteric rise of respiration, tomato fruit soften, the yellow color intensifies (loss of chlorophyll and increase in carotenoids) and fruit aroma (volatiles) increases. The peak of respiration rate usually represents the time at which tomatoes are considered ripe for consumption. Afterwards, respiration gradually decreases as the fruit senesces. Respiration rate varies among commodities but also within commodities. The maturity of a plant at harvest also influences the respiration rate and commodities harvested during active growth have high respiration rates (Saltveit, 2005).

15 Respiration rate is tightly related to the metabolism rate of the fruit or vegetable and is generally proportional to the rate of deterioration (Kader, 2002). 3.2.1.2. MONITORING METABOLIC ACTIVITY Since measuring respiration is a non-destructive way of monitoring the metabolic and physiological state of the produce, it can be used to evaluate the stored fruits and vegetables response after harvest. It provides information on the loss of substrate, the synthesis of new compounds and the release of heat energy (Saltveit, 2005). Some climacteric fruit, like tomatoes, are harvested prior to maturity. The storage facilities have to be optimized to allow the commodities to reach their best quality through respiration, like the synthesis of pigments (lycopene and -carotene in tomatoes) and volatiles, the loss of chlorophyll and the conversion of starch to sugar for sweetness (Saltveit, 2005). 3.2.2. RESPIRATION QUOTIENT DEFINITION To evaluate the respiration process and provide an indication of metabolic activity, it is necessary to determine the ratio between the amounts of CO2 produced (Rx) over the amount of O2 consumed (Ry) by the plant material. This ratio is referred to as the respiratory quotient (RQ) (Plasse, 1986; Saltveit, 2005).
RQ = Rx CO2 produced = Ry O2 consumed

( 3.3 )

Where: Rx Ry = = CO2 produced, %; O2 consumed, %.

Both must be given in the same units, either moles or volume of gas, at the same temperature and pressure. With regards to the substrate being oxidized, RQ values for fresh commodities may range from 0.7 to 1.3 for aerobic respiration (Saltveit, 2005). If the

16 respiratory process is normal and sugars are metabolized, RQ should be equal to one. RQ values greater than one indicates that the organism is burning carbohydrates to produce fat or there is oxygenated substrate utilization in respiration, such as organic acids (proteins). A RQ value less than one may indicate several possible situations, but it generally indicates that the oxidation reaction is not complete (Plasse, 1986) or that the lipids (fats) are aerobically respired (Saltveit, 2005). A very high value of RQ would indicate an anaerobic process (Saltveit, 2005). 3.2.3. RELATIONSHIP METABOLISM Metabolic activity is an important factor to determine the rate of deterioration of the harvested fruit or vegetable. As metabolic activity increases the physiological state of the tissues changes and may accelerate senescence and ripening. Reducing the respiration rate to the minimum that still permits normal cellular function will delay ripening and increase the produces shelf life (Kader, 2002). 3.2.4. ENVIRONMENTAL FACTORS AFFECTING RESPIRATION RATE AND RESPIRATION QUOTIENT There are many factors affecting respiration including light, chemicals, radiation, water, growth regulators and pathogens. But the most important factors to consider are temperature, atmospheric composition and physical stress (Saltveit, 2005). Temperature is the leading factor because it has a profound effect on the rates of biological reactions in the commodities. The rate at which the respiration process takes place is directly related to temperature: the higher the temperature the higher the respiration rate. Its effect leads to overactive respiration at high temperatures causing phytotoxic symptoms and even complete tissue collapse (Saltveit, 2005). On the other hand, it may induce metabolic disturbance, even BETWEEN RESPIRATION RATE AND

RESPIRATION QUOTIENT AND THEIR EFFECT ON PRODUCT

17 physiological injury, when the temperature of storage is too low (Plasse, 1986). Chilling stress may induce dramatic increases in respiration rate as the commodity is returned to a non-chilling temperature. It reflects the cells efforts to detoxify the metabolism and repair damages to membranes and other subcellular structures (Saltveit, 2005). Many conservation techniques rely on the low availability of oxygen in the atmosphere to reduce the metabolic activity, as reflected by a reduction in starch degradation and sugar consumption, of the stored produce (Plasse, 1986). Most fruits and vegetables respond to a reduced oxygen concentration. The primary metabolic response to low O2, between 1 and 3 kPa, is a general metabolic suppression through the inhibition of respiration. The secondary metabolic response to low O2, around 6 kPa, is the suppression of ripening through the inhibition of ethylene action (Mir and Beaudry, 2001). Even if the produce metabolism responds to low O2, reduced O2 has not been widely used for storage of fruits which are highly susceptible to decay, like tomato and blueberry. Low O2 atmospheres are limited by the development of decay since O2 partial pressures have little effect on decay organisms (Mir and Beaudry, 2001). Increasing the CO2 level around commodities also reduces respiration, delays senescence and retards fungal growth (Saltveit, 2005). The effect of CO2 on respiration relies on the inhibition of some enzymatic activities and the decrease in the synthetic reactions of ripening (Plasse, 1986). Carbon dioxide is a soluble gas. As its concentration increases in the storage atmosphere, the quantity dissolved or combined with other constituents also increases. The effect that these changes in gas concentration have on the respiration rate differs between different types of produce (Plasse, 1986). For example, the respiration rate of tomato does not slow down until the CO2 concentration in the atmosphere gets up to 9%. Also, the oxygen level can decrease to 12% without having any effect on metabolic activity of the tomato (Henig and Gilbert, 1975).

18 Physical stresses are indirect, secondary factors, which have an important impact on respiration and may cause a substantial rise in respiration rate, often associated with an increased ethylene evolution (Saltveit, 2005). These factors stimulate respiration in an indirect way and their effects are not readily observable. Physical stresses are encountered prior to storage, such as water stress, heat stress, shortage or excesses in nutrients, and other preharvest horticultural practices. Those factors can induce physiological or pathological disorders to the stored commodity, such as the inhibition or the promotion of senescence or a decrease in the rate of degradation (Fennir, 1997). 3.2.5. RQ VALUES AND THEIR SIGNIFICANCE The respiration rate and respiration quotient are very closely related. When the respiration process is normal and sugars are metabolized, the commodity undergoes robic respiration. For example, RQ value recorded in an open steady state system with atmospheric oxygen levels would present a fairly stable RQ curve, slightly rising over time, as the commodity consumes its carbohydrates. Initial values of RQ should range between 0.8 and 0.9 and can rise to values up to 1 within hours. But these values are dependent on the principal substrate that the plant is using in the respiratory process and can range from 0.8 to 1.33 with fats or organic acids, respectively (Lencki et al., 2004). At some point, the commodity will have used all of its resources and can no longer consume oxygen. Oxygen available in the commodity will drop below the critical value and induce fermentation. The respiration process will become anrobic and starts producing more carbon dioxide. From that moment, RQ values present a drastic change and start going up. This transition zone between robic and anrobic respiratory processes is referred to as the extinction point (EP). This term was first proposed by Thomas and Fidler (1933). It was also considered by Turner (1951) as the lowest concentration of oxygen at which the RQ remains about 1. The definition was further redefined as the lowest concentration of oxygen at which alcohol production ceases (Kubo et al., 1996). From the EP, carbon dioxide production increases and ethanol accumulates, inducing off-

19 flavours and tissue breakdown in the commodity. Previous results have demonstrated that recording the oxygen and the carbon dioxide concentration in a storage facility provides information on RQ and allows the determination of the EP (Kubo et al., 1996). RQ values are rarely measured in experiments (Lencki, 2004); however, the extinction point, associated to RQ, is the most efficient way to determine the transition between robic and anrobic respiration and predict how long the commodity can be stored under specific conditions before irreversible physiological damage is observed (Kubo et al., 1996). Beaudry (1993) presented values of RQ based on different partial pressures of oxygen and carbon dioxide on blueberry (Vaccinium sp.) fruit. At retail temperature, 15C, RQ values increased at CO2 partial pressure above 20 kPa. Partial pressures between 15 and 20 kPa enhanced storability, but those above 25 kPa were injurious. Beaudry (1993) presented the EP as the breakpoint or the O2 lower limit where the oxygen partial pressure causes a 20% change in the RQ relative to the aerobic RQ. RQ values between 0.85 and 1.10 were maintained for periods up to 30 days. Large swings in RQ are not typical and should be examined more closely and questioned (Lencki, 2004). 3.2.6. METHODS TO MEASURE RESPIRATION RATE AND RQ Different techniques are available to determine respiration rate. It can be evaluated by measuring one or many of the following constituents: water production, loss of substrate, loss of O2, increase in CO2 concentration or the production of heat (Saltveit, 2005). The most common method is to measure the CO2 released and the O2 uptake with either a static (closed chamber or closed loop) or a dynamic system (open chamber or open loop). The static system consists of placing commodities in a sealed container and measuring the CO2 increase in time. The dynamic system allows a flow of air to pass through the commodities at a known rate. After the system reaches equilibrium, the difference in gas concentration between the inlet and the outlet is measured. The

20 production rate is calculated by multiplying the difference in concentration by the flow rate and dividing by the total weight of the commodity (Saltveit, 2005). The dynamic system has an advantage over the static system since there are no effects of gas accumulation. An open steady-state system is independent of the loading and the produce pH as opposed to an unsteady-state closed chamber (Lencki, 2004). RQ is simply obtained by dividing RRco2 by RRo2. 3.3. PHYSICAL TREATMENT 3.3.1. HEAT Mild pre-storage heat treatment has been used for insect control and has been proven effective against many storage diseases and physiological disorders (Terry and Joyce, 2004), and shown to improve the eating qualities of stored fruits and vegetables (Mulas and Schirra, 2007), and even to protect certain phytochemicals like the red colour pigments in postharvest tomatoes, melons and mangoes (Fallik, 2004). Heat treatments can be applied through vapour heat, hot water dipping, or very short water rinse and brushing (Terry and Joyce, 2004). 3.3.1.1. THERMOTOLERANCE Studies on the thermotolerance of different fruits are summarised in Lu et al. (2007). Heat treatments enable fruits and vegetables to develop resistance to injuries caused by low-temperatures. Tomatoes submitted to 38C air for 2-3 days were then stored for up to a month at 2C without developing chilling injuries (Lurie and Sabehat, 1997). This beneficial effect was also observed on pomegranate (Punica granatum L.), peach (Prunus persica (L.) Batsch), orange (Citrus sinensis (L.) Osbeck) and avocado (Persea sp.) (Lu et al., 2007). On the other hand, exposure to inappropriate heat can cause damage. Tomatoes were found to be very sensitive to temperatures over 38C. Treatment with a temperature of 42 or 46C for 24 hours caused both external and internal damage to the fruit (Lurie and Sabehat, 1997). To be beneficial, heat treatment needs to be well understood with respect to the heat-damage tolerance of the

21 species which could change within 1 to 2C, the cultivar, the harvest maturity, the growing conditions and handling methods (Lu et al., 2007). 3.3.1.2. EFFECTS ON DISINFECTION AGAINST HUMAN PATHOGENS In the last couple of years, cantaloupes and melons (vars. of Cucumis melo), leafy vegetables and tomatoes were largely linked to foodborne illness in North America (Delaquis and Austin, 2007). Most of the outbreaks were due to contamination with human pathogens like Salmonella, E. coli and Listeria. Thermal treatments directed at the surface of the produce were an attempt to properly eradicate any pathogen on the produce surface. 3.3.1.2.1. CANTALOUPE AND MELONS The immersion of cantaloupes in water at 76C for 2-3 min did reduce Salmonella enterica but did not completely eradicate inocula (Annous et al., 2004). The quality attributes of the fruits were not adversely affected and fungal decay rates and overall microbial populations were lowered. Immersion in water at temperatures up to 96C for 2 min were also tested and compared to untreated fruits to determine the efficacy of heat as a pasteurizing treatment (Ukuku, 2006). In that particular study, Salmonella grew faster on the cantaloupes that had received the heat treatment. It was concluded that sanitized produce are susceptible to recontamination if exposed to human bacterial pathogens during subsequent handling.

3.3.1.2.2. LEAFY VEGETABLES Heat treatments can help control and delay the appearance of quality defects that limit the shelf-life of fresh-cut lettuce. Immersion in chlorinated solution in the range of 47 to 50C inhibits phenylpropanoid metabolism and delay the appearance of edge-browning (Delaquis et al., 2004). However, pathogens have been found to grow at a faster rate on heat-treated compared to conventionally-

22 processed fresh-cut lettuce (Li et al., 2001; Delaquis et al., 2002). The same phenomenon was reported on broccoli (Brassica oleracea L., Italica group) florets and cut green beans (Phaseolus vulgaris L.) that have been immersed in water at 52C for 90 seconds prior to packaging (Stringer et al., 2007). 3.3.1.2.3. TOMATOES Tomatoes are prone to human pathogen contamination during production, from the planting, flowering, to the harvesting period. It was recognised that Salmonella can survive long periods on the fruit and plant surfaces. Bacterial infiltration can occur through the stem or flower prior to harvest (Guo et al., 2001), and post harvest through stem, scar and abrasion or puncture injuries of the thin skin (Yuk et al., 2007). Contamination can also occur through the stem scar by the immersion of warm fruits in water of lower temperature (Wei et al., 1995). Heat treatments at 50C and up to 60C for short periods were tested on fresh tomatoes. The treatments did improve the storage stability of tomatoes but were marginally effective against human pathogens E. coli or Salmonella spp. (Delaquis and Austin, 2007). Heat treatments seem inappropriate for the disinfection of fresh fruits and vegetables to be stored before being processed or sent to the consumer market, and for fresh-cut produce prior to packaging (Delaquis and Austin, 2007). Even though heat treatment has been proven to be beneficial in terms of crops physiology and to be efficient on the control of some insect and fungal invasions, the non-uniformity and slow rate of heat transfer through the fruit or vegetable, by hot air or water, are probably the major obstacles to the industrialisation of heat treatments (Lu et al., 2006). 3.3.2. UV-C Short-wave ultra violet light, UV-C ranging between 190 and 280 nm wavelength, have been used as physical methods to control postharvest diseases (Wilson et al., 1997). The most effective wavelength to produce a germicidal effect is approximately 260 nm (Shama, 2005). Although high dosage of UV light is

23 generally harmful to living systems, low doses can induce disease resistance, slow down ripening and improve quality attributes of horticultural crops (Charles and Arul, 2007). As heat shock or other physical stress, UV light alters the chemistry of plants and in some cases enhances the nutraceutical potential in the plant (Shama and Alderson, 2005). Studies have reported changes in the physiological compounds of the irradiated fruits as it increased their levels of phenols, flavonoids and phytoalexins (Ben-Yehoshua, 2003). 3.3.2.1. EFFECTS ON DISEASE Effective control of several pathogens was achieved by low-doses of UV irradiation prior to storage. Low dosage of irradiation, ranging from 0.25 to 8.0 kJ m-2, has been found to control many storage rots (Terry and Joyce, 2004). However, UV light is only effective for disinfection of the surface and it has very low penetrability into the solid material. Surfaces are to be smooth and exempt of impurities (Shama, 2005). From most studies, it is recognised that the reduction of disease incidence in the UV-C treated commodities is due to the enhancement of the natural resistance of the product (Charles and Arul, 2007). Germicidal effect of UV-C light is essentially limited to the time of exposure to the UV source ranging from fraction of seconds to tens of seconds. It is considered a direct effect. The hormetic effect of UV-C treatment occurs after exposure for germicidal treatment, at periods of time ranging from hours to days (Shama, 2007). 3.3.2.2. HORMESIS EFFECTS IMPROVEMENTS OF QUALITY ATTRIBUTES 3.3.2.2.1. POSITIVE CHANGES Hormetic UV doses range from 0.125 to 9 kJ m-, with respect to the crop variety. For most commodities, a single dose at low dosage is sufficient but others require a range of doses. UV-C light treatment improved firmness of strawberry, peach, apple (Malus domestica Borkh.), peppers (Capsicum annuum L.) and tomato (Charles and Arul, 2007) and it delayed the colour changes of peppers

24 (Vincente et al., 2005), broccoli (Costa et al., 2006), and tomato (Charles and Arul, 2007). All these attributes contribute to an increase in shelf-life. UV-C also has an impact on the respiration pattern of some fruits and vegetables. Tomato ripening and senescence were delayed by a hormic dose of UV-C (200-280 nm, 3.7 kJ m-) but a higher dosage (24.4 kJ m-2) impaired ripening and caused abnormalities in fruit development (Maharaj et al., 1999). In addition, the respiration rate and the ethylene production were also reduced. UV also induced rapid accumulation of photooxidation products as the plants react by stimulating their defence mechanisms against oxidation (Shama and Alderson, 2005). It showed particularly promising results in increasing the resveratrol content in table grape (Vitis vinifera L.) (Cantos et al., 2001). 3.3.2.2.2. ADVERSE EFFECTS UV dosage induced skin discolouration in tomatoes, browning and drying in strawberries (Fragaria ananassa Duchesne) and mangoes (Mangifera indica L.), brown rot in peaches, premature ripening of mangoes and a prolonged exposure of tomatoes accelerated ripening and senescence (Shama and Alderson, 2005). UV rays are also known to destroy vitamins C and B but enhance vitamin D production. They cause oxidative deterioration of oils and fats leading to rancidity and cause browning of some vegetables (Shama, 2005). Further, UV exposure had a negative impact on carotenoids, especially lycopene, in tomatoes (Charles and Arul, 2007; Jagadeesh et al., 2009) and pepper (Vincente et al., 2005). Considering these results, UV-C might not be a valuable treatment to enhance the beneficial properties of tomatoes, as lycopene is a highly valued phytochemical mostly present in tomatoes. 3.3.2.3. EFFICACY OF UV TREATMENT UV-C treatment needs to be adapted to a particular commodity, after thorough investigation. Each variety, with respect to the time of harvest and the intended target, needs a specific treatment length and intensity. It is therefore important to determine the appropriate dosage to induce protection and not deteriorate fresh

25 produce (Shama, 2007). Further, UV-C treatment is not a systemic treatment and its efficacy is very much dependent on the geometry and skin type of the commodity (Charles and Arul, 2007). Disease resistance will only be induced in tissue directly exposed to rays (Shama, 2007). Some produce are very difficult to treat, like grapes, as the exterior will get an overdose before the inside gets the needed dosage. One should also consider, prior to commercialisation of any system, the danger that UV-C rays exposure represent on human health and be aware that special considerations have to be given to protect workers (Shama, 2007). 3.3.3. PRESSURE Pressure treatment is one of the techniques that can meet the consumer demand in aiding the supply of high quality foods that are not genetically modified or irradiated (Cardello, 2003) and are microbiologically safe with an extended shelflife (Patterson, 2005). Pressure treatment consists of applying pressure beyond atmospheric pressure to fresh or processed foods (Anon., 2000; Ahmed and Ramaswamy, 2006). Exposure to the pressure can range from a millisecond pulse to a treatment time of over 1200 s (Anon., 2000). These treatments offer homogeneity as they act instantaneously and uniformly throughout the entire mass of food, independently of its size, shape or composition (Ahmed and Ramaswamy 2006). Considering the large scale of pressures applied to produce, pressure treatments need to be categorized (Fig. 3.1). Treatments can be divided into two categories: low pressure treatment that can be hypobaric and/or hyperbaric, and high pressure. Low pressure treatment (0 to 1 MPa) is applied to fresh produce and high pressure (above 100 MPa) is generally applied to processed food. In the range of 1 MPa and 100 MPa, pressure might be too high to treat pressure sensitive fresh horticultural crops without damaging them, and too low to have a significant effect on microorganisms reduction and enzymes inactivation.

26

Pressure Treatment Hypobaric 0-0.1 MPa Hyperbaric 0.1-1 MPa High > 100 MPa Processed food Pressure

Fresh horticultural crop

Absolute zero pressure

Atmospheric pressure

Figure 3.1 : Representation of pressure range treatment and the type of produce on which the treatment can be applied.

27 3.3.3.1. HIGH PRESSURE PROCESSING (HPP) High pressure processing (HPP), also referred to as high hydrostatic pressure (HHP) or ultra high pressure (UHP) processing, consists of subjecting liquid and solid foods, with or without packaging, to pressures between 100 and 800 MPa (Anon., 2000), and even up to 1200 MPa (Ahmed and Ramaswamy, 2006). Exposure to the pressure can range from a millisecond pulse to a treatment time of over 1200 s (20 min) (Anon., 2000). There is an increase in interest around the world in the application of HPP treatment for food because of the advantages of this technology over other processing and preservation methods. Until now, thermal processing was the most widely used technology since it allows efficient inactivation of both pathogenic and spoilage microorganisms. Unfortunately, these treatments alter organoleptic and nutritional qualities of the food (Ludikhuyze et al., 2003). Many studies have demonstrated that HPP can (1) significantly or totally inactivate micro organisms and (2) increase functional and nutritional retention of ingredients (Estrada-Girn et al., 2005). High pressure processing offers many advantages but the main drawback is the high capital cost of the commercialscale equipment (Ahmed and Ramaswamy, 2006). 3.3.3.1.1. EFFECTS ON PATHOGEN High pressure processing has been used in the production of processed fruits and vegetables primarily to reduce micro-organisms and inactivate enzymes that would, upon storage, cause deterioration of the product or endanger consumers health. Yeasts, moulds and vegetative cells are pressure sensitive and can be inactivated by treatments between 300 and 600 MPa. On the other hand, bacterial spores are highly resistant to pressure treatment and over 1200 MPa is necessary for their inactivation. (Garcia and Barrett, 2002). Bacterial spores, because of their extreme resistance to pressure, are sometimes very difficult to control by a unique pressure treatment (Ludikhuyze et al., 2003).

28 It was demonstrated that HPP treatment can inactivate yeasts (Saccharomyces cerevisiae and Listeria innocua) on diced apples, and whole processed blueberries, strawberries and grapes (Chauvin et al., 2005). No significant effect was observed between pressures of 300 MPa for 20 to 100 s and 375 MPa for 30 to 180 s at 21C. Both treatments were suitable to inactivate the micro organisms and preserve the fresh appearance and texture of the fruits. Vegetative cells were reduced by six-fold in apple juice using a pressure of 200 MPa for 60 min or 300 MPa for 5 min (Voldrich et al., 2004). High pressure of 600 MPa at 20C was also applied to orange juice to reduce microbial load to a nondetectable level after a 4 week period of storage (Bull et al., 2004). 3.3.3.1.2. EFFECTS ON ENZYMES Many studies tested the efficiency of HPP treatment on the control of enzymes and pathogenic organisms in fresh fruits and vegetables and fresh fruit juices. Natural enzymes in fruits and vegetables cause changes in colour, flavour, texture and nutritive value; thus enzymatic reactions are a major problem. Some enzymes can be inactivated at room temperature and low pressures whereas others can withstand high temperatures and pressures (Ahmed and Ramaswamy, 2006). Heat treatments (high temperature and ultra high temperature), have been widely used to kill pathogenic micro organisms, inactivate anti-nutritional substances, promote certain sensory properties and increase storage life (Wennberg and Nyman, 2004). But high temperatures are also partly responsible for the loss of nutrients, such as minerals and vitamins, and the formation of off-flavours and degradation of colour and texture (Lambert et al. 1999; Wennberg and Nyman, 2004). In some cases, HPP treatments have been combined with thermal treatment of temperature ranging from below 0C to 100C, over different lengths of time and varying storage periods. This combination of treatment has achieved an increased rate of inactivation of enzymes and pathogens while maintaining the beneficial properties of the food. A pressure treatment of 450 MPa at 50C for 30 to 60 min, completely inactivated enzymes in apple and orange juice, even

29 upon storage (Bayindirli et al., 2006). Similar results were obtained with 500 MPa at 50C in orange juice (Nienaber and Shellhammer, 2001) and 600 MPa at 60C in apple juice (Voldrich et al., 2004). Bull et al. (2004) used a pressure of 600 MPa at 85C for 25 s (pulsed treatment). This treatment reduced enzymes significantly and did not affect the orange juice quality parameters such as viscosity, browning and color. Ludikhuyze et al. (2003) reviewed studies on enzymes inactivation by pressure ranging from 0.1 to 1000 MPa, and/or temperature ranging from -20C to 100C, and proposed a model to optimise pressure-temperature processes in future studies. 3.3.3.1.3. EFFECTS ON PHYSICAL PROPERTIES AND QUALITY OF PROCESSED FRUITS AND VEGETABLES High pressure processing has been investigated for its ability to promote sensory and physical properties of fruits and vegetables compared to heat treatment. The aromatic properties of premium strawberry coulis were evaluated under heattreatment and HPP compared to the raw strawberry (Lambert et al., 1999). No significant changes in the aromatic compounds were noticed after HPP treatments of 200 and 500 MPa at 20C for 20 min. On the other hand, changes appeared after sterilisation, 800 MPa at 20C for 20 min followed by 120C for 20 min. Suthanthangjai et al. (2005) assessed the impact of HPP at room temperature on the colour pigments in raspberry (Rubus idaeus L.) puree after different storage periods. The highest stability for a 9 day storage period was found with pressure treatments of 200 and 800 MPa stored at 4C and the lowest stability was found under the 400 and the 600 MPa treatments stored at 30C. 3.3.3.2. HPP COMBINED WITH MILD THERMAL TREATMENT The use of HPP as pre-treatment to thermal processing (mild heat treatment) was also found to be beneficial in reducing the texture degradation of chopped carrots (Daucus carota L.). Pressures from 200 to 500 MPa at temperatures of 20, 40 and 60C were compared. All pressures associated with the highest temperature (60C) improved significantly carrot texture (Sila et al., 2004). On the

30 other hand, tests were performed comparing HPP with fresh, frozen, steam blanched carrots for tissue damage. Only pressures of 100 MPa applied for 2 min did not damage carrot tissue (Araya et al., 2004). Chopped cabbages (Brassica oleracea L., Capitata group) were subjected to HPP treatments of 400 and 500 MPa at 20, 50 and 80C. All treatments were efficient to reduce solubilisation of dietary fibre (Wennberg and Nyman, 2004). HPP of 600 MPa at 25C, induced changes in physico-chemical properties and did not induce loss of beneficial substances on fresh crushed or liquid extracts of carrots, tomatoes and broccoli (Butz et al., 2002), apples, peaches, tomatoes, frozen berries and mixed juices (Butz et al., 2003). HPP resulted in a better retention of flavour and superior sensory characteristics in orange juice treated at 600 MPa and 40C compared to thermal pasteurisation (Polydera et al., 2005). The effect of conventional sterilisation of tomato puree on lycopene content was compared to that of HPS (high pressure sterilisation) treatment. Pressures of 700 MPa at temperature of 90C for 30 s improved colour and retained lycopene content compared to a 40% loss after conventional sterilisation (Krebbers et al., 2003). Tomato puree was compared in terms of lycopene stability to a solution made of artificial lycopene (in hexane) when these were subjected to different HPP treatments and storage temperatures. The lycopene solution did not retain lycopene content under all treatments. The maximum stability of lycopene content was obtained when tomato puree was pressurised at 500 MPa and stored at 4C. High storage temperature (24C) resulted in the higher loss of lycopene (Qiu et al., 2006). 3.3.3.3. HPP COMBINED WITH LOW-TEMPERATURE TREATMENT There is also a potential for the pressure-supported freezing and thawing of fruits and vegetables. High pressure - low temperature processing (250 MPa at -27C) was used to determine the effect on cell membrane, texture, colour and visual appearance of potato (Solanum tuberosum L.) tissue, compared to freezing alone. Results after thawing showed that a considerable improvement was

31 obtained using a HPP-low temperature combination treatment compared to conventional freezing (Luscher et al., 2005). 3.3.3.3.1. EFFECTS ON RESPIRATION RATE OF FRUITS AND VEGETABLES UPON STORAGE The basis of all post harvest treatment is to decrease respiration rate to increase shelf life and maintain produce quality. Eggleston and Tanner (2005) tested the effect of HPP on respiration rate of carrots sticks. Pressure of 600 MPa was applied for periods of 2, 5, and 10 minutes. In this study, respiration rate decrease was observed as treatment duration increased. However, postharvest life of fresh mume (Prunus mume Siebold & Zucc.) fruits was evaluated based on the ethylene production after pressurisation (5 to 200 MPa) and substantial physiological injuries were observed at pressures greater than 5 MPa (Baba et al., 1999). Deterioration and unacceptable commercial quality was observed at pressures of 100 MPa and higher. Also, ethylene production decreased after all pressure treatments. Based on ethylene reduction, a second study evaluated the effect of low pressure on the same fruit. Mume fruits were subjected to pressures of 0.5 to 5 MPa for 10 minutes and maintained at 0.5 MPa for 5 days (Baba and Ikeda, 2003). Treatment of 0.5 MPa decreased CO2 and ethylene production during storage, decreased weight loss, and showed some benefits against chilling injuries. These new processes often result in heterogeneous interpretation and terminology. Benet et al. (2004) presented a paper with definitions and standardized nomenclature for the different pressure and low temperature combinations. Luscher et al. (2005) used these terminologies to identify the treatments they applied to improve the preservation of potato products. 3.3.3.4. HYPOBARIC AND HYPERBARIC PRESSURE TREATMENT 3.3.3.4.1. HYPOBARIC PRESSURE TREATMENT Novel methods for controlling postharvest rots and improve food properties include biocontrol agents, salts, natural substances and physical treatments

32 (Romanazzi et al., 2001). Among the physical means for controlling postharvest decay of fruits and vegetables, the use of sub-atmospheric pressure treatments has been reported to delay ripening of some climacteric fruits (Burg and Burg, 1966) and increase shelf life (Apelbaum et al., 1977). Sub-atmospheric pressure, also called hypobaric storage, is an emerging postharvest storage technique which can rapidly remove the heat, reduce the oxygen level and expel harmful gases over time during storage (Wang et al., 2001). Hypobaric storage can adjust the inside temperature and composition of the atmosphere reliably and consistently (Li et al., 2006). 3.3.3.4.2. EFFECTS ON PATHOGENS AND DISEASES Postharvest physiological disorders and diseases can be induced during storage by poor environmental conditions and/or by wound on the fruit or vegetable. Mould and rot production during storage decrease the market value of the product. Hypobaric pressure of 0.005 MPa used as a post storage treatment was compared to air storage alone to reduce scald and decrease production of oxidative product in fresh apples after storage for 0.5 to 6 months (Wang and Dilley, 2000). Apples subjected to hypobaric conditions after one month of storage did not develop scald. Hypobaric storage at 0.040 to 0.050 MPa reduced decay of loquat (Eriobotrya japonica (Thunb.) Lindl.) fruits by 87% compared to a control stored at 2C (Gao et al., 2006). Pressures of 0.050 MPa applied for 4 hours were the most effective treatment against mould and rots on sweet cherries (Prunus avium L.) and strawberries, as 0.025 MPa for 24 hours decreased the incidence of mould on table grape (Romanazzi et al., 2001). Table grapes were also wounded and inoculated after hypobaric treatment and it was determined that hypobaric decreased infection and diameter of lesions significantly compared to the untreated control fruits. The use of a fungicidal chitosan (crab-shell chitosan) coating prior to storage is also a known technique to prevent decay of fruits and vegetables. Differences

33 between fungicide treatment, hypobaric pressure and a mix of both treatments were tested on sweet cherries prior to storage to control the production of mould and rots. Pressures of 0.025 and 0.050 MPa for 4 hours were applied prior to 14 days of storage at 0C followed by 7 days of shelf life. The fungicide treatment was done by dipping the cherries into a 1% chitosan solution prior to storage and shelf life studies. Both independent treatments significantly reduced mould and rots. The combined treatment was the most effective in controlling decay (Romanazzi et al., 2003). 3.3.3.4.3. EFFECTS ON FRUITS AND VEGETABLES CONSERVATION For different fruits and vegetables, hypobaric storage has been compared to refrigerated storage and controlled atmosphere storage with respect to its ability to increase shelf life. Pressures less than 0.1 MPa seem to increase shelf life but desiccation seems to be a consequence of hypobaric pressure storage in many cases. Apelbaum et al. (1977) tested the effect of hypobaric pressure storage on mango fruits. It was observed that at pressure below 0.05 MPa, mangoes undergo desiccation and even upon ripening, did not develop the proper red and orange color. However, fruits submitted to pressures of 0.013 and 0.01 MPa had an extended shelf life of 25 and 35 days respectively. Fresh green asparagus (Asparagus officinalis L.) were subjected to hypobaric pressure of 0.035 to 0.045 MPa. Asparagus were sprayed with water upon storage to prevent desiccation. Their storage life was extended to 50 days compared to 25 days under refrigerated storage and 6 days under room temperature. Furthermore, hypobaric treatment significantly inhibited respiration, prevented loss of chlorophyll, vitamin C and acidity, improved sensory qualities and delayed postharvest senescence (Li et al., 2006). Similar results were observed on loquat fruits (Gao et al., 2006) in addition to a significant decrease in enzyme activity and flesh leatheriness.

34 Cucumbers (Cucumis sativus L.) were placed at 0.07 MPa for 6 hours in the dark, to simulate air flight transportation, and placed subsequently in cold storage facilities at 0.1 MPa. Results showed that cucumbers submitted to the hypobaric simulation had significantly more open stomata, compared to the control, after 96 hours of subsequent storage (Laurin et al., 2006). Water can be sprayed to resolve the problem of insufficient relative humidity, causing desiccation during hypobaric storage. It is likely that desiccation experienced by fresh fruit and vegetable subjected to hypobaric conditions is due to an increase in transpiration rate enhanced by the properties and action of stomata (Laurin et al., 2006), lenticels, cuticle and epidermal cells (Ben-Yehoshua and Rodov, 2003). 3.3.3.4.4. HYPERBARIC PRESSURE TREATMENT Hyperbaric atmosphere, or high O2 atmosphere, or superatmospheric O2 concentration, represents an environment with an oxygen concentration greater that 21 kPa around and within the commodity (Kader and Ben-Yehoshua, 2000). This definition is also similar in the medical field where hyperbaric oxygen therapy is defined as the inhalation of oxygen at increased pressure (Gupta et al., 2005). Hyperbaric treatments have been confused with high pressure processing. But as pressure treatments have been defined as the processing of fruits and vegetables under high air pressure, the definitions of hyperbaric atmosphere do not fulfill the criteria of pressure treatments. In the case of high oxygen atmosphere, the proportion of gas in the air is altered for the benefit of oxygen only, but in the HPP treatments, the proportion of each gas in the air is maintained. 3.3.3.4.5. EFFECTS ON FRUITS AND VEGETABLES QUALITY Apelbaum et al. (1977) tested the effect of hyperbaric pressure storage on mango fruits. Pressures, from 0.25 to 0.7 MPa, did not result in a shelf life any greater than commercial storage (16 days). Mume fruits were subjected to pressures of 0.5 to 5 MPa for 10 minutes and maintained at 0.5 MPa for 5 days (Baba and Ikeda, 2003). Treatment of 0.5 MPa decreased CO2 and ethylene

35 production during storage, decreased weight loss, and showed benefits against chilling injuries. However, although results from these two studies are encouraging, very little information is available on the effect of hyperbaric pressure treatment of fresh fruits and vegetables during postharvest handling and preservation. 3.3.3.5. BENEFICIAL SUBSTANCES AND FUNCTIONAL PROPERTIES OF FRUITS AND VEGETABLES INDUCED BY PRESSURE TREATMENT During the last decade, physical treatments used to improve preservation of biological products and promote improvement of nutritional and functional properties of fruits and vegetables have been investigated (Wennberg and Nyman, 2004; Butz et al., 2002). Non-thermal treatment seems to be an attractive alternative compared to conventional processing methods for the retention of vitamins and other low molecular food components, as well as flavour and taste. The nutritional effects can be related to the physico-chemical properties of the fruit or vegetable matrices. For example, dietary fibres are associated with a number of positive physiological effects in the human body. But when the food is subjected to an increased temperature, dietary fibres will undergo solubilisation or extensive degradation, representing a loss of beneficial effect (Wennberg and Nyman, 2004). Also, some substances responsible for physical properties of fruits and vegetables have health promoting effects. For example, lycopene is the predominant carotenoid found in tomatoes and is responsible for the redness of ripe tomatoes and tomato products. It is also one of the major carotenoids present in human serum and organs and has been shown to be associated with decreased risk of chronic diseases. But this substance is greatly affected by the actual sterilisation process of tomato products (Qui et al., 2006).

36 3.4. TOMATO Tomato is a fruit widely consumed in many countries around the world and its consumption is predicted to grow in the future. Improving the beneficial properties of tomatoes could represent a global improvement of the human health (Hanson et al., 2004). 3.4.1. PHYSIOLOGY OF TOMATO 3.4.1.1. COMPOSITION Total solids and water make up the composition of a tomato. Generally, a tomato is about 95% water and 5 to 7.5% dry matter with about 1% in cuticle and seeds and 4 to 6% in soluble solids. In ripened tomatoes, reducing sugars, such as glucose and fructose, represent 50% of the dry matter content while proteins, pectin, cellulose, hemicellulose, organic acids, minerals, pigments, vitamins and lipids represent the other 50%. Minerals account for 8% of the dry matter (Dorais et al., 2001) while acids (citric and malic) account for 15% (Cox, 2001). There is usually an inverse relation between yield and total solids content (Dorais et al., 2001). Tomato is the 16th among all fruits and vegetables as a source of vitamin A and 13th in vitamin C. It contains significant amounts of lycopene, -carotene, magnesium, niacin, iron, phosphorus, potassium, riboflavin, sodium and thiamine (Cox, 2001). It also contains small quantities of selenium, copper and zinc as well as folates, fibers, various flavonoids and phenolic acids (Astley and Elliott, 2005). 3.4.1.2. CHARACTERISTICS Tomato is a climacteric fruit and is considered a chilling-sensitive commodity (Kader, 2002). The optimum temperature for tomato ripening is between 20 and 25C. The ideal storage and transit temperature ranges between 10 and 15C. Temperatures over 30C and below 10C induce temperature injuries. The optimal air quality for storage conditions should be 90-95% humidity, 3-5%

37 oxygen, and 0-3% carbon dioxide (Cox, 2001). The respiration rate of tomato is considered to be moderate with a production of 10-20 mg CO2 kg-1 h-1. Some compositional changes in produce may continue to occur after harvest. They may be desirable or undesirable and their impact may be different considering whether it is a fruit or a vegetable. The development of pigment with maturation is a good example; the loss of chlorophyll is desirable in fruits but not in vegetables and the development of carotenoids and anthocyanins is favourable in fruits. But some changes are not as easy to classify. Changes in anthocyanins and other phenolic compounds may provoke some browning of the tissues which is undesirable for quality appearances but contributes to the total antioxidant capacity of the commodity and is beneficial to human health (Kader, 2002). 3.4.2. QUALITY PARAMETERS Quality is a combination of attributes of a fruit or vegetable that determines its degree of acceptability by the user and its value or worth. Physical features are used to assess the quality of various fruits and vegetables. Size, shape and surface characteristics are widely used to evaluate the marketable value of harvested commodities. Some characteristics, such as color and texture, might be visible to a naked eye but require expensive equipment for proper evaluation. Parameters to note chemical changes of the fruits and vegetables are most difficult to assess as they imply profound changes in the composition of the produce. They are evaluated by destructive sampling and chemical analysis, e.g. total soluble solids or sugar to acid ratio. The relative importance of each factor depends upon the commodity and its intended use (e.g. fresh market vs. processing). Many parameters influence the composition and quality of fresh commodities: growing environment, maturity at harvest, harvesting method and postharvest procedures.

38 3.4.2.1. MATURITY The maturity at harvest is the most important factor determining quality and storage life of the commodity. Maturity and ripening stages of tomatoes are presented in Fig. 3.2. A fruit harvested fully ripe had time to develop all its eating qualities. However, it will generally not survive postharvest handling and storage. Fruits have to be harvested mature but not ripe. To withstand some storage and handling steps, tomatoes are harvested green and mature. This stage is called the near breaker stage. Horticultural crops are classified according to their perishability and potential storage life preserved at optimal temperature and RH (Kader, 2002). Perishability ranges from very high to very low. Tomatoes harvested partially ripe (green and mature) are considered to have a high perishability index with a potential storage life of 2-4 weeks. Tomatoes harvested ripe have even higher perishability index with a potential storage life less than 2 weeks. 3.4.2.2. COLOR Color is an important factor associated with the evaluation of all food products as it is the first impression provided to consumers. In tomates, the degree of color represents an important figure of the total quality of the fruit. Chlorophyll pigments are responsible for the green color in tomatoes. As tomato starts to ripen, chlorophyll is degraded and carotenoids are synthesized (Arias et al., 2000). Their concentration increases with maturity causing the development of the red color. The most abundant carotenoid in tomato is lycopene which represents approximately 83% of the pigments of the fruit. Fruit picked green and ripened in storage are very much lower in carotene than vine-ripened fruit (Gould, 1992). Carotenoids are important not only because of the color they impart but also for their recognized benefits on human health.

39

GREEN The tomato surface is completely green.

PINK Pink or red color shows on over 30%.

BREAKER There is a definite break of color.

LIGHT RED Pinkish-red or red color shows on over 60%.

TURNING Tannish-yellow, pink or red color shows on over 10% to 30%.

RED Red means that more than 90% of the tomato surface is red.

Figure 3.2 : Maturity and ripening stages of tomatoes.

40 3.4.2.2.1. COLOR MEASUREMENT Measurements using a chroma meter provide skin color readings of the tomato. The ratio of a*/b* is used, where the a* parameter represents the intensity of the red color. It ranges from -10 to 30 as a consequence of the depletion of chlorophyll and synthesis of lycopene (Arias et al., 2000). The b* parameter shows the yellow discolouration as a result of the synthesis of -carotene, the second most important carotenoid in tomato. It increases through the first four stages of maturity of tomatoes and then decreases. The a*/b* value for the light red stage is between 0.60 and 0.95. The red stage ranges between 0.95 and 1.21 (Batu, 2004). 3.4.2.3. TEXTURAL QUALITY Firmness, fibrousness, toughness, succulence, juiciness and textural qualities are all part of sensory attributes. Texture values are very important for marketing. They are greatly influenced by fruit internal structure and firmness. Texture is composed of several properties, including those that are mechanical (firmness, chewiness, viscosity, geometry, internal particle size and shape), and chemical (moisture and fat content) (Dorais et al., 2001). Firmness is very important as firmer produce can better withstand postharvest handling and transportation. But most of the time, firmer produce represent under ripened tomatoes that will be very firm and stiff and not well received by consumers. On the other hand, inferior texture or softer tomatoes are better received because it is often associated with improved flavor. Overripe tomatoes have a loose, watersoaked texture, sometimes grainy. The ideal texture perceived by consumers is slightly firm and smooth (Cox, 2001). 3.4.2.4. TOMATO SOLIDS Tomato solids are measured to give an idea of the product consistency and overall quality. It provides information about the degree of maturity and water stress. The total solids content represents the degree of solids concentration of the fruit expressed in degree Brix or as measured by a refractometer (Gould,

41 1992). This measure is affected by temperature. If a sample is subjected to high temperatures, it will expand and become less dense and the refractive index will decrease. 3.4.2.5. FLAVOUR QUALITY Saltiness, astringency, bitterness, aroma (odour) and sensory evaluation are all part of the flavour qualities. The interaction between sugars and acid levels largely determines the perceived flavor in tomatoes. Fructose and glucose are the two primary sugars produced by tomatoes and are the source of the tomatos sweetness. Citric and malic acids are the source of the tart flavor. A combination of high sugar and high acid provides a flavour in tomatoes that most people are looking for and is considered good. In contrast, a combination of low sugar and low acid in tomatoes is considered bland by consumers. High acidity along with low sugar content gives the tomato a tart flavour (acidic). A tomato with high sugar content and low acidity is considered sweet (Cox, 2001). A minor factor also influencing flavor is the multiple volatile compounds present in tomatoes. These compounds are not detected by the tongue but by the olfactory nerve in the nose and are detected only in minute amounts. Hundreds of volatiles are present in tomatoes, but only 30 occur in quantities greater than one part per billion. Of these 30, only 16 have been considered to have a significant contribution to tomato flavor (Schultz Nelson, 2007). 3.5. LYCOPENE 3.5.1. LYCOPENE IN TOMATO Lycopene is a carotenoid, an acyclic isomer of -carotene. In tomato, lycopene is present in the chromoplasts of the fruit (Cox, 2001). It is the pigment responsible for the red color and constitutes 75 to 83% of the total carotenoid content whereas -carotene represents 3 to 7% (Dorais et al., 2001). Lycopene concentration in tomatoes varies based on cultivar, stage of ripeness and growing condition (Cox, 2001), and is reported to range between 43.6 to

42 181.2 g per g of fresh weight (Dorais et al., 2001). Red light stimulates lycopene production and influences chlorophyll breakdown in the tomatoes. Blue light enhances carotenoid synthesis, but slows lycopene production (Dorais et al., 2001). Tests were performed by Cox (2001) on tomatoes to measure the effect of light exposure on lycopene development. Tomatoes were subjected to 8 hours or 24 hours photoperiods, under cool white fluorescent lights. Lycopene content reached 0.29 mg g-1 of dry matter under an 8 hour photoperiod and up to 0.52 mg g-1 of dry matter in the 24 hours photoperiod ripened tomatoes. Past studies determined that light quality affected the red colour development. Fruits ripened under cool white fluorescent bulbs were 16 times redder and fruits under wide spectrum bulbs were 32 times redder than dark-ripened controls (Cox, 2001). In the case of tomato, redness translates almost directly to lycopene content (Chen, 2008). Lycopene synthesis in the fruit is optimum at 24C and is severely impaired at temperatures above 30C or below 10C (Cox, 2001). The lycopene molecule is very stable in the tomato. However, when extracted from the fruit, it is very sensitive to heat, light and O2. Analysis of fresh tissue has to be performed within a very narrow time frame (Nguyen and Schwartz, 1999). 3.5.2. LYCOPENE AND HUMAN HEALTH Lycopene is the most important carotenoid in North American and European diets and over 85% comes from tomato and tomato products. It is the most predominant carotenoid in human plasma (0.5 to 0.8 mol L-1 of plasma) and it accounts for 50% of the total carotenoids (Agarwal and Rao, 2000). Its bioavailability to the human body depends on the processing practices and what other foods are consumed along with it. Heating tomatoes disrupts the cells and makes lycopene readily oxidized and bioavailable for absorption in the intestine. Additionally, lycopene is highly lipophilic and eating tomatoes with fat will increase lycopene uptake. However, some dietary fibers reduce the bioavailability of lycopene and therefore, reduce its uptake (Riedl et al. 2001;

43 Agarwal et al., 2001). Once in the body, lycopene is concentrated in different tissues: 1 nmol g-1 in adipose tissue and up to 20 nmol g-1 in the testes, adrenal glands and prostate (Stahl and Sies, 1996). Its persistence in the body varies between studies from a half life of 2-3 days to 12-33 days (Cox, 2001). Lycopene is considered the most effective quencher of singlet O2 and free radicals among all carotenoids (Dorais et al., 2001). It prevents oxidative damage to phospholipids of cell membranes, vital proteins and DNA (Di Mascio et al., 1989). It was proven to be effective in reducing/preventing cancers of the prostate, lung and stomach, and suggested to reduce cancers of the pancreas, colon and rectum, oesophagus, oral cavity, breast and cervix (Giovanucci, 1999). It is also recognised to effectively reduce the risk of cardiovascular disease (Agarwal and Rao, 2000). Even exercise-induced asthma was found to be ameliorated by lycopene consumption of 30 mg per day in 55% of the subjects (Neuman et al., 2000). 3.6. SUMMARY Tomato is a nutritious fruit. Its physiological properties are most beneficial to human health. Studies have demonstrated that there is a tendency towards healthy eating by consumers, which has led to development and research on ways to maintain beneficial nutrients through storage and all the way to the consumers table. Furthermore, preservation methods, free of chemicals, have been investigated to prevent rotting of fruits and vegetables through handling and storage. Many studies are focusing on the improvement of the quality attributes of fresh fruits and vegetables. These improvements are then associated with functional foods and are sold with higher market values. Tomatoes already have these properties but are somehow difficult to store and retrieve fresh on the market counter, year-around, having all their beneficial properties fully developed.

44 Hyperbaric treatment is not a surface sterilising treatment but it has the advantage of being uniform. It can be used to create a hormic stress to the commodity being treated. The response reaction to a hormic stress would be a plant defence reaction. Considering the present review, it can be assumed that tomatoes would produce more lycopene in response to a hormic stress and it should be further investigated.

CHAPTER 4 4. CONCEPTUALIZATION, DESIGN AND EVALUATION OF A HYPERBARIC RESPIROMETER

4.1. INTRODUCTION For many years, consumers demand for fresh fruits and vegetables has been a challenge for producers. The numbers associated with postharvest losses being as high as 50% have encouraged development of technologies that prolong shelf life of fresh produce (McLaughlin and OBeirne, 1999). A major factor contributing to postharvest losses is high respiration rate of the commodities. Many techniques have been described to reduce respiration rate and improve conservation period of fresh horticultural produce. Hyperbaric treatment is one that may contribute to storage and be well accepted by consumers as it reduces O2 consumption; it does not require chemicals and it may help suppress infection by pathogens. Hyperbaric atmosphere, high O2 atmospheric, or super atmospheric O2 concentrations represent an environment with an O2 concentration greater than 21 kPa around and within the commodity (Kader and Ben-Yehoshua, 2000). This definition is also similar in the medical industry where the hyperbaric O2 therapy is defined as the inhalation of O2 at increased pressure (Wang and Dilley, 2000). But, in the case of high O2 atmosphere, the proportion of gas in the air is disrupted for the benefit of O2 only, whereas in hyperbaric treatments the proportion of each gas in the air is maintained. Many studies have evaluated the effects of temperature and gas concentration on the respiration rate during storage of fruits and vegetables (ASHRAE, 2006). The investigation on gas concentration effects led to the development of Controlled Atmosphere (CA) storage and Modified Atmosphere Packaging (MAP) as a way of reducing respiration rate and increasing shelf life (Nakamura et al.,

46 2004). The idea of respiration suppression under high CO2 and low O2 conditions has long been suggested (Ranson et al., 1960; Garipy et al., 1991; DeEll et al., 2006). These CA and MAP approaches were important development in increasing storage life. However, one has to be careful with O2 concentration that are too low, as they can result in anrobic respiration and diminish the quality of fruits or vegetables, while excessive CO2 concentration can cause injuries like softening, off-flavours and/or discoloration (Nakamura et al., 2004). Some techniques have been associated with the reduction of respiration by applying a wax coating or polymeric films on the surface of the produce. The difficulty with this technique is to maintain equilibrium in gas exchanges (Kader and Saltveit, 2003). Respiration is equivalent to the biological oxidation of the produce. An inverse relationship exists between the respiration rate and the postharvest life of fruits and vegetables (Kader, 2002). Thus, the measurement of respiration is very important as it provides information on the metabolic activity of the plant tissue: loss of dry weight, O2 consumption, CO2 production, heat production or loss of energy (Saltveit, 1995). Many techniques have been developed to measure respiration rate and most of them involve the estimation of CO2 production by the product which is the easiest and most accurate measure to obtain. The two main systems to measure the respiration rate are static and dynamic systems (Saltveit, 1995). The static system consists of a sealed container in which the accumulation of carbon dioxide created by the produce is measured over a period of time. The disadvantage using a static system is that it is susceptible to air leaks if it is not properly sealed. Also the increase in carbon dioxide concentration or oxygen depletion in time may affect the respiration rate of the produce being tested. The dynamic system uses a continuous flow of gas through the mass of produce, and has the advantage that leaks are not as critical and that gas of various compositions can be used to ventilate the container, allowing measurement of respiration rate (RR) under different controlled conditions. The dynamic system is

47 more suitable for long-term experiments since air is flowing thru the system allowing a stable gas concentration around the produce being tested. Also, pressure and gas composition can be modified as desired during a test without stopping data acquisition. Calegario et al. (2001) measured the respiration rate of tomatoes by continuously monitoring CO2 level in a dynamic system. This kind of flowing system was presented by Kader and Saltveit (2003) as a method for the measurement of respiration rate for intact tissues. While numerous dynamic systems exist to measure RR, none of them have shown the capacity to measure RR under pressure. Apelbaum et al. (1977) measured the effect of storage under hyperbaric pressure ranging from 0.25 to 0.7 MPa on mango fruits. The treatment did not enhance shelf life beyond that of commercial storage (16 days). Mume fruits were subjected to pressures ranging from 0.5 to 5 MPa for 10 minutes and maintained at 0.5 MPa for 5 days (Baba and Ikeda 2003). A 0.5 MPa pressure maintained for five days decreased ethylene production during storage, decreased weight loss and showed benefits against chilling injury. Although these two studies exist in the literature, very little information has been published on the effect of hyperbaric pressure treatment on fruits and vegetables as a postharvest treatment. An exhaustive literature search did not reveal additional scientific publications on this subject. However, hyperbaric pressure affects respiration and can be physiologically detrimental to any living organism subjected to these pressures. Baba et al. (1999) tested mume fruits placed under high pressure to extend their postharvest life. The pressures tested ranged between 5 and 200 MPa. These high pressures were above the threshold for irreversible tissue damage and caused substantial injuries to the fruits. Pressure treatment might be an alternative to chemical treatment for maintaining fruit quality and may avoid the problem generated by lack of uniformity encountered with many physical treatments (Lu et al., 2006). But it is important to have a better understanding of how a pressure treatment can affect storage life.

48 This can be evaluated by measuring how it affects the respiration rate of the treated produce. The objective of this project was to design and test a computer-controlled dynamic system to measure the RR of fresh horticultural produce under absolute pressures ranging from 2 to 7 atmospheres absolute pressure. 4.2. PRELIMINARY WORKBENCH A preliminary workbench was designed to determine the feasibility of the experiment, to evaluate the respirometers design and to explore different pressure treatments. 4.2.1. RESPIROMETER DESIGN The test chamber consists of two main parts, the outer chamber and inner chamber (Fig. 4.1). The purpose of the outer chamber was to resist pressures up to 15 atm. It was built from a soil moisture test apparatus made of stainless steel (Soil Moisture Equipment Co.TM, Santa Barbara, California, USA). It was 125 mm high by 300 mm inside diameter and is closed with a bolted steel cover. The internal volume of the outer chamber was 8.836 L. An O-ring rubber seal of 6.35 mm in diameter was placed between the cover and the chamber to ensure air tightness (Fig. 4.2). Two compression fittings were fastened on the side of the chamber to connect air flow inlet and outlet using plastic tubes of 3.2 mm inner diameter. The inlet of the outer chamber (Fig. 4.1) was connected to a cylinder of compressed medical air with a certified CO2 concentration of 325 ppm. The cylinder was equipped with a manometer which regulates the pressure to the desired value. The air flow passed thru a humidifier to increase the humidity level of the air in order to avoid drying of the produce being tested. An airtight connector was used to insert a T-type thermocouple inside the chamber to measure temperature.

49
Medical air cylinder

Manometer

Respiration chamber

CO2 gas analyzer Data acquisition card and computer

Humidifier

Flow meter and control valve

Pressure transducer

Pressure Flow meter and sensor control valve

Outer chamber

Humidifier

Manometer

CO2 gas analyser

Inner chamber T-type thermocouple

Medical air cylinder

------ Gas flow path

Figure 4.1 : Pictorial and schematic view of the hyperbaric respirometer. Dotted lines represent the gas flow pathway thru the dynamic respiration system.

50

Outer chamber air inlet Air outlet O-ring

Produce O-ring

Calibrated inlet

Figure 4.2 : Chamber and equipment details of the dynamic respiration system developed.

51 The inner chamber is used to reduce the volume of air surrounding the produce (Fig. 4.1). By reducing the volume, the time required to reach equilibrium of CO2 concentration inside the inner chamber was also reduced as expressed by the general dilution Eq. 4.1.

C initial V Dt = ln Q C final
Where : Dt V Q Cinitial Cfinal = = = = = Dilution time, min;

( 4.1 )

Void volume of gas to be diluted in the chamber, mL; Ventilation rate of the chamber, mL min-1; Initial concentration of the gas to be diluted, ppm; Final concentration of diluted gas, ppm.

In order to measure the respiration rate of a specific produce, the volume and shape of the inner chamber may be adjusted to the produce shape and size to maintain the void volume as small as possible. The inner chamber (Fig. 4.1) placed inside the outer chamber was adjusted to fit one medium-sized tomato fruit. It was built using a plastic (ABSTM) tube, 100 mm high by 75 mm in diameter. The inner chamber had a volume of 0.442 L. The inner chamber was sealed using ABS glued fittings. An O-ring rubber seal of 3.2 mm in diameter was placed in the cover to ensure air tightness (Fig. 4.2). Two compression fittings were fastened on the side of the inner chamber to connect air flow inlet and outlet. The inlet of the inner chamber was open to the inside of the outer chamber. A calibrated orifice inlet 0.01 mm in diameter was placed at the inlet of the inner chamber creating a sufficiently fine diffusion channel to avoid CO2 dissipation from the inner chamber to the outer chamber. The outlet of the inner chamber was connected directly to the outlet of the outer chamber (Fig. 4.2). A pressure sensor, an air flow meter equipped with a control valve and an infrared

52 CO2 gas analyzer were placed in series along the outlet air pathway (Fig. 4.1). A pressure sensor (Bourdon HeanniTM model CTX355H211, Vendme, France) was used to measure the pressure inside the inner chamber within a range of 0 to 6 0.06 atm. A flow meter - control valve (BronkhorstTM model F-201C-FBB22-V, Ruurlo, Netherlands) was used to measure and regulate the air flow within a range of 5 to 250 2.5 mL min-1. An infrared gas analyzer (CrowconTM model Deltagas, Abingdon, England) was used to measure the CO2 concentration within a range of 0 to 2000 20 ppm of CO2. The flow meter, control valve, pressure transducer, CO2 gas analyzer, and type T thermocouple were all connected to a data acquisition and control card (DATA shuttle ExpressTM, Strawberry TreeTM, Sunnyvale, USA) and a personal computer. A software interface was programmed using WorkBenchTM version 3.0. (Strawberry TreeTM , Sunnyvale, USA). The interface allowed a control of the air flow rate through the proportional valve of the flow meter, based on a PID (Proportional, Integral, Derivative) algorithm system. The interface recorded the pressure, air flow rate, CO2 level and the temperature inside the chamber, computed the respiration rate in real time and provided a display of the process parameters. The respiration rate (Eq. 4.2) was computed by multiplying the percentage increase of CO2 concentration by the air flow rate, divided by the fresh weight of tissues of the tested produce. The increase in CO2 concentration was obtained by computing the CO2 concentration difference between the inlet and outlet of the chamber.

RR =

CO2 * Q m

( 4.2 )

where, CO2 = difference between the CO2 concentration, ppm leaving and entering the system,ppm; Q m = = flow rate, mL h-1; mass of produce, kg.

53 4.2.2. CALIBRATION OF THE RESPIROMETER Calibration of the gas analyzer and the verification of the air tightness of the system were performed by circulating 50 mL min-1 of the calibration gas having a certified CO2 concentration of 1478 ppm. The calibration gas was circulated directly through the inner chamber of the respirometer and the gas analyzer. This process was repeated three times on different days for a period of 120 minutes. The first period of 30 minutes was used as a flushing period to allow the calibration gas to meet its provided concentration. The next 90 minutes were recorded to evaluate CO2 concentration. The 30-minute delay was chosen based on Eq. 4.1 using a volume of 442 mL with an airflow rate of 50 mL min-1, which resulted in a theoretical flushing of 96.7% (Fig. 4.3). Since it is a single point calibration, the air flow rate used to measure the respiration rate during a test should be adjusted to obtain a CO2 gas concentration of approximately 1478 ppm at the outlet. All the operational parameters of the system were evaluated and calibrated to determine the feasibility of using such a design to perform hyperbaric treatments on fruits and vegetables. 4.2.3. EVALUATION OF THE EFFICACY OF THE SYSTEM TO MEASURE THE RESPIRATION RATE OF LIVING PRODUCE Preliminary tests using living plant organisms were performed to establish (1) the sealing of the system under pressure; (2) the feasibility of recording respiration in real time; (3) the protocol for pressurizing living plant organisms. Tests were performed using tomatoes. The tomatoes were conditioned at 13C for a period of at least 16 hours prior to testing; some tomatoes were used after 16 hours and were considered as mature green and others were used the following day and were closer to the breaker stage. The RR measurement was performed on two different masses of produce depending on the chamber used. When the inner chamber was used, the mass of tomato tested was 200g 5g with an airflow rate of 50 mL min-1. It was possible to remove the inner chamber to use the entire space of the outer chamber. In that case, the mass of tomato tested was 1218g 5g with an airflow rate of 110 mL min-1. The airflow rate was adjusted in

54

100 90 80 70 % of flushing 60 50 40 30 20 10 0 0

99.9% 96.7%

10

20

30 Time (min)

40

50

60

Figure 4.3 : Theoretical % of flushing during the calibration as a function of time using the general dilution equation (Eq. 4.1). The volume used was 442 mL and an airflow rate of 50 mL min-1.

55 order to get a CO2 concentration of approximately 1478 ppm at the outlet of the chamber; the air flow rate was 165 mL min-1. Tests were performed over a period of time long enough to reach equilibrium of CO2 concentration in the system (Figs. 4.4 & 4.5). The software recorded in real time the respiration rate at 1 min intervals. For the experiment, two pressure levels were evaluated with 3 replicates for each pressure level. The pressure levels used for evaluation purposes were 2 and 7 atmabs. The point of doing replicates was to evaluate the curve profile for respiration. Since tomatoes were not exactly all at the same stage of maturity, the amplitude of the respiration was shifted but the profiles of the curves were comparable. 4.3. RESULTS AND DISCUSSION 4.3.1. RESPIROMETER DESIGN Measurement error using the respirometer was calculated as a function of the RR and the mass of produce (Fig. 4.6). The error was computed using a precision of 1% of the scale for both the gas analyser and the gas flow meter. As the RR increased, the airflow rate required to maintain the CO2 concentration at the calibration level (1478 ppm) increased, thus reducing the reading error (Fig 4.6). For a small quantity (0.2 kg) of a low RR commodity, such as apple, producing about 5 mL CO2 kg-1 h-1, the error would be as high as 17.3%, while a larger quantity (0.8 kg) of a high RR commodity, such as strawberry, producing about 22 mL CO2 kg-1 h-1, the error would be inferior to 1.0%. This result demonstrated the importance of maintaining the CO2 production sufficiently high to maintain an acceptable precision. The mass of produce should be increased to fill the respirometer chamber when measuring the respiration rate of a low-RRcommodity. Furthermore, increasing the production rate of CO2 by increasing the mass of produce increased the air flow rate (Fig. 4.7) required to maintain the CO2

56

2/kg-hr) Respiration rate (RR, mL CO

10 9 8 7 6 5 4 3 2 1 0 0 5 10 Time (min) 15 20 25
Rep 1 Rep 2 Rep 3

Figure 4.4 : Respiration rate pattern of 200 g tomato stored inside of the 442 mL airtight chamber pressurized at 2 atmabs and maintained at 13C with an airflow rate of 50 mL min-1.

57

3.0 R espiration rate(mL C O2 /kg-hr) 2.5 2.0 1.5 1.0 0.5 0.0 0 200 400 600 Time (min) 800 1000 Rep 1 Rep 2 Rep 3

Figure 4.5 : Respiration rate pattern using the outer chamber having a volume of 8863 mL with 1218 g of tomato at 13C and pressurized at 7 atmabs and an airflow rate of 110 mL min-1.

58

40% 35% 30% Error % 25% 20% 15% 10% 5% 0% 0 5 10 15

0.2 kg 0.4 kg 0.8 kg

20

25

Respiration rate (mL CO2/kg-hr)

Figure 4.6 : Error of the respiration rate (RR) reading as a function of the RR for different mass of produce.

59

300

Airflow rate (mL min )

-1

200

100

0 0.0 0.1 0.2 0.3 CO2 production rate (mL CO2 min-1)
Figure 4.7 : Air flow rate (mL min-1) required to maintain the CO2 concentration of the exhausting gas at 1478 ppm as a function of the commodity respiration rate (mL of CO2 min-1).

60 concentration of the exhausting gas at 1478 ppm, increased the air change per unit of time and the dilution process of the gas inside of the chamber. This increase of the dilution process decreased the waiting period required to precisely measure the RR of a given produce. 4.3.2. CALIBRATION OF THE RESPIROMETER The results of the calibration trials using the 1478 ppm CO2 calibration gas are presented in Figure 4.8. The average concentration for the 270 readings over three periods of 90 min was 1476.7 ppm of CO2 and the experimental error remained fairly stable over the entire period, showing a standard deviation of 4.8 ppm. The precision of the gas analyser meets the specifications of the company (<20 ppm error, or 1% of the full scale, 2000 ppm), corresponding to four times the standard deviation calculated from the calibration readings. However, the first and second degree regression curves calculated on the calibration data showed a slight but significant increase in the reading of the CO2 concentration as the measurement progressed, clearly demonstrating an effect of the time on the reading. This increase in the reading of the gas concentration may be attributable to the flushing procedure used during the calibration. Since the calibration gas was injected at 50 mL min-1 through the inner chamber of the respirometer, the dilution effect of this flushing process allowed some gas to remain in the inner chamber for a longer time. The remaining gas may take hours to be completely flushed out as it happens in the flushing process in storage rooms. Theoretically, based on the general dilution equation (Eq. 4.1), the time to completely eliminate the gas contained in the chamber is infinite. However, after a certain time, the remaining gas should be negligible compared to the precision of the instrument used for the measurement. The 30 min used as flushing period might not have been sufficient to establish a negligible effect on the CO2 concentration. It is clearly shown in Fig. 4.3 that theoretically, after 30 minutes, the plateau of the calibration curve is not yet

61

1500
2

CO2 = -0.000928 t + 0.173 t + 1471.4 R = 0.2509 CO 2 concentration (PPM)

1475

1450 0 20 40 Time (min) 60 80

Figure 4.8 : Calibration curves obtained using the dynamic respiration system developed and a calibration gas containing 1478 ppm of CO2.

62 reached, with a 96.7% dilution. But after 60 minutes, 99.9% dilution is attained. These results are comparable to the calibration test performed using a synthetic gas with CO2 concentration of 1478 ppm presented in Fig. 4.8. The average concentration for the three tests was 1476.74.9 PPM of CO2 and was stable over a period of 90 min. Again the precision of the gas analyser meets the specification of the company (<20 ppm error or 1% of the scale). Considering the tendency of the curve, the 30 minutes delay was not enough for the dilution to be completed and for the CO2 concentration to attain 1478 ppm. Based on these results, it was concluded that an initial delay of 60 minutes should be maintained between the beginning of the flushing process and the first measure of gas concentration. The mean CO2 concentration reading after 60 min was 1478.3 4.4 ppm, which is very close to the standard gas concentration used (1478 ppm of CO2). 4.3.3. EVALUATION OF THE EFFICACY OF THE SYSTEM TO MEASURE THE RESPIRATION RATE OF LIVING PRODUCE The respiration pattern obtained by the developed dynamic system followed a standard pattern (Figs. 4.4 & 4.5). The CO2 concentration in the outflow from the container increased until as much CO2 left the containers as was produced by the plant tissue, i.e., equilibrium conditions. The respiration rate was obtained once the equilibrium concentration was reached. The time to reach equilibrium in respiration rate using the developed system, with tomatoes at 13C, ranged from 12 to 1000 min, depending on the set up used. The time required to reach equilibrium was clearly related to the pressure applied, respiration rate, the airflow rate, the mass of produce and the void volume of the respirometer. Some of these parameters could be adjusted to a certain extent to shorten or extend the time required to reach equilibrium. In this particular experiment, the main factor influencing equilibrium was the void volume of the chambers. The inner chamber reduced the void volume during the 2 atm experiment. The respiration rate for pressure treatments of 2 and 7 atmabs are presented in Table 4.1. The difference between the respiration rate at 2 and 7 atmabs was

63 significant (p 0.05, SAS). The mean respiration rates obtained were respectively 9.07 mL kg-1 h-1 and 3.01 mL kg-1 h-1. Complete results are presented in Table 4.1. The respiration rate at 2 atmabs was comparable to the respiration rate of tomatoes under atmospheric pressure but the respiration rate at 7 atmabs was significantly reduced. The statistical equation (4.3) is used to determine the minimal difference ( x - y ) between two populations (x and y) one could significantly identify based on a desired level of confidence and a fixed number of replicates.
2 2 + Sy ) t 2 (S x (x y) N 2

( 4.3 )

Where :

x, y
t2

= =

mean values of the population x and y, respectively; two-tailed Student t-test value based on the required interval of confidence of 95% and 99% or = 0.05 and 0.01, t2= 3.842 and 6.636, respectively;

2 2 , Sy = Sx

standard deviation of the population x and y, respectively; number of samples required to produce a significant difference.

Using the experimental results in Eq. 4.3, the minimal differences in respiration rate that could be significantly identified with intervals of confidence of 95% and 99% are 1.16 and 1.52 mL kg-1 h-1 at 2 atmabs, and 0.16 and 0.21 mL kg-1 h-1 at 7 atmabs, respectively. The respiration rates were 9.07 and 3.01 mL kg-1 h-1 at 2 and 7 atmabs, a difference of 6 mL kg-1 h-1. Knowing that under the condition tested, a minimal difference of 1.52 mL kg-1 h-1 could be significantly detected and assuming a linear relationship between RR and pressure, the pressure difference could be divided in 4 steps (each 1.25 atmabs) and still produce significant differences.

64 Table 4.1 : Respiration rate (RR) of tomato fruits exposed to a pressure of 2 and 7 atmabs and a temperature of 13C.

RR (mL CO2 kg-1 h-1) Repetition 1 2 3 Average Standard deviation Coefficient of variation Minimal difference detectable with three replicates and = 0.05 2 atmabs 8.92 9.86 8.43 9.07 0.727 8.01% 1.16 7 atmabs 2.94 2.96 3.12 3.01 0.099 3.28% 0.21

65

4.4. CONCLUSION The dynamic respirometer is reliable to measure the respiration rate of fruits or vegetables under pressure conditions varying from 2 and 7 atmabs. Preliminary results showed that pressure treatment can potentially improve the storage life of living produce by reducing their respiration rate. Physiological differences were observed between the tomatoes treated at two pressure levels. Further studies are necessary to determine which level of pressure treatment is suitable to reduce RR without causing physiological damage to the produce. A more efficient system should be designed to evaluate the effect of hyperbaric treatments on the physiological changes and the respiration rate of post harvest produce, and establish the impact that it might have on post harvest quality and future technologies 4.5. REFERENCES ASHRAE. 2006. Thermal properties of foods. In: Refrigeration. American Society of Heating, Refrigerating and Air Conditioning Engineers, Inc. Atlanta, Georgia. Chapter 9. Apelbaum, A., G. Zauberman, and Y. Fuchs. 1977. Subatmospheric pressure storage of mango fruits. Scientia Horticulturae 7(1977): 153-160. Baba, T., G. Como, T. Ohtsubo, and F. Ikeda. 1999. Effects of high-pressure treatment on mume fruit (Prunus mume). J. Amer. Soc. Hort. Sci. 124(4): 399-401. Baba, T. and F. Ikeda. 2003. Use of high pressure treatment to prolong the postharvest life of mume fruit (Prunus mume). Acta Hort. 628: 373-377. Calegario, F.F., R.G. Cosso, F.V. Almeida, A.E. Vercesi, and W.F. Jardim. 2001. Determination of the respiration rate of tomato fruit using flow analysis. Postharvest Biology and Technology 22(3): 249-256. DeEll, J.R., P.M.A. Toivonen, F. Cornut, C. Roger, and C. Vigneault. 2006. Addition of sorbitol with KMnO4 improves broccoli quality retention in modified atmosphere packages. Journal of Food Quality 29(1): 65-75.

66 Garipy, Y., G.S.V. Raghavan, F. Castaigne, J. Arul, and C. Willemot. 1991. Precooling and modified atmoshpere storage of green asparagus. Journal of Food Processing and Preservation 15(3): 215-224. Kader A.A. (ed). 2002. Postharvest technology of horticultural crops. 3rd edition. Coop. Ext. Uni. of Ca. Division of Agriculture and Natural Resources. Univ. of CA, Davis, CA. Publ. no. 3311. 535p. Kader, A.A. and S. Ben-Yehoshua. 2000. Effects of superatmospheric oxygen levels on postharvest physiology and quality of fresh fruits and vegetables. Postharvest Biology and Technology 20(2000):1-13. Kader, A.A., and M.E. Saltveit. 2003. Respiration and gas exchange. In Postharvest physiology and pathology of vegetables, eds. J.A. Bartz and J.K. Brecht, Chapter 2, New York: Marcel Dekker, pp. 7-29. Lu, J., B. Goyette, M.T. Charles, C. Vigneault, V. Orsat, and G.S.V. Raghavan. 2006. Effect of non-uniform heat treatment on tomato ripening and chilling injury. CSBE Paper no. 06-196S. McLaughlin, C.P. and D. OBeirne. 1999. Respiration rate of a dry coleslaw mix as affected by storage temperature and respiratory gas concentrations. Journal of Food Science 64(1): 116-119. Nakamura, N., D.V.S. Rao, T. Shiina, and Y. Nawa. 2004. Respiration properties of tree-ripe mango under CA condition. JARQ 38(4): 221-226. Ranson, S.L., D.A. Walker, and I.D. Clarke. 1960. Effects of carbon dioxide on mitochondrial enzymes from Ricinus. J. of Biochem. 79: 216-219. Saltveit M.E. 1995. Measuring Respiration. Internal report. Univ. of CA, Davis, CA. 5p. Wang, Z. and D.R. Dilley. 2000. Hypobaric storage removes scald-related volatiles during the low temperature induction of superficial scald of apples. Postharvest Biology and Technology 18(2000):191-199.

67

CONNECTING TEXT
A hyperbaric respirometer was conceptualized, designed and built. Preliminary tests were performed using tomato fruits. The effect of void volume and air flow rate on the response time was evaluated. The precision of the reading was determined. The tests demonstrated that hyperbaric treatment can affect the respiration rate. In the next chapter, a multi chamber respirometer study based on the design proposed in chapter 4 and the effect of hyperbaric treatments on respiration rate and respiratory quotient of tomato fruits will be presented.

CHAPTER 5 5. EFFECT OF HYPERBARIC TREATMENT ON RESPIRATION RATE AND RESPIRATORY QUOTIENT OF TOMATO
5.1. INTRODUCTION Fruits and vegetables are living organisms. They go through the process of maturation either attached to the plant or after harvest. When the fruit is still attached to the plant, the process of photosynthesis provides the oxygen necessary for the development of the plant nutrients which are transferred to the fruit. After harvest, the plant no longer provides nutrients to the fruit; however the fruit is still very much alive and continues its physiological activities. The fruit undergoes respiratory processes by itself, using its self-contained sources of energy and surrounding oxygen to maintain vital processes. When any fruit or vegetable uses all of its energy reserves, the living status is exhausted, respiration shifts from robic to anrobic and the commodity shows loss of flavour, especially sweetness, loss of dry weight, and overall reduction of food value and quality for the consumer (Kader, 2002). The process of respiration involves a biochemical reaction by which complex substrate molecules like carbohydrates, proteins and fats are broken down into simpler molecules such as CO2 and H2O (Eq. 5.1). Along with this reaction, energy and intermediate molecules are produced (Kader, 2002).
C6 H 12 O6 + 6 O2 6 CO2 + 6 H 2 O + 686 kcal / mole

( 5.1 )

Respiration rate could be used as an indicator of the potential storage life of a fruit or vegetable; the higher the respiration rate, the shorter is the storage life and the faster is the quality deterioration. To ensure an extended shelf life, the respiration rate has to be reduced to the lowest possible level, just enough to

69 maintain the essential living activities. This way, the stored reserves of the crop are conserved and the postharvest life of the fruit is extended to a maximum. A volumetric respiration rate could be calculated using Eq. 5.2 (Lencki et al., 2004).

Respiration rate =

volume of CO2 released or O2 consumed kg of commodity time

( 5.2 )

Beside the respiration rate, a respiratory quotient (RQ) can also be used as an indication of the metabolic activity (Eq. 5.3). The RQ is the ratio between the amounts of CO2 released (Rx) over the amount of O2 consumed (Ry) by the plant material (Plasse, 1986; Saltveit, 2005).

RQ =

Rx CO2 released = Ry O2 consumed

( 5.3 )

RQ values for fresh commodities range from 0.7 to 1.3, for robic respiration (Saltveit, 2005). The normal process of robic respiration should produce a RQ close to one; for each mole of CO2 produced, one mole of O2 is consumed. A RQ value greater than one may indicate that the organism is burning carbohydrates to produce fat or, that there is an oxygenated substrate utilized in the respiration process, like organic acid. A RQ value less than one may indicate several possibilities, principally that the reaction of oxidation is not complete (Plasse, 1986) or, that lipids (fats) are robically respired (Saltveit, 2005). A very high value of RQ would indicate an anrobic process (Saltveit, 2005). There are many factors affecting respiration but the ones considered the most important are the temperature, the atmospheric composition (i.e., controlled or modified atmosphere), and physical stress (Saltveit, 2005), temperature being the leading factor. Many researchers have evaluated the effect of temperature and controlled or modified atmosphere on RR and RQ of fruits or vegetables. However, only a few studies have dealt with the effect of physical stress on RR and RQ, especially with respect to hyperbaric treatment.

70 The objectives of this research were (1) to conceptualize, design, build and test a system that could monitor and record, in real-time, RR and RQ under hyperbaric condition and (2) to determine the effect of hyperbaric treatment on RR and RQ on tomato fruits. 5.2. MATERIAL AND METHOD 5.2.1. HYPERBARIC RESPIROMETER DESIGN A dynamic hyperbaric respirometer system was designed and built to measure the effect of hyperbaric pressure on RR and RQ. The design of the respirometer was based on the results presented in Chapter 4 but was adapted for the purpose of this experiment. It consisted of five vessels used as respiration chambers. The respiration chambers were connected as an open circuit through which compressed air was flowing to supply O2 uptake and dilute the CO2 released by the commodity (Fig. 5.1). The vessels were made from a stainless steel painting apparatus (GracoTM, Minneapolis, Mn), 300 mm high by 300 mm inside diameter and closed with a bolted steel cover. The total volume of each vessel was 10.75 L and was large enough to treat 20 tomato fruits at a given time. A flat ring rubber seal of 12.7 mm in diameter was placed between the cover and the chamber to ensure air tightness. Two SMCTM pneumatic fittings were fastened at the cover of the respiration chambers to connect air flow inlet and outlet using plastic tubes of 3.2 mm inside diameter (Fig. 5.2). Each cover of the respiration chamber was equipped with a pressure gauge and a pressure regulator to individually regulate the pressure in each respiration chamber from 1 to 9 atmabs. A safety relief valve was also installed on the cover to prevent overload pressure. An airtight connector was used to insert one T-type thermocouple inside each respiration chamber to record temperature during the treatment. The air flowing through the respiration chamber was manually adjusted using a needle valve at a flow rate of 205 mL min-1. The air leaving each respiration chamber was collected in a recuperation bag, 20 L in capacity, prior to gas

71

Respiration chambers Pressure regulator Compressed air cylinder Relief valve

Recuperation bags Flow control valve

Relief valve Bubble flow meter Gas analyser

Figure 5.1 : Schema of the automated respirometer developed to measure the respiration rate and the respiration coefficient using a continuous flow through system.

72

Qin

Qout V

Figure 5.2 : Detail of the closed container unit showing the air inlet for injecting Qin, the air outlet through which the airflow Qout exhausts from the system, and the inside volume (V) and the gas (G) produced or used by the produce.

73 analysis (Fig. 5.1). The recuperation bags were made from aluminized double polyethylene plastic, having an O2 permeability of 2.4 mL h-1 m-2 atm-1 (Vigneault et al., 1991). A relief valve was installed on the recuperation bag to prevent blow up. The CO2 and O2 concentrations were measured at the inlet and outlet of each respiration chamber. Twice a day, air samples were taken from the recuperation bag and analysed using a gas analyser (Ultramat 23, Siemens, Montral, Canada) equipped with an infrared CO2 sensor and having a paramagnetic property of O2 measuring cell. At the same time, air flow rate was measured using a bubble flow meter and a chronometer. After each gas analysis, the gas contained in the recuperation bag was pumped out to start a new sampling period. 5.2.2. BIOLOGICAL MATERIAL The effects of hyperbaric pressure on RR and RQ were tested on tomato fruits (Lycopersicon esculentum Mill. cv. DRW 453) obtained from a local greenhouse producer. Prior to each experiment, tomato fruits were selected for their color uniformity which reflects their degree of maturity. Acceptable color was established as mature green tomato near breaker stage (USDA, 2007). Breaker stage is a noticeable break in color with less than 10% of other than green color (Lopez Camelo and Gomez, 2004). All tomato fruits were weighed and had to be within the range of 200 g 10 g. 5.3. EXPERIMENTAL DESIGN Tomato fruits were submitted to absolute pressures of 1, 3, 5, 7, or 9 atmabs. The experiments were repeated 4 times at different dates. For each treatment, 100 tomato fruits were selected and surface sterilized for 3 min in a chlorine solution (1000 ppm), then rinsed with tap water for another 3 min, and finally left on filter paper to drain (Polenta et al., 2006). The fruits were randomly divided in five uniform lots for pressure treatment. Each lot was placed in one of the 5 respiration chambers. The respiration chamber were sealed and kept pressurized

74 throughout the experiment at the selected treatment level. Temperature and humidity were stabilized throughout the experiment at 130.2 C and 952.5 %, respectively. Temperature and humidity were recorded using a U-23-001 HoboProTM sensor (OnsetTM, Massachusetts, USA), placed inside each chamber of the respirometer. The CO2 and O2 gas concentrations were measured twice a day for the entire experiment duration. Since it is a dilution process, the CO2 concentration in the flow leaving the respirometer increases in time until as much CO2 leaves the respirometer as is produced by the tomatoes. It is the time required to reach equilibrium. Generally, equilibrium is reached after 75 to 160 h at a set hyperbaric pressure. Profiles of respiration rate and respiration quotient were analysed and compared through linear regression analysis. 5.4. RESULTS AND DISCUSSION The RR and RQ were calculated based on the air flow rate and the difference in CO2 and O2 gas concentrations measured at the inlet and outlet of the chamber. RRCO2, RRO2 and RQ were computed using Eqs. 5.4, 5.5, and 5.6, respectively.

RRCO2 =

CO2 Q m O2 Q m
RRCO2 RRO2

( 5.4 )

RRO2 =

( 5.5 )

RQ =

( 5.6 )

Where: CO2 O2 Q m = difference in CO2 concentration between the inlet and outlet, ppm; = difference in O2 concentration between the inlet and outlet, ppm; = flow rate, mL h-1; = mass of produce, kg.

75 Results are presented as the variation in RR based on CO2 released (RRco2), O2 uptake (RRco2), and the respiratory quotient (RQ) as a function of time for tomato fruits exposed to different CO2 partial pressure (Figs. 5.3, 5.4 & 5.5). As seen in the respiration profiles (Figs. 5.3 & 5.4), at the beginning of the experiment, RR values recorded for pressurized tomatoes were low and then gradually increased. This type of result is generally due to a dilution process happening in any flow through system with a fluid generation or degeneration. Thus, in the present case during the dilution process, the data of RR obtained did not correspond to the real RR. The RR apparently increased until the system reached equilibrium and the amount of CO2 released by the tomato equalled the amount of CO2 leaving the respiration chamber. After equilibrium, RR linearly decreased as a function of time until physiological or pathological problem(s) arose. The linear portion was considered as the real RR and was used for data analysis (Figs. 5.6 & 5.7). The time required to reach equilibrium ranged from 75 to 160 h depending on the pressure. The relation between the time to reach equilibrium and the absolute pressure can be explained by the fact that the ambient-pressure-equivalent void volume of the respiration chamber is directly proportional to the absolute pressure inside the container. Hyperbaric pressure significantly affected respiration rate based on CO2 and O2 concentrations. As pressure increased, respiration rate decreased or slowed down. However, respiration rate monitored under 9 atmabs pressure conditions, indicated that respiration had become anrobic and that the tomatoes had undergone fermentation. Anrobic RR results were not included in further data analysis since anrobic respiration is a totally different respiration process and represents different respiration profiles that are not comparable. 5.4.1. RESPIRATION RATE CALCULATION To determine RR, a linear regression analysis was performed on the linear portion of the RR data obtained as a function of time (Figs 5.6 & 5.7), eliminating

76

9 8 7
RR (mL CO2/kg-h)

stabilisation

data used to compute RR

0.022 atm CO2, 1 atm total pressure 0.063 atm CO2, 3 atm total pressure 0.083 atm CO2, 5 atm total pressure 0.093 atm CO2, 7 atm total pressure 0.094 atm CO2, 9 atm total pressure

6 5 4 3 2 1 0 0 50 100 150 200 250 300 350 400 450

time (h)

Figure 5.3 : Respiration rate (RRCO2) of tomato based on CO2 production as a function of time for various hyperbaric pressure and equivalent CO2 partial pressure.

77
0.022 atm 0.063 atm 0.083 atm 0.093 atm 0.094 atm CO2, CO2, CO2, CO2, CO2, 1 atm 3 atm 5 atm 7 atm 9 atm total total total total total pressure pressure pressure pressure pressure

9 8 7 RR (mL O2/kg-h) 6 5 4 3 2 1 0 0 50 100 150 200 250 300 350

400

450

time (h)

Figure 5.4 : Respiration rate (RRO2) of tomato based on O2 production as a function of time for various hyperbaric pressure and equivalent CO2 partial pressure.

78

2.5

Anaerobic respiration

0.023 atm CO2, 1 atm total pressure 0.064 atm CO2, 3 atm total pressure 0.083 atm CO2, 5 atm total pressure 0.093 atm CO2, 7 atm total pressure 0.096 atm CO2, 9 atm total pressure

2.0

1.5

RQ
1.0 0.5 0.0 0 50 100 150 200 250 300 350 400 450

time (h)

Figure 5.5 : Respiration quotient (RQ) of tomato as a function of time for various hyperbaric pressure and equivalent CO2 partial pressure.

79
0.022 atm 0.063 atm 0.083 atm 0.093 atm 0.094 atm CO2, CO2, CO2, CO2, CO2, 1 atm 3 atm 5 atm 7 atm 9 atm total total total total total pressure pressure pressure pressure pressure

7 6 5 RR (mL CO2/kg-h) 4 3 2 1 0 150

160

170

180

190

200 time (h)

210

220

230

240

250

Figure 5.6 : Linear portion of respiration rate data based on CO2 used for linear regression analysis.

80
0.022 atm CO2, 1 atm total pressure 0.063 atm CO2, 3 atm total pressure 0.083 atm CO2, 5 atm total pressure 6 0.093 atm CO2, 7 atm total pressure 0.094 atm CO2, 9 atm total pressure 5 RR (mL O2/kg-h)

0 150

160

170

180

190

200 time (h)

210

220

230

240

250

Figure 5.7 : Linear portion of respiration rate data based on O2 used for linear regression analysis.

81 the anaerobic respiration data obtained at 9 atmabs pressure. Results of the linear regression parameters for RRco2 are presented in Table 5.1. The intercepts were considered as the RR at the beginning of the experiment and the slope of the curve as RR decrease with time. From these two parameters, a linear relation was obtained by plotting RR and RR decrease (RRD) with time as a function of the CO2 partial pressure (Figs. 5.8 & 5.9). Similarly, the intercepts and the slopes of the curves based on RRo2 presented in Fig. 5.7, were calculated (Table 5.2), and plotted as a function of the CO2 partial pressure (Figs. 5.10 & 5.11). From these linear regressions, RRco2 (Eq. 5.7) and RRo2 (Eq. 5.8) were calculated as a function of CO2 partial pressure and time, based on the Eqs. 5.4 & 5.5.

RRCO2 = 22.324 p + 0.0112 pt 0.0112t + 8.317

RRO2 = 22.595 p 0.0080 pt 0.0092t + 7.881
5.4.2. MODEL FOR PREDICTING THE RESPIRATION RATE

( 5.7 ) ( 5.8 )

When using a dynamic respirometer, the CO2 concentration (C) inside the respiration chamber can be calculated based on the equation for general exhaust ventilation (Burgess et al., 2004). The difference in volume of CO2 inside the respiration chamber (v) was calculated (Eq. 5.9) as the difference between the volume of CO2 generated by the mass of produce (G) and the volume of CO2 removed by the exhausting airflow (Q) times the CO2 concentration of the exhausting gas (C) during a time differential of t.

v = G t Q C t

( 5.9 )

The change in CO2 concentration (C) as a function of time (Eq. 5.10) was expressed by dividing both side of the equation by the void volume (V). Rearranging Eq. 5.10, the differential equation (Eq. 5.11) was obtained. Further, by integrating Eq. 5.11, the change in concentration (Eq. 5.10) between time 1 (t1) and time 2 (t2) was obtained (Eq. 5.12).

82 Table 5.1 : Parameter of the linear regression analysis of respiration rate based on CO2 production as a function of time for various CO2 partial pressures (Fig 5.6). RR linear regression Pressure Partial CO2 pressure atmabs 1 3 5 7 atm 0.022 0.063 0.083 0.093 mL CO2/kg-h2 -0.0110 -0.0106 -0.0103 -0.0102 mL CO2/kg-h 7.79 6.94 6.55 6.14 0.8418* 0.6693* 0.8026* 0.9718* Slope Intercept R2

*Significant at 0.01 level

83

9 8 7 RR (mL CO2/kg-h) 6 5 4 3 2 1 0 0 0.02 0.04 0.06 0.08 0.1 partial CO2 pressure (atmabs ) RR co2= -22.324p + 8.3171 R2 = 0.9883

Figure 5.8 : Respiration rate based on CO2 released as a function of CO2 partial pressures. The respiration rate presented in this figure is obtained from the intercept of the linear regression analysis presented in Table 1.

84
-0.008 RRD co2= (0.0112p - 0.0112)t R2 = 0.9938 -0.009 RRD (mL CO2/kg-h)

-0.01

-0.011

-0.012 0 0.02 0.04 0.06 0.08 0.1 partial CO2 pressure (atmabs )

Figure 5.9 : Decrease of respiration rate in time based on CO2 released as a function of CO2 partial pressures. The respiration rate presented in this figure is obtained from the slope of the linear regression analysis presented in Table 1.

85

Table 5.2 : Parameter of the linear regression analysis of respiration rate based on O2 production as a function of time for various CO2 partial pressures (Fig 5.7). RR linear regression Pressure Partial CO2 pressure atmabs 1 3 5 7 atm 0.022 0.063 0.083 0.093 mL O2/kg-h2 -0.0093a -0.0098a -0.0097a -0.0099a mL O2/kg-h 7.39 6.44 6.00 5.78 0.7859* 0.7019* 0.8729* 0.9962* Slope Intercept R2

*Significant at 0.01 level

86

8 7 6 RR (mL O2/kg-h) 5 4 3 2 1 0 0 0.02 0.04 0.06 0.08 0.1 CO2 partial pressure (atm) RRo2 = -22.595p + 7.8811 R2 = 0.9994

Figure 5.10 : Respiration rate based on O2 released as a function of CO2 partial pressures. The respiration rate presented in this figure is obtained from the intercept of the linear regression analysis presented in Table 2.

87
0 -0.002 RRD (mL O2/kg-h2) -0.004 -0.006 -0.008 -0.01 -0.012 -0.014 0 0.02 0.04 0.06 0.08 0.1 CO2 partial pressure (atm) RRD o2 = (-0.0080p - 0.0092)t R2 = 0.8292

Figure 5.11 : Decrease of respiration rate in time based on O2 released as a function of CO2 partial pressures. The respiration rate presented in this figure is obtained from the slope of the linear regression analysis presented in Table 2.

88

C =

v G t Q C t = V V V V C = t G QC

( 5.10 )

( 5.11 )

2 1 V dC = dt G QC C1 t1

C2

( 5.12 )

Solving with the initial concentration which was assumed to be zero (C1 = 0) at t1=0, Eq. 5.13 was obtained. Since G was the CO2 generation rate, the Eq. 5.13 was equivalent to Eq. 5.14 and was rearranged to obtain Eq. 5.15, allowing one to describe the RRmeasured using RRreal.

C=

G Q t ( 1 exp ( )) Q V

( 5.13 )

C=

RRreal m Q t ( 1 exp ( )) Q V CQ Q t = RRreal ( 1 exp ( )) m V

( 5.14 )

RRmeasured =

( 5.15 )

In a dynamic respirometer, the concentration of CO2 leaving the respirometer increases until as much CO2 leaves the respirometer as is produced by the commodity. In the first few hours, the measured RR (RRmeasured) did not represent the real RR (RRreal). Theoretically, RRreal can be obtained anytime during the experimentation by rearranging Eq. 5.15 to calculate RRreal as a function of RRmeasured (Eq. 5.16).

RRreal =

RRmeasured Q t ( 1 exp ( ) V

( 5.16 )

89 Using Eq. 5.16, the dilution process was taken into account and the RRreal was obtained in the first few hours of the experiment. However, this equation (Eq. 5.16) can be used only if the dilution process is steady and no other phenomenon takes place, like solubilisation of gases within the produce. Equation 5.17 was obtained by replacing RRreal of Eq. 5.16 by Eq. 5.7, where RRreal was expressed as a function of pressure (p) and time (t):

RRmeasured = (8.3171 - 22.324p - 0.0112t + 0.0112pt) ( 1 exp (

Q t )) V

( 5.17 )

Figure 5.12 shows the comparison between the theoretical RR (Eq. 5.17) and experimental RR. At the beginning of the experiment, the curve did not fit the theoretical RR. Theoretical RR overestimated the RR obtained experimentally. This means that dilution was not the only phenomenon occurring at the beginning of the experiment. Since air was compressed in the respiration chamber, CO2 partial pressure changed drastically at the beginning of the experiment and a CO2 pressure gradient was created between the tomatoes and the surrounding air. This pressure gradient forced the solubilisation of CO2 in the liquid contained in the tomatoes. Part of the CO2 released by the tomatoes was solubilised instead of being rejected out to the surrounding air. This process reduced the CO2 concentration difference when using Eq. 5.4, thus underestimating the RR value. However, the solubility of CO2 in a tomato fruit is not well known and more research would be required to specifically study this phenomenon during the transient period at the beginning of the test, and further correct the equation and respiration rate accordingly during this unstable period. 5.4.3. RQ VALUES AND THEIR SIGNIFICANCE Results of RQ as a function of time for different CO2 partial pressures are presented in Fig. 5.5. At the beginning of the experiment RQ values ranged from 0.5 to 0.98 and were inversely proportional to pressure. Then RQ values had a constant increase in time. RQ should be stable in time, at a value closed to unity,

90

0.022 atm CO2 partial pressure 0.063 atm CO2 partial pressure 0.083 atm CO2 partial pressure 0.093 atm CO2 partial pressure 0.094 atm CO2 partial pressure

RR (mL CO2/kg h

0 0 50 100 Time (h) 150 200 250

Figure 5.12 : Comparison between the theoretical RR calculated using Eq. 5.17 (continuous lines) and experimental RR data measured (data points).

91

unless anrobic respiration takes place. The increase of RQ was likely due to solubilisation of CO2 in tomato cells instead of a specific metabolic state. Based on Henry's Law, the solubility of CO2 in water is directly proportional to the CO2 partial pressure of the environment around the surface of that water. As CO2 solubilised in plant cells, RQ artificially increased. The driving force for solubilisation of CO2 in the tomato cells is the difference between the actual quantity of CO2 solubilised and the quantity of CO2 solubilised at saturation. As CO2 solubilised in tomato, the solubilisation rate of CO2 decreased in time until the saturation occurred. For this particular experiment, time to obtain stable RQ values was approximately 120 hours. In order to evaluate the amount of CO2 solubilised in tomato, a linear regression was performed on the RQ data obtained during the first 120 hours (Fig. 5.13). The intercept of these curves, which represented the RQ at time 0, and the slope, which represented the rate of increase of RQ in time, are presented in Table 5.3. From these parameters, a linear relationship was established between the intercept and the slope of the curves as a function of the CO2 partial pressure. From this linear regression analysis, an equation of RQ for the first 120 hours (RQ120) as a function of the partial CO2 pressure (p) and time (t), was obtained (Eq. 5.18). The results of the regressions are graphically presented (Fig. 5.14).

RQ 120 = 1.13 6.187 p 0.00075t + 0.0539 pt

( 5.18 )

The volume of CO2 (Vco2) solubilized in the tomato cells can be obtained if the real RQ (RQreal) of the process is known. It would be calculated by integrating the difference between RQreal and the measured RQ, multiplied by the RRo2 (Eq. 5.5) for the solubilisation time. In the present study, replacing the terms of Eq. 5.5 by their values would result in Eq. 5.19, for the period of time between 0 and 120 hours.

92

1,2

0,8

RQ

0,6

0.022 atm CO2, 1 atm total pressure 0.063 atm CO2, 3 atm total pressure 0.083 atm CO2, 5 atm total pressure 0.093 atm CO2, 7 atm total pressure 0.094 atm CO2, 9 atm total pressure

0,4

0,2

0 0 20 40 60 time (h) 80 100 120

Figure 5.13 : Apparent RQ results calculated for the first 120 hours after submitting tomato fruits to different absolute pressures ranging from 1 to 9 atmabs.

93

Table 5.3 : Parameter of the linear regression analysis of RQ as a function of time for various CO2 partial pressures (Fig. 13). RQ linear regression Pressure atmabs 1 3 5 7 9 Partial CO2 pressure atm 0.022 0.063 0.083 0.093 0.094 Slope h-1 0.00035 0.00295 0.00402 0.00387 0.00376 0.983 0.739 0.602 0.558 0.500 0.2930* 0.8298* 0.8647* 0.8878* 0.8886* Intercept R2

*Significant at 0.01 level

94

RQ = -6.1867p + 1.1276 R2 = 0.9984

0.8

0.6 RQ 0.4 0.2 0 0 0.02 0.04 0.06 0.08 0.1 partial CO2 pressure (atmabs )

Figure 5.14 : Respiration coefficient (RQ) measured after CO2 gas reached equilibrium during pressure tomato fruit treatments at different absolute pressures ranging from 1 to 9 atmabs.

95

VCO = ( RQreal RQmeasured ) ( RRO 2 ) dt 2

0

120

( 5.19 )

This equation assumes that O2 solubilization is negligible compared to CO2 solubilization; which is an acceptable assumption considering the solubility in water of these two gases (Anon., 1957). However, RQreal is unknown for that part of the test and more experiments are required to elucidate this phenomenon. 5.4.4. EFFECT OF HYPERBARIC TREATMENT ON RR AND RQ Hyperbaric treatment affected the RR of tomato fruits (Tables 5.1 and 5.2). Respiration rates under 7, 5 and 3 atmabs were reduced by 20%, 16% and 11%, respectively, compared to the control (1 atm). This RR reduction is an indication that, even at a pressure as low as 3 atmabs, the tomato could be preserved for a longer period of time. In hyperbaric treatments, the partial pressure of CO2 and O2 are increased. Many fruits and vegetables respond to controlled atmosphere by having a higher CO2 partial pressure and tomato is one such commodity. It is not surprising that hyperbaric treatments reduced RR since the CO2 partial pressure was increased during the treatment. Henig and Gilbert (1975) reported that the respiration rate of tomato did not slow down until the CO2 concentration in the atmosphere reached 0.09 atm partial pressure. The results obtained in this study are in contradiction with these previous results since the CO2 partial pressure at 3 atmabs was 0.063 atm and it still reduced RR. Under atmospheric pressure, initial values of RQ should range between 0.8 and 1.33, depending on the principal substrate that the plant is using in the respiration process. When the respiration process is normal and sugars are metabolized, the commodities undergo robic respiration and the respiration quotient (RQ) is normally very close to the unity. For example, RQ values recorded in an open steady state system under normal atmospheric oxygen

96 condition present a fairly stable RQ curve, with values going up slightly in time, as the commodity uses its carbohydrates. In the case of our hyperbaric treatments, RQ at the beginning of the experiment ranged from 0.50 at 9 atmabs to 0.98 at 1 atmabs (control). A RQ value of 0.5 was low compared to what was expected from the literature (Henig and Gilbert, 1975), but, as observed in the case of RRco2, solubilisation of CO2 affects the measure of RQ. The RQ values obtained at the beginning of the experiment did not represent the real RQ. As solubilisation decreased, RQ values tended towards 1, in all cases, except for the treatment at 9 atmabs, where anrobic respiration was observed. 5.5. CONCLUSION A dynamic respirometer was designed and built to measure the effect of hyperbaric pressure on RR and RQ of plant tissues. Hyperbaric treatments were tested on tomato fruits. When working with a dynamic respirometer, it is important that the CO2 and O2 gas concentration reach equilibrium inside the respiration chamber in order to get reliable data. An attempt was made to calculate the real RR and RQ from the data measured at the beginning of the hyperbaric treatment, in the transient stage. But the solubilisation of the gas within the internal liquid of the tomato fruits is a phenomenon that needs to be further studied to elaborate proper ways to measure the RR and RQ during the transient stage. The effect of hyperbaric treatments on RR and RQ of tomato fruits was measured. There was a significant impact on the RR and RQ of pressurized tomatoes compared to the non-pressurized control. Tomato fruit subjected to 7, 5 and 3 atmabs treatments showed a RR reduction of 20%, 16% and 11%, respectively. Tomatoes fruits subjected to a hyperbaric pressure of 9 atmabs underwent irreversible damage caused by anrobic respiration.

97 The measured RQ was affected by the solubilisation of CO2 during hyperbaric treatment. When the system was in equilibrium, RQ was not affected by the hyperbaric treatment, with its values remaining close to 1. The hyperbaric respirometer was effective in monitoring and recording RR and RQ, in real-time, and should be further studied to establish the effect of hyperbaric pressure on quality attributes of tomatoes. 5.6. REFERENCES Anon. 1957. Handbook of Chemistry and Physics. 39th ed. Chemical Rubber Publishing Co. Cleveland, Ohio. Burgess, W.A., M.J. Ellenbecker, and R.D. Treitman. 2004. Ventilation for control of the work environment. Second Edition. New York, NY, John Wiley & Sons, Inc. 464 pp. Henig, Y.S. and S.G. Gilbert. 1975. Computer analysis of the variables affecting respiration and quality of produce packaged in polymeric films. Journal of Food Science 40(1975): 1033-1035. Kader, A.A. 2002. Postharvest biology and technology: an overview. In: Post Harvest Technology of Horticultural Crops, Third Edition. Ed. A.A. Kader. University of California, Davis, CA: Division of Agriculture and Natural Resources. Publication 3311, 535 pp. Lencki, R.W. 2004. Comparison of unsteady- and steady-state methods for produce respiration rate determination 2. Reexamination of the literature. Postharvest Biology and Technology 31(2004): 239-250. Lencki, R.W., M. Zhu, and C.-L. Chu. 2004. Comparison of unsteady- and steady-state methods for produce respiration rate determination 1. Model development and validation. Postharvest Biology and Technology 31(2004): 229-238. Lopez Camelo, A.F. and P.A. Gomez. 2004. Comparison of color indexes for tomato ripening. Hortic. Bras. 22(3): 534-537.

98 Plasse, R. 1986. Vegetable storage, respiration and design criteria in a membrane storage system. Masters Thesis. Department of Agricultural Engineering, Macdonald Campus of McGill University, Ste-Anne-deBellevue. 215 pp. Polenta, G., C. Lucangeli, C. Budde, C.B. Gonzalez, and R. Murray. 2006. Heat and anaerobic treatments affected physiological and biochemical parameters in tomato fruits. LWT 39(2006): 27-34. Saltveit, ME. 2005. The commercial storage of fruits, vegetables, and florist and nursery crops Respiratory metabolism. Saltveit Agricultural Handbook Number 66, USDA/ARS. Available on www.ba.ars.usda.gov/hb66/019respiration.pdf Accessed April 6th, 2009. USDA. 2007. U.S. Standards for Grades of Greenhouse Tomatoes. USDA, Agr. Mktg. Serv., Washington, DC. (http://www.ams.usda.gov/standards/stanfrfv.htm) Accessed January 2010. Vigneault, C., R.L. Granger, and G.S.V Raghavan. 1991. Mini-chambers for labscale research on controlled atmosphere storage. Appl. Eng. Agricult. 7(5): 617-621.

99

CONNECTING TEXT
Although the previous chapter demonstrated that hyperbaric treatments reduce respiration rate of tomato, it is important to evaluate the effect of such treatments on the quality parameters. The next chapter deals with the effect of hyperbaric treatments on quality attributes of tomato fruits. The quality attributes that will be evaluated are: weight loss, firmness, color, lycopene content, total soluble solids (TSS), titratable acidity (TA) and TSS/TA ratio.

CHAPTER 6 6. EFFECT OF HYPERBARIC TREATMENT ON QUALITY ATTRIBUTES OF TOMATO

6.1. INTRODUCTION Fruits and vegetables are parts of the daily diet around the world. They are grown in large varieties, under different climates, for different purposes and with different techniques. Their shelf-life period varies widely according to their maturity, physiological and pathological conditions at harvest, the handling method and the storage facilities used. Fast cooling prior to storage is one of the most important factors to reduce their metabolic activity and respiration rate (Rennie et al., 2003). The reduction of metabolic activity of harvested produce is an important step in the preservation of fruits and vegetables freshness and quality attributes. Physical treatments, such as cooling and controlled atmosphere (Raghavan et al., 2005), efficiently reduce respiration rate, retard some microbial growth and prolong produce quality for long periods. However, under these storage conditions, not all diseases and pathogens are inhibited. The commodities have to be exempt of cuts and bruises and preferably, sanitized before storage (Kader, 2002). Operational parameters of the controlled atmosphere system have to be well adapted to the selected fruit or vegetable and to its physiological stage at the time of storage to be beneficial rather than detrimental (Vigneault and Arts Hermndez, 2007). Chemical pre-treatments are often necessary to prevent rot and microorganism growth over long storage periods; however, they are prohibited in many countries and should be replaced by chemical-free treatments (Lu et al., 2008). Considering the increased worldwide importance of healthy eating, organic foods and the growing unacceptability of pesticides to the public, it was necessary to investigate other avenues (Anon., 2005). Physical treatments such as irradiations

101 (Lacroix and Vigneault, 2007), low dosage of UV-C light (Charles and Arul, 2007) and heat treatments (Lu et al., 2007) have been tested. The primary mode of action of physical treatments is the disinfection of the fresh commodity to prevent microorganism growth and decrease decay during storage. However, physically stressing some species stimulated natural disease resistance of harvested produce and led to an increase in resistance against future infection (Terry and Joyce, 2004). In some cases, an increase in quality attributes of the fruit or vegetable was observed (Charles and Arul 2007). Increased bioactive compounds and phytochemicals and their impact on human health have brought a new approach to postharvest treatments. Low dosage of UV-C light has been used to reduce losses due to fungal attack and premature senescence (Charles and Arul, 2007). Dosage should be appropriate for the targeted microorganism, as well as for the different produce, so as not to induce adverse effects. The geometry of the produce to be treated is also an important factor and determines the uniformity of the treatment. In most produce, UV treatment does not produce a systemic effect and disease resistance is only induced in tissue directly exposed to UV-C light (Shama, 2007). To be optimum, radiation and UV treatments have to be performed on produce with a smooth surface, exempt of any impurities and over the entire surface (Shama, 2005). Heating fruit or vegetables is another method to disinfect the produce surface and maintain its quality through storage. Heat treatments can be performed by hot air, dry air, hot water or microwaves. The problem is often in achieving treatment uniformity (Lu et al., 2007). The thermal properties of each horticultural crop are different and affected by the produce tissue and moisture content (Wang and Tang, 2007). Fruits and vegetables need to be rotated in some heating system to prevent burning injuries and insure treatment is uniform, as it is only a superficial and not a systemic treatment (Lu et al., 2007). Heating is also an operation which requires large amounts of energy (Wang and Tang, 2007).

102 Physical treatment such as controlled atmosphere using a high O2 concentration is a technique that was explored to extend shelf life of fruits and vegetables and control fungal growth and decay incidence in soft fruits (Zheng and Wang, 2007). Hyperbaric treatment consists of subjecting fruits and vegetables to an environment with an O2 concentration greater that 21 kPa (atmospheric pressure) around and within the commodity (Kader and Ben-Yehoshua 2000). Hyperbaric treatments are different than high pressure treatments where high pressure treatment consists of subjecting food to pressures between 100 and 1200 MPa (Goyette et al., 2007). Fresh fruits and vegetables cannot withstand these pressures without suffering irreversible damage (Ahmed and Ramaswamy, 2006). Postharvest hyperbaric treatment is a pressure treatment in which the proportion of each gas in the air is maintained or disrupted to the benefit of O2 only, and that at pressures ranging between 1 and 10 atmospheres. Unlike heat or UVC processing, where temperature and radiation gradients are established, pressure treatment offers homogeneity as it acts instantaneously and uniformly around each single item of produce or throughout the entire mass of food, independently of its size, shape or composition. Very little information is available on the effect of hyperbaric pressure treatment of fresh fruits and vegetables as a postharvest treatment for preservation (Goyette et al., 2007). Tomato fruits are of great interest as they contain naturally-occurring beneficial ingredients and they represent the second most important vegetable crop grown worldwide, next to potato (Food and Agriculture Organization of the United Nations, 2004). Tomatoes are also a rich source of several nutrients. They have high levels of vitamin C content, vitamin A and vitamin K and different minerals such as molybdenum, potassium, manganese, chromium. In addition, tomatoes are a good source of vitamin B1 and B6, folate, copper, niacin, vitamin B2, magnesium, iron, pantothenic acid, phosphorus, vitamin E and protein (Health Canada, 2008). All the physical attributes or characteristics of the tomato are related to their degree of purity, flavor and maturity that contributes to consumers acceptance

103 (Gould, 1992). However, color is one of the most important quality attributes of a fruit or vegetable. It is the first sign observed by consumers and it provides preconceived ideas of the general quality of the produces flavor, freshness, and aroma. In the case of tomato, the color practically represents the measure of total quality in the eye of the consumer. Color is the result of the presence of carotenoid pigments, such as lycopene, a non-nutritive bioactive plant substance (Basu et al., 2007). Lycopene is a natural pigment synthesized by plants and microorganisms but not by animals. An acyclic isomer of -carotene, it is also one of the most potent antioxidants and that most predominant in human plasma. Lycopene has been associated with decreased risks of chronic diseases such as cancer and cardiovascular disease, by protecting critical cellular bio molecules like lipids, lipoproteins, proteins and DNA (Agarwal and Rao, 2000). Fresh tomato fruits and tomato products contain higher levels of lycopene than any other fruit or vegetable (Anon, 2005). The objective of this study is to quantify the effect of hyperbaric treatment on quality attributes of tomato fruit such as firmness, lycopene level, sugar/acid ratio, weight loss and color. 6.2. MATERIALS AND METHODS 6.2.1. HYPERBARIC SYSTEM A dynamic hyperbaric apparatus was designed and built to test the effect of pressure on quality attributes of tomato fruits (Fig. 6.1). The concept of this hyperbaric system was based on the design presented in Chapter 4. It consisted of five vessels connected in a closed circuit through which compressed air was flowing to supply O2 uptake and dilute the CO2 released by the commodity. Each vessel was large enough to treat 20 tomato fruits at a given time. The vessels were made from a stainless steel painting apparatus (GracoTM, Minneapolis, Mn), 300 mm high by 300 mm inside diameter, closed with a bolted steel cover.

104

Relief valve Pressure regulator Pneumatic fitting

Data acquisition and control system card

Pressure transducer Compressed air cylinder Flow meter

Flow control valve Vessel

Figure 6.1 : Hyperbaric system used to test the effect of pressure on tomato.

105 A flat ring rubber seal having a width of 12.7 mm was placed between the cover and the chamber to ensure air tightness. Each cover was equipped with a pressure regulator and a needle valve to individually regulate the pressure and airflow in each of the vessel. A safety relief valve was also installed on the cover to prevent overload pressure. Two SMCTM compression fittings were fastened on the cover of the vessel to connect air flow inlet and outlet using plastic tubes of 3.2 mm inner diameter. An airtight connector was also used to insert a T-type thermocouple inside each vessel to record temperature. The inlet of the vessel was connected to a compressed air cylinder for compressed air supply. A flow meter (BronkhorstTM model F-201C-FBB-22-V) was used to measure the air flow in the range of 5 to 2000 mL/min. An infrared gas analyzer (CrowconTM model Deltagas) was used to measure the CO2 concentration in the range of 0 to 2000 ppm of CO2. A pressure sensor (Bourdon HeanniTM model CTX355H211) was used to measure the pressure inside the vessels in the range of 1 to 9 atmabs. The compressed air cylinder was equipped with a manometer which regulated the pressure up to 10 atmabs. The air flowing through the vessels was circulated to a manifold that redirected the airflow using three-way valves. The air flow was further redirected out of the container or through the pressure sensor, the air flow meter and the CO2 gas detector, depending on whether the vessel was in the measuring or normal mode. The flow meter, control valve, pressure transducer, CO2 gas analyzer, and type-T thermocouple were all connected to a data acquisition and control card (DATA shuttle ExpressTM, Strawberry TreeTM) and a personal computer (Fig. 6.1). A software interface was programmed using WorkBenchTM version 3.0. The interface recorded the pressure, air flow rate, CO2 level and the temperature inside the chamber, computed the respiration rate in real time and provided a display of the process parameters. The respiration rate was computed by multiplying the percentage increase of CO2 concentration of the air by the flow rate and dividing by the fresh weight of tissues (Eq. 6.1).

106

Respiration rate =

volume of CO2 released kg of commodity time

( 6.1 )

6.2.2. BIOLOGICAL MATERIAL The biological material used was tomatoes (Lycopersicon esculentum Mill. cv. DRW 453) obtained from a local greenhouse producer. Prior to each experiment, tomatoes were selected to be uniform in size and shape and exempt from bruises. Acceptable color was established as mature green tomatoes near breaker stage (USDA, 2007). Breaker stage is a noticeable break in color with less than 10% of color other than green (Lopez Camelo and Gomez, 2004). All selected tomatoes were sanitized for 3 min in a chlorine solution (1000 ppm), then rinsed with tap water for another 3 min, and finally left on filter paper to drain (Polenta et al., 2006). All tomatoes weighed 200 g 10 g. 6.2.3. EXPERIMENTAL SET UP The effect of two system parameters: pressure (atmabs) and duration of the treatment (days), were evaluated on different quality attributes of tomato. The tomato fruits were subjected to five different pressures, 1, 3, 5, 7, or 9 atmabs exposed for three different periods of time, 5, 10 or 15 days. Each combination of pressure and duration was repeated three times. For each pressure/duration combination treatment, 55 tomato fruits were selected and sterilized. Five tomatoes were used for initial quality evaluation prior to treatment. The remaining fruits were randomly divided in five lots of 10 tomatoes for pressure treatment and placed in one of the 5 vessels. The vessels were sealed and continuously pressurized throughout the experiment according to the specific treatment. Respiration rate was recorded in real time over the entire duration of the experiment. Temperature was maintained at 13C and relative humidity (RH) equilibrated at 95%2.5%. Temperature and RH were recorded using a U-23-001 Hobo-ProTM sensor (OnsetTM, Massachusetts, USA), placed inside each chamber of the respirometer. At the end of the pressure treatment,

107 depressurizing the chambers required special care to prevent injuring the tomatoes. The quality attributes were individually measured on 5 tomatoes from each vessel at the end of the pressure treatment. The remaining 5 fruits were stored in a room maintained at 20C and 85%2.5% RH for 12 days of maturation. After this maturation period, tomatoes were analyzed for quality attributes as well. 6.2.3.1. DECOMPRESSION Decompression is a very important factor in any pressurized system. This is well established in medicine for humans where compression/decompression can have detrimental effects when not performed properly (Landolfi et al., 2006). Time is a very important factor in any decompression protocol. Depressurizing has to be performed slowly, step-by-step, to allow time for living organisms to adjust to new environmental factors such as CO2, O2 and N2 levels. The decompression protocol for tomatoes was established after a few preliminary trials. Depressurizing tomatoes was automated and performed over a 24 hours period. Every 6 hours, the pressure was reduced by 2 atmospheres. During the preliminary test, this protocol was effective for all treatments and no visual differences were observed. However, the tomatoes pressurized at 7 and 9 atmabs for 15 days became very fragile at the opening of the chambers. Their skin was very thin and the tomatoes had to be handled carefully to prevent them from bruising or cracking. After the storage period of 12 days, these tomatoes were no longer marketable. Respiration analysis (Chapter 5) demonstrated that under pressures of 7 and 9 atmabs applied for more than 10 days, tomatoes undergo anrobic respiration and start a fermentation process. At the opening, these tomatoes were visually acceptable but had already undergone irreversible physiological damage and were no longer living organisms. This aspect will be considered in the analysis of the data for tomatoes subjected to 10 days at 9 atmabs and the ones subjected to 15 days at 7 and 9 atmabs hyperbaric pressures followed by 12 days of storage. Finally, the results of these specific treatments were not included in the statistical analysis.

108 6.2.4. EVALUATION OF QUALITY PARAMETERS Quality evaluation was undertaken at three different times: before hyperbaric treatment, that is an initial evaluation (IE); after hyperbaric treatment or at the opening of the chambers; and after 12 days of storage at 20C. Investigation of quality consisted of measuring the weight loss, the firmness, the total soluble solids (TSS) and the total acidity (TA), and evaluating the color change. An evaluation of lycopene concentration was also performed upon opening the hyperbaric chambers and after 12 days of storage. 6.2.4.1. WEIGHT LOSS The balance between water content and the various constituents of a ripe tomato is very dependent on its genotype, its nutritional treatment and the environment in which it was grown. A general indication of the weight composition of a ripe tomato fruit varies around 6.5% of dry matter, 14% of various constituents, and 79.5% of water (Hobson and Grierson, 1993). Water loss of fruits and vegetables is the main cause of deterioration since it represents a direct quantitative loss of stable weight as well as a loss in appearance (wilting and shrivelling), texture (softening, flaccidity, limpness, loss of crispiness and juiciness), and of nutritional quality (Kader, 2002). Since the evaporation of water or transpiration is influenced by external factors such as temperature, humidity and atmospheric pressure, it is important to record it in the process of hyperbaric pressure treatments. Tomatoes were labelled and weighed prior to treatment, after chamber opening and after the storage period, using an electronic scale. Weight loss was computed. 6.2.4.2. FIRMNESS Firmness is an important factor to determine product quality and consumers acceptance. A universal testing machine (Lloyd InstrumentsTM, Leicester, England LRX), equipped with a 6 mm diameter round stainless steel probe with a flat end connected to a 100 N load cell, was used to measure the firmness of the

109 fruit. The downward speed of the probe for this test was 25 mm/min. Measurements were expressed in N required to compress the tomato skin by a distance of 3 mm. This technique was referred to as a non destructive method in which the force to deform the tissue by a certain distance was determined (Batu, 1998). Mean values of four readings around the equatorial region was used for the statistical analysis. 6.2.4.3. COLOR AND LYCOPENE Color in tomato is one of the most important characteristic for the consumers purchase decision. It is also a very good indicator to determine fruit ripeness (Lopez Camelo and Gomez, 2004). Color was measured using Commission Internationale de lclairage (CIE) L*, a* and b* color space coordinates with a Minolta chromameter (model CR-400; Konika Minolta, Japan). Color values were evaluated at four locations around the equatorial region of each fruit. From the a* and b* numerical values, a mathematical relationship was used to determine color (Eq. 6.2). The hue angle (H) represents the level of maturation of the treated tomato.

b* 1 = H tan a*

( 6.2 )

Hue angle is a good indicator of color categories in term of human perception (Lopez Camelo and Gomez 2004). A hue angle of 180 represents a pure green color and a hue angle of 0 represents pure red. A table of relationships between visual colors of tomatoes and different ripening indexes is reported in Lopez Camelo and Gomez (2004) from INTA E.E.A Balcarce, Argentina (2001). Values related to Hue angle are presented in Table 6.1. Lycopene level was calculated using the a*/b* ratio with respect to the correlation established by Arias et al. (2000) between the lycopene content measured by HPLC and the chromaticity values of the tomato surface measured with a portable Minolta chromameter. The equation used to predict lycopene content

110

Table 6.1 : Ripening index values for tomato fruits at different color stages. Adapted from Lopez Camelo and Gomez, 2004.

Visual color Mature green Breaker Turning Pink Light red Red

Hue 113.3 109.1 93.2 78.1 64.9 59.3

111 was obtained from the linear regression of the correlation of lycopene measured by HPLC and Chromaticity (R2 = 0.905) (Eq. 6.3):

b* Lycopene, mg / 100 g = 8.7073 a*

( 6.3 )

This correlation was established for Laura tomatoes but considering that lycopene content does not vary between cultivars but mainly between seasonal picking dates, the relationship was considered appropriate (Hernandez et al., 2007). The use of the chromameter to predict lycopene content is an easy and non-destructive method. 6.2.4.4. TOTAL SOLUBLE SOLIDS (TSS) AND TITRATABLE ACIDITY (TA) Total acidity represents a mixture of acids naturally present in the fruit and sometimes, of acids produced by the action of mircroorganisms. Acids are largely responsible for the tart or sour flavour. To measure the total soluble solids (TSS) and the Titratable acidity (TA), a 40mm-wide strip was cut off at the equatorial region on five tomato fruits for each treatment. The whole strip was blended with a homogenizer for 15 seconds, and filtered through gauze to remove seeds, skin, and membrane. The juice obtained was used to evaluate the TA and TSS. Titratable acidity was determined using a Titrino 719S automatic titrator (Metrohm, Switzerland) with 1 mL of tomato juice diluted in 30 mL of distilled water. Titration was done with 0.1 N sodium hydroxide of pH 8.1. Titratable acidity was expressed as g of citric acid mL-1 of tomato juice. Total soluble solids content of the juice were determined by the AOAC method using an electronic refractometer (Reichert Analytical Instruments, USA) at room temperature. A drop from a homogeneous juice blend was placed on the

112 refractometer prism and readings were taken directly. TSS is expressed as percentage on the Brix scale. Once the TSS and the TA had been determined, the values were used to compute the sugar to acid ratio. 6.2.5. STATISTICAL ANALYSIS The different quality attributes were measured on each sampling unit consisting of each of the 5 fruits contained in the vessels. The harvesting dates presented a significant effect on some of the quality attributes, as presented by Hernandez et al. (2007). The difference obtained in the measurement of the attributes of the individual fruit included a confounding effect between the replicates in time, which was affected by the time of harvest and the season. Thus, the experimental design was considered as a factorial design including 3 factors (5 pressures, 3 duration, and 3 harvesting dates). The ANOVA analysis was performed on mean values using SAS software, version 9: SAS Institute Inc. Treatment effects reported were significant according to an F test. Significant differences between results were compared using the Least Significant Difference (LSD) with an interval of confidence of 95% (p < 0.05) (Steel and Torrie, 1987). 6.3. RESULTS AND DISCUSSION All quality attributes of the tomatoes were measured prior to the experiment to establish basic values and confirm homogeneity between sampling units. Repetitions were made over time in three groups. There was no significant difference between sampling units. A group of sampling units was placed inside a chamber at 13C and subjected to local ambient atmospheric pressure. These sampling units were considered as control values and used for comparison when necessary. The respiration data analysed in Chapter 5 demonstrated that under the 9 atmabs hyperbaric pressure, the extinction point of tomatoes was reached within 15 days

113 and hyperbaric pressure caused the collapse of tomato cell walls. Consequences of these internal disorders were visually observed at the opening of the chambers after 15 days and the tomatoes from this treatment were not included in the analysis. Under the 7 atmabs hyperbaric pressure maintained for 15 days, tomatoes had no visual signs of damages at the opening of the chambers but were rotten and not acceptable after 12 days of storage. These tomatoes were also removed from the statistical analysis. 6.3.1. WEIGHT LOSS All tomatoes were weighed prior to the hyperbaric treatment, at the opening of the hyperbaric chambers and after the storage period. The weight difference, with respect to the initial value, was considered for analysis as weight loss. 6.3.1.1. OPENING Fruit weight loss was significantly affected (p0.05) by hyperbaric treatments (Fig. 6.2). The average weight loss for the control across the three treatment durations was 2.73%. The average weight loss for the 3, 5, 7 and 9 atmabs hyperbaric treatments across the three treatment durations were significantly different and were respectively 0.82%, 0.47%, 0.32% and 0.24%. For the control, the rate of weight loss was 0.36% per day for 5 days for a total of 1.8% and then dropped to 0.23% per day for 15 days for a total of 3.4%, and they were significantly higher than those measured under all hyperbaric pressure treatments. The rate of weight loss under 3 atmabs was stable in time at 0.08% per day; under 5 atmabs and higher pressures, the rate of weight loss varied between 0.06 to 0.04% per day. The vapour pressure deficit between the tomatoes and the surrounding air was lower as the air pressure increased. In general, there is an increase in water loss with treatment time for the 1 and 3 atmabs treatments with a significant increase in water loss as treatment time increased. At 5 atmabs, water loss stabilized between the 10 and 15 days

114

10 9 8

5 days 10 days 15 days

Weight loss %

7 6 5 4 3 2 1 0 1 3 5 7

b c

a f g e d h f e f h g f g g h h h

h h
9

Pressure (atmabs)

Figure 6.2 : The percentage of tomato weight loss after 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. Vertical bars indicate standard deviation. Means with the same letters are not significantly different at = 0.05.

115 treatments and for the 7 and 9 atmabs, there was no significant difference between treatment durations. 6.3.1.2. AFTER 12 DAYS OF STORAGE Weight loss kept increasing over the 12 days of storage at 20C for tomatoes that were initially maintained at atmospheric pressure (Fig. 6.3). At opening, the 9 atmabs treatments had undergone the least weight reduction with 0.24% but after 12 days of storage, the average weight loss was the largest with 6.53%, which was 27 times greater than at opening. Tomatoes subjected to hyperbaric pressure of 9 atmabs maintained for 10 days had the largest increase in weight loss of all tested tomatoes with a final value of 7.69%. The least weight loss was observed for treatments of 5 and 3 atm for 5 days with values of 3.16% and 3.22% respectively. Values of 3.51% and 3.99% were not significantly different between 5 atmabs treatment kept for 15 or 10 days, respectively. After the 12 days of storage at room temperature, the rate of weight loss for the control kept decreasing to values of 0.24% to 0.22% per day. For the hyperbaric treated tomato, the average rate of weight loss was still lower than the control after 12 days of storage, passing from 0.19% for the 5 and 10 days of hyperbaric treatments, to 0.16% to 0.13% after 15 days. 6.3.2. FIRMNESS Marketable tomatoes usually have a firmness value above 1.45 N mm-1, although consumers still consider the firmness value of 1.28 N mm-1 acceptable (Batu, 2004). Generally, the hyperbaric treatments had a positive effect on the firmness of tomatoes (Figures 6.4, 6.5).

116

5 (+12 days mat) 10 9 8 10 (+12 days mat) 15 (+12 days mat)

Weight loss (%)

7 6 5 4 3 2 1 0

b b e f

d e e f f g

e f f g g

b c c d d e

b c

Pressure (atmabs )

Figure 6.3 : The percentage of tomato weight loss after 12 days maturation at 20C and 80% RH for 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. Vertical bars indicate standard deviation. Means with the same letters are not significantly different at = 0.05.

117 6.3.2.1. OPENING Under ambient atmospheric pressure, the control tomatoes shrivelled rapidly (Fig. 6.4). On average, the control had lost approximately 50% of the initial firmness at opening. The average firmness of the tomato decreased from an initial value of 4.78 N mm-1 to 2.47 N mm-1 over the three treatment duration. But the tomatoes were still marketable. The loss in firmness under ambient atmospheric pressure was significantly higher than under any of the hyperbaric treatments. Pressures sustained for 15 days resulted in significant firmness reduction compared to the 5- and 10-day treatments (Fig. 6.5). On average, pressures sustained for 5, 10 and 15 days above atmospheric pressure resulted in firmness reduction with respect to initial values of 29.5%, 34.2% and 39.3%, respectively. Higher firmness values were obtained for pressures of 9 and 7 atmabs applied for 5 days with reductions of 27.3% and 27.6% respectively from the initial values. 6.3.2.2. AFTER 12 DAYS OF STORAGE Differences in firmness values of all tomatoes are not very large after 12 days of storage at 20C but are determinant in separating marketable tomatoes, consumer acceptable tomatoes and tomatoes that are too shrivelled for consumption. Under atmospheric pressure, firmness decreased with increased treatment duration (Fig. 6.5). For the 5 and 10 days treatments, firmness was under the marketable value (1.45 N mm-1) with values around 1.35 N mm-1. For the 15 days treatment, firmness was under the consumers acceptance level (1.28 N mm-1) with a firmness value of 1.19 N mm-1. Under hyperbaric pressure, firmness decreased with increased hyperbaric pressure. All pressurized tomatoes had firmness values still acceptable for consumers (>1.28 N mm-1) and they were even marketable (>1.45 N mm-1) in some cases; tomatoes subjected to the 3, 5 and 7 atmabs for 10 days as well as

118

6 5

Firmness (N/mm)

4 3 2 1 0

d e

a b b c c d e f g

a a c b b d c e

initial 5 days 10 days 15 days

a a

e f

a f

Pressure (atmabs)

Figure 6.4 : Initial tomato firmness and after 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. The firmness is expressed in N mm-1 required to penetrate the tomato by 3 mm using a 6 mm diameter flat punch. Vertical bars indicate standard deviation. Means with the same letters are not significantly different at = 0.05.

119

6 5

5 (+12 days mat) 10 (+12 days mat) 15 (+12 days mat)

Firmness (N/mm)

4 3 2 1 0 1 3 5 7 9

a b b c c d d

a a b b a c

a b c a a

a b c a

a c b d c

Pressure (atmabs)

Figure 6.5 : Tomato firmness after 12 days maturation at 20C and 80% RH for 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. The firmness is expressed in N mm-1 required to penetrate the tomato by 3 mm using a flat 6 mm diameter punch. Vertical bars indicate standard deviation. Means with the same letters are not significantly different at = 0.05.

120

the 5 atmabs for 5 days had firmness values of 1.45, 1.48 1.48 and 1.47 N mm-1 respectively. 6.3.3. COLOR AND LYCOPENE Photographs were taken of tomatoes before the treatment (tomatoes used to determine initial values), at the opening of the chambers and after 12 days of maturation (for 5 days treatments only). These pictures are presented for each treatment as a reference for the reader (Appendix A). This gives a visual indication of color indices and maturation. 6.3.4. COLOR Hue values for mature green and breaker stage tomatoes are considered to be

113.3 and 109.1, respectively (Table 6.1). Initial Hue values of the samples
were 110.5 on average and were thus considered the near breaker stage . 6.3.4.1. OPENING The Hue values of the tomatoes within the control group were significantly different from those of pressure-treated tomatoes, and with respect to treatment duration (Fig. 6.6). The Hue values at 10 and 15 days were < 59.3 and the color stage was considered red. The 5 days treatment had a hue value of 67.5 and was considered pink. The color of tomatoes under atmospheric pressure for 5 days was not significantly different than those of the 3 atmabs treatment sustained for 10 or 15 days. Hyperbaric pressure had a significant effect on color development. As pressure increased, the development of red color was delayed compared to the control. Treatment time is also significant. Hue values decreased as treatment time increased, except for the 9 atmabs treatment. There was no significant difference between the 7 atmabs treatment for 5 days and the 9 atmabs for a 5 or 10 days

121

initial 120 100 5 days 10 days 15 days

Color (hue)

80 60 40 20 0

f h i

d e f

c g

b d e

c c

a ba

Pressure (atmabs)

Figure 6.6 : Initial Tomato color and after 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. Vertical bars indicate standard deviation. Means with the same letters are not significantly different at = 0.05.

122 duration. These had hue values around 90 and were considered as turning. There was no significant difference between the 5 atmabs treatment for 5 days and the 7 atmabs treatments for 10 or 15 days. These had hue values around 84 and were also considered as turning. The 3 atmabs 5 days duration and the 5 atmabs, 10 days duration treatments had values of roughly 77 and were considered as pink. 6.3.4.2. AFTER 12 DAYS OF STORAGE Even if there was a significant effect of pressure treatment and duration on color development, these differences were small, and all tomatoes were considered red after 12 days of storage, with hue values 59.3, varying from 41.05 to 45.22 (Fig. 6.7). The greatest hue values were obtained for tomatoes under atmospheric pressure. For pressurized tomatoes, color development was inhibited during hyperbaric treatment but was accelerated during subsequent storage. Color development was not significantly different between tomatoes between 1, 3, 5 and 7 atmabs for 5 days duration and the 3 atmabs for 10 days treatments. A darker red color developed for the 5, 7 and 9 atmabs for 10 days and for a 9 atmabs for 5 days treatments. 6.3.5. LYCOPENE The lycopene content was calculated based on the a* and b* values (Eq. 6.3). and increased as the Hue value decreased (Figs. 6.8 & 6.9). All tomatoes that were considered turning (maturity stage) with respect to the Hue value had comparable and very low lycopene contents. Hyperbaric pressure significantly inhibited color development and lycopene synthesis. 6.3.5.1. OPENING Lycopene content of the control under atmospheric pressure significantly increased in time, as tomatoes ripened (Fig. 6.8). Control tomatoes had significantly higher lycopene contents than tomatoes subjected to hyperbaric

123

5 (+12 days mat) 120 100 80 60 40 20 0 1 3 5 7 9 10 (+12 days mat) 15 (+12 days mat)

Color (hue)

b a a

b b a

c b d d

b c d

c d d

Pressure (atmabs)

Figure 6.7 : Tomato color after 12 days maturation at 20C and 80% RH for 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. Vertical bars indicate standard deviation. Means with the same letters are not significantly different at = 0.05.

124
5 days 14 10 days 15 days

Lycopene (mg/100g)

12 10 8 6 4 2 0 1

a b

f e g

c f e g f

f f f g g g

g g

Pressure (atmabs)

Figure 6.8 : Lycopene concentration of tomato after 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. Vertical bars indicate standard deviation. Means with the same letters are not significantly different at = 0.05.

125

5 (+12 days mat) 10 (+12 days mat) 15 (+12 days mat) 14 12

Lycopene (mg/100g)

c c d

a b a

b a c

a a b b

10 8 6 4 2 0 1 3 5 7 9

Pressure (atmabs)

Figure 6.9 : Lycopene concentration of tomato after 12 days maturation at 20C and 80% RH for 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. Vertical bars indicate standard deviation. Means with the same letters are not significantly different at = 0.05.

126 pressures, except for the 3 atmabs 15 days, which developed more lycopene than the control 5 days duration treatment. Under hyperbaric pressure, lycopene content significantly increased in time for the 3 and 5 atmabs treatments. There was no significant difference for the 7 and 9 atmabs, for any duration. These tomatoes were all turning and had low lycopene content. Compared to the control, lycopene synthesis was reduced by 57%, 71% and 77% by hyperbaric treatments of 3, 5, and 7 and 9 atmabs respectively. 6.3.5.2. AFTER 12 DAYS OF STORAGE Lycopene concentration between treatments was correlated to the results obtained for Hue values (Fig. 6.9). No increase in lycopene content was observed between the opening and the end of the storage period for tomatoes at atmospheric pressure for 15 days and only 1.3 times more lycopene was recorded for the tomatoes subjected for 10 days duration. It is obvious from these results that hyperbaric pressure has an impact on lycopene synthesis in tomatoes. Under hyperbaric pressure lycopene stayed steady during treatments at 13C and induced lycopene production during storage at 20C, compared to the control group of tomatoes. Under 5 atmabs, 15 days and 7 atmabs for 10 days, there was a five-fold increase in lycopene content over the storage period and the final value was significantly higher (by 28%) than the control . It is interesting to observe Hue values and lycopene content development in the control group, at atmospheric pressure and a low temperature of 13C. Compared to the results presented by Arias et al. (2000), lycopene did not develop to its full potential for tomatoes subjected to a low temperature. The longer the exposure to low temperature (13C instead of 20C), the less lycopene was synthesized. Javanmardi and Kubota (2006) evaluated lycopene development in tomatoes subjected to storage temperatures of 4, 12 and 18C for 14 days. They reported

127 that the storage temperature had a tremendous effect on lycopene development. Low temperatures, 4C and 12C, significantly reduced lycopene development compared to storage at room temperature. That is in line with the literature where it is cited that the formation of lycopene occurs at temperatures between 12 and 32C, with optimal conditions between 16C and 26C (Trk et al., 1994). In the present study, lycopene formation was significantly influenced by the exposure to low a temperature (13C). It appears that the longer tomatoes were kept at 13C, the less lycopene they were able to synthesize during storage at 20C. Long term exposure to temperature of 13C alone seems to inhibit the physiological processes that synthesize lycopene. Previous research did not extend the exploration on lycopene content after the low temperature storage period, providing no comparatives for the present results. The conservation temperature recommended for storage of tomatoes is 13C (Kader, 2002). But considering the results obtained now, that temperature might be too low to allow optimal lycopene synthesis after the storage period. 6.3.6. TITRATABLE ACIDITY Titratable acidity was significantly influenced by the hyperbaric treatments. 6.3.6.1. OPENING On average, the control presented 12.9% less acidity than the initial value (Fig. 6.10). There was a significant difference between the control and the hyperbaric treatments; the acidity was reduced by 7.1% under hyperbaric pressure, compared to the control. On average, there was no significant difference between the 3, 5, 7 and 9 atmabs pressure treatments (Fig. 6.10). Furthermore, there was no significant difference between the acidity content after 5 and 10 days at atmospheric pressure and 3 or 5 atmabs after 5 days. However, treatment duration significantly influenced the acidity level, which decreases as treatment duration increased.

128

initial 6.0

Titratable acidity (mg/ml)

5 days 10 days 15 days

5.5 5.0 4.5 4.0 3.5 3.0 1 3 5

aa

c d

c d a e f e f

a c b d d e e f

c b d c f

c d ec f d

Pressure (atmabs)

Figure 6.10 : Initial TA of tomato and after 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. Vertical bars indicate standard deviation. Means with the same letters are not significantly different at = 0.05.

129 6.3.6.2. AFTER 12 DAYS OF STORAGE There were significant differences, between the values at the onset and after 12 days of storage, among durations, for all treatments. Acid content generally decreased over the storage period. On average the values were 4.25 (Fig. 6.10) to 3.25 (Fig. 6.11), which corresponded to reductions of 24%. 6.3.7. TOTAL SOLUBLE SOLIDS (TSS) Generally, TSS contents were significantly higher than the initial value after 5 days (Fig. 6.12 & 6.13). After 10days, it decreased to values not significantly different from the initial TSS content. After 15 days treatments, TSS content had decreased significantly compared to the initial value. 6.3.7.1. OPENING On average, the TSS content of the control was higher by 3.1% than the TSS content of tomatoes exposed to hyperbaric pressures (Fig. 6.12). TSS content is influenced by hyperbaric pressure treatments compared to the control and by the treatment duration: it decreased with increases in time and hyperbaric pressure, but on average, there was no significant difference between the TSS obtained under the different hyperbaric pressures. Individual treatments showed very little difference in TSS. After the 5 and 10 days duration treatments, the 1, 3, 5 and 7 atmabs did not give significantly different results. Treatment duration significantly influenced the TSS content. On average, it decreased by 4.7% with every 5 days of treatment duration. After 15 days of hyperbaric treatment, the TSS was lower than what was measured at the initial value. 6.3.7.2. AFTER 12 DAYS OF STORAGE In general, the TSS increased over the storage period (Fig. 6.13). There was a significant difference among treatments and generally, TSS increased as pressure increased, except for the pressures maintained for 15 days, where TSS

130

6.0

5 (+12 days mat) 10 (+12 days mat) 15 (+12 days mat)

Titratable acidity (mg/ml)

5.5 5.0 4.5 4.0 3.5 3.0 1 3 5 7 9

a b c c d d a e e f

a b c a d b f

d e e e f f f

b c a d b e

a b c a d b e c

Pressure (atmabs)

Figure 6.11 : TA of tomato after 12 days maturation at 20C and 80% RH for 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. Vertical bars indicate standard deviation. Means with the same letters are not significantly different at = 0.05.

131

initial 5 days 10 days 15 days 5.5

Soluble solid ( Brix)

5.0 4.5 4.0 3.5 3.0

be acf g

ad be f g h

a c b d eg h

a c b d e h

b c d f g

Pressure (atmabs)

Figure 6.12 : Initial TSS of tomato and after 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. Vertical bars indicate standard deviation. Means with the same letters are not significantly different at = 0.05.

132

5 (+12 days mat) 10 (+12 days mat) 15 (+12 days mat)

5.5

Soluble solid (oBrix)

5.0 4.5 4.0 3.5 3.0

a b d d d d e

c c d d e

a b

a b c

Pressure (atmabs)

Figure 6.13 : TSS of tomato after 12 days maturation at 20C and 80% RH for 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. Vertical bars indicate standard deviation. Means with the same letters are not significantly different at = 0.05.

133 content was significantly lower than for all other treatment conditions, and were even lower than the initial values.

6.3.8. TSS TA RATIO There was no significant difference among the treatment durations of 5, 10 and 15 days (Fig. 6.14 & 6.15). Also, there was no significant difference between the 4 hyperbaric pressures. After 12 days storage, the TSS/TA ratio decreased over the treatment duration for the 3, 5, 7 and 9 atmabs pressure treatments. The highest ratios were obtained with pressures of 3, 5, 7 and 9 atmabs for 5 days and 5 atmabs for 10 days. 6.4. GENERAL DISCUSSION The results presented here should help determine the best possible hyperbaric treatment to improve the quality attributes of the fruits or vegetables while allowing conservation of the stored commodities. Consumers acceptance makes a big difference in the determination of the quality of a fruit. But it is even more interesting if the treatment can provide a longer storage life to marketable produce. Hyperbaric pressures of 3, 5, 7 and 9 atmabs were tested for different durations, 5, 10 and 15 days (at 13C), followed by a storage period of 12 days at 20C. After the 12 days storage period, all control tomatoes were no longer acceptable for consumption, on the basis of their firmness values. Hyperbaric pressure sustained for 15 days was too much in most of the cases; weight loss was higher, firmness values were below marketable values and, 15 days duration of treatment associated with high hyperbaric pressures such as 7 and 9 atmabs, caused irreversible physiological damage to the tomatoes.

134

16 15

initial 5 days 10 days 15 days

Sugar/acid ratio

14 13 12 11 10 9 8

b c d d e e f f g g g

a a b b c e c d f e g

c d e f g

a b c d

a b c d e f

c d a e b f g

a b c d e f

a f g

Pressure (atmabs)

Figure 6.14 : Initial TSS/TA ratio of tomato and after 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. Vertical bars indicate standard deviation. Means with the same letters are not significantly different at = 0.05.

135
5 (+12 days mat) 10 (+12 days mat) 15 (+12 days mat) 16 15 14

b c c c d d d

a a b b c c d e d e

a b d e

a b c d

Sugar/acid ratio

13 12 11 10 9 8 1 3 5 7 9

Pressure (atmabs)

Figure 6.15 : Initial TSS/TA ratio of tomato after 12 days maturation at 20C and 80% RH for 5, 10 and 15 days of continuous hyperbaric treatment at a temperature of 13C and 95% RH. Vertical bars indicate standard deviation. Means with the same letters are not significantly different at = 0.05.

136 Firmness values considered marketable after 12 days maturation were obtained for treatments of 3, 5 and 7 atmabs. These values should be associated with weight loss. The lowest weight loss values were observed with treatments of 3 and 5 atmabs for 5 days and 5 atmabs for 10 days. Along with what is considered marketable, the antioxidant content of the fruits and vegetables is a factor increasing in importance with consumers, as it is well recognized that they are beneficial substances for human health. Lycopene content of the tomatoes was improved by hyperbaric pressure. The highest value of lycopene is obtained for tomatoes subjected to 5 atmabs for 10 or 15 days. For safety and economic considerations, parameters of the hyperbaric chambers should be chosen with respect to the lowest pressure giving the best quality values. Considering the quality values of the tomatoes, hyperbaric pressure of 5 atmabs maintained for 10 day duration treatments followed by 12 days of storage provides tomatoes still marketable, with a darker red color, the highest level of lycopene and a TSS/TA ratio not being significantly different than what would be obtained with untreated fruits. 6.5. CONCLUSION Hyperbaric pressure has an impact on the quality attributes of tomatoes. Overall, there was no significant impact on the TSS/TA ratio. Hyperbaric pressure contributed significantly to a decrease in the weight loss normally encountered with untreated tomatoes, and improved firmness so as to maintain it within marketable values, and increased color development and lycopene synthesis. Over the treatment periods of 5, 10 and 15 days at 13C and 95% RH, there were significant differences observed in the development of lycopene content in the tomatoes. The control group of tomato developed lycopene rapidly over the treatment period but it did not continue developing lycopene over the storage period of 12 days at 20C. It seems that temperature had an effect on the

137 synthesis of lycopene and the low temperature definitely inhibited the synthesis of lycopene for the control tomatoes. Lycopene development was inhibited by hyperbaric pressure but, over the storage period of 12 days at 20C, lycopene developed to levels higher than what was observed in the control tomatoes. Recommendations for the design of a hyperbaric pressure system should be based on the results obtained for quality attributes, on respiratory quotient, as well as safety and economic considerations. All considered, the 5 atmabs hyperbaric pressure maintained for 10 days, seems to represent the most suitable combination to attain these objectives. 6.6. REFERENCES Agarwal, S. and A.V. Rao. 2000. Tomato lycopene and its role in human health and chronic diseases. Canadian Medical Association Journal 163(6): 739744. Ahmed, J. and H.S. Ramaswamy. 2006. High pressure processing of fruits and vegetables. Stewart Postharvest Review 1(8): 1-10. Anon. 2005. Canadian Food Trends to 2020. A long range consumer outlook. Report prepared for Agriculture and Agri-Food Canada by Serecon Management Consulting, July 2005. 113 pp. Apelbaum, A., G. Zauberman, and Y. Fuchs. 1977. Subatmospheric pressure storage of mango fruits. Scientia Horticulturae 7(1977): 153-160. Arias, R., T.C. Lee, L. Logendra, and H. Janes. 2000. Correlation of lycopene measured by HPLC with the L*, a*, b* color readings of a hydroponic tomato and the relationship of maturity with color and lycopene content. J. Agric. Food Chem. 48(2000): 1697-1702. Basu, S.K., J.E. Thomas, and S.N. Acharya. 2007. Prospects for growth in global nutraceutical and functional food markets: a Canadian perspective. Australian Journal of Basic and Applied Sciences 1(4): 637-649.

138 Batu, A. 1998. Some factors affecting on determination and measurements of tomato. Journal of Agriculture and Forestry 22(1998): 411-418. Batu, A. 2004. Determination of acceptable firmness and color values of tomatoes. Journal of Food Engineering 61(2004): 471-475. Charles, M.T. and J. Arul. 2007. UV treatment of fresh fruits and vegetables for improved quality: a status report. Stewart Postharvest Review 3(6): 1-8. Food and Agricultural Organization of the United Nations. 2004. Agricultural data FAOSTAT. http://faostat.fao.org/ Gould, W.A. 1992. Tomato production, processing and technology. 3rd Ed. CTI Publications inc. Baltimore, USA. 536 pp. Goyette B, M.T.Charles, C.Vigneault and G.S.V. Raghavan. 2007. Pressure treatment for increasing fruit and vegetable qualities. Stewart Postharvest Review. 3(3): 5.1-5.6 Health Canada. 2008. Nutrient value of some common foods. HC Pub.: 4771. Cat.: H164-49/2008E-PDF. 68 pp. Hernandez, M., E. Rodriguez, and C. Diaz. 2007. Free hydroxycinnamic acids, lycopene, and color parameters in tomato cultivars. J. Agric. Food Chem. 55(2007): 8604-8615. Hobson, G. and D. Grierson. 1993. Tomato. In: Biochemistry of Fruit Ripening. Seymour, G.B., J.E. Taylor, and G.A. Tucker Eds. Chapman & Hall, London, UK. Javanmardi, J. and C. Kubota. 2006. Variation of lycopene, antioxidant activity, total soluble solids and weight loss of tomato during postharvest storage. Postharvest Biology and Technology 41(2006): 151-155. Kader, A.A. 2002. Postharvest biology and technology: an overview. In: Post Harvest Technology of Horticultural Crops, Third Edition. Ed. A.A. Kader. University of California, Davis, CA: Division of Agriculture and Natural Resources. Publication 3311, 535 pp. Kader, A.A. and S. Ben-Yehoshua. 2000. Effects of superatmospheric oxygen levels on postharvest physiology and quality of fresh fruits and vegetables. Postharvest Biology and Technology 20(2000): 1-13.

139 Lacroix M. and C. Vigneault. 2007. Irradiation treatment for increasing fruit and vegetable quality. Stewart Postharvest Review. 3(3): 7.1-7.8 Landolfi, A., Z.J. Yang, F. Savini, E.M. Camporesi, F. Faralli, and G.Bosco. 2006. Pre-treatment with hyperbaric oxygenation reduces bubble formation and platelet activation. Sport Sci Health. 1(2006): 122-128. Lopez Camelo, A.F. and P.A. Gomez. 2004. Comparison of color indexes for tomato ripening. Hortic. Bras. 22(3): 534-537. Lu, J., C. Vignault, M.T. Charles, and G.S.V. Raghavan. 2007. Heat treatment application to increase fruit and vegetable quality. Stewart Postharvest Review 3(4): 1-7. Lu J., P. Delaquis, C. Vigneault, M.T. Charles, G.S.V. Raghavan, V Toussaint and J.W. Austin. 2008. Multidisciplinary approach to postharvest heat treatment of fruits and vegetables. In : C. Stevens and V.A. Khan (Eds). Recent Advances in Agriculture. Research Signpost, Kerala, India. Chapter 8, p.189-210. Polenta, G., C. Lucangeli, C. Budde, C.B. Gonzalez, and R. Murray. 2006. Heat and anaerobic treatments affected physiological and biochemical parameters in tomato fruits. LWT 39(2006): 27-34. Raghavan, G.S.V., C. Vigneault, Y. Garipy, N.R. Markarian and P. Alvo. 2005. Refrigerated and controlled/modified atmosphere storage. In D. Barrett, L. Somogyi and H. Ramaswamy (eds). Processing Fruits, Science and technology. 2nd edition. CRC Press. 23-52 Rennie, T., C. Vigneault, J. R. DeEll and G.S.V. Raghavan. 2003. Cooling and storage. In: A. Chakraverty, A. S. Mujumdar, G. S. V. Raghavan and H. S. Ramaswamy (eds.), Handbook of Postharvest Technology: Cereals, Fruits, Vegetables, Tea, and Spices. Marcel Dekker Inc. New York, NY. 505-538. Shama, G. 2005 Ultraviolet light. In: Hui, Y.H. (Ed.), Handbook of Food Science, Technology and Engineering. CRC Press, Boca Raton, USA, pp. 122-1-12214.

140 Shama, G. 2007. Process challenges in applying low doses of ultraviolet light to fesh produce for eliciting beneficial hormetic responses. Postharvest Biology and Technology 44(2007): 1-8. Steel, R.G.D. and J.H. Torrie. 1987. Principle and procedures of statistics. A biometrical approach (2nd ed.). New York: McGraw-Hill Book Company. 633 pages. Terry, L.A. and D.C. Joyce. 2004. Elicitors of induced disease resistance in postharvest horticultural crops: a brief review. Postharvest Biology and Technology 32(2004): 1-13. Thomas, M. and J.D. Fidler. 1933. Studies in zymasis. 6. Zymasis by apples in relation to oxygen concentration. Biochem. J. 27(1933): 1629-1642. Trk, R., V. Seniz, N. zdemir, and M.A. Szen. 1994. Changes in the chlorophyll carotenoid and lycopene contents of tomatoes in relation to temperature. Acta Horticulturae (368): 856-862. USDA. 2007. U.S. Standards for Grades of Greenhouse Tomatoes. USDA, Agr. Mktg. Serv., Washington, DC. (http://www.ams.usda.gov/standards/stanfrfv.htm) Vigneault C. and F. Arts Hermndez. 2007. Gas treatments for increasing phytochemical content of fruits and vegetables. Stewart Postharvest Review. 3(3): 8.1-8.9 Wang, S. and J. Tang. 2007. Heating uniformity and differential heating of insects in almonds in radio frequency systems. ASABE Paper No. 076019. St. Joseph, Mich.: ASABE. Zheng, Y. and C.Y. Wang. 2007. Effect of high oxygen atmoshperes on quality of fruits and vegetables. Stewart Postharvest Review 2(1): 1-4.

CHAPTER 7 7. GENERAL SUMMARY AND CONCLUSIONS

The use of physical treatments has been investigated to elaborate alternative methods for postharvest decay control of fruits and vegetables in order to minimise chemical treatments. Early senescence, chilling injuries and pathogens can be controlled by different physical means such as heat or UV-C. But the major problem using these physical treatments is that it is very difficult to treat the fruits or vegetables uniformly. Physical treatments have also shown some effects on the fruits and vegetables physiology and biochemistry. In some cases, hormic stress treatments enable the fruit or vegetable to develop its own mechanism of resistance against pathogens. In fact, fruits and vegetables contain phytochemicals and their concentration level can be increased by stimulating their production with hormic stress. A dynamic, hyperbaric respirometer was designed and built to investigate the effect of hyperbaric hormic stress on respiration rate (RR), respiratory quotient (RQ) and quality attributes of tomato. The respirometer was a reliable tool to measure, in real time, RR and RQ of fruits or vegetables under pressure conditions. In light of the results obtained, pressure treatment can potentially improve the storage life of living produce by reducing their respiration rate. In fact, tomato fruit subjected to 7, 5 and 3 atmabs treatments showed reductions of RR of 20%, 16% and 11%, respectively. But tomato fruit subjected to a hyperbaric pressure of 9 atmabs experienced irreversible physiological damage caused by anaerobic respiration. This observation was confirmed by their RQ value that rose over time to reach a value as high as 1.8. An important phenomenon to take into account while using a hyperbaric respirometer is the solubilisation of CO2 in plant tissues. Solubility induces an error in RR and RQ measurement resulting in underestimating their values. Solubilisation of CO2 in the tomato cells is a

142 phenomenon that needs to be studied to elaborate proper ways to measure the RR and RQ during solubility period. Hyperbaric pressure has an impact on the quality attributes of tomatoes. Hyperbaric pressures of 3, 5, 7 and 9 atmabs were tested for different durations of 5, 10 and 15 days at 13C, followed by a storage period of 12 days. After the 12 days storage period, all control tomatoes were no longer acceptable for consumption on the basis of firmness values. Hyperbaric pressure applied for 15 days was too much in most of the cases; weight loss was higher, firmness values were below marketable criteria, and in the case of high hyperbaric pressures (7 and 9 atmabs,) they caused irreversible physiological damage to the tomatoes. Firmness values considered marketable were obtained for treatments of 3, 5 and 7 atmabs maintained for 10 days, and 5 atmabs maintained for 5 days. These values are mainly to be associated with weight loss. The lowest weight loss values were observed with treatments of 3 and 5 atmabs for 5 days and 5 atmabs for 10 days. Another important factor that affects the marketability of fruits and vegetable is the presence of phytochemicals. The antioxidant content of the fruits and vegetables is a factor increasing in importance with consumers, as they are well recognized as a beneficial substance to human health. The lycopene content of tomatoes is improved by hyperbaric pressure. The highest value of lycopene was obtained for tomatoes subjected to 5 atmabs for 10 or 15 days. Overall, there is no significant impact on TSS/TA ratio. Hyperbaric pressure contributed significantly to a decrease in weight loss normally encountered with untreated tomatoes, an improved firmness within marketable values and, an increase in color development and lycopene synthesis. Over the treatment periods of 5, 10 and 15 days at 13C and 95% RH, there were significant differences observed in the development of lycopene content in the tomatoes. The control group of tomato developed lycopene rapidly over the treatment period but it did not continue developing lycopene over the storage period of 12 days at

143 20C. It seems that temperature had an effect on the synthesis of lycopene and the lower temperature definitely inhibited the synthesis of lycopene for the control tomatoes. For safety and economic considerations, parameters of the hyperbaric chambers should be chosen with respect to the lowest pressure giving the best quality values. Considering the quality values of the tomatoes, a hyperbaric pressure of 5 atmabs maintained for 10 day duration treatments followed by 12 days of storage at 20C produced tomatoes which were still marketable, had a darker red color, higher levels of lycopene and a TSS/TA ratio not significantly different from those of untreated fruits. The next step would be to investigate the use of hyperbaric treatment under less refrigeration. Cooling and keeping cold a large storage room, to the right temperature, can be a difficult task. If cooling is not done in an appropriate time span, the commodity can be affected in an irreversible way and its market value can be reduced. Also, during transport or in under developed countries, keeping commodities cold, at the right temperature, without fluctuation, is a very difficult, if not an impossible task to achieve. Investigation on reducing refrigeration needs while using hyperbaric treatment should be performed in the near future. Hyperbaric treatment improving the quality of mature green tomatoes or other commodities along with a reduction in the refrigeration needs would be a major discovery in the postharvest field.

CHAPTER 8 8. RECOMMENDATIONS FOR FUTURE STUDIES

Based on the results obtained in this study, many aspects of the hyperbaric treatment should be further investigated in order to have a better understanding of the response obtained to this physical stress by the commodities. Here is the list:

Determine the effect of CO2 partial pressure inside the respiration chamber on
RR and RQ for commodities subjected to different hyperbaric pressures.

Determine the CO2 solubility coefficient in tomato in order to get a more

accurate RR and RQ response when using a hyperbaric respirometer.

Determine the optimum temperature/pressure combination to synthesise

lycopene after hyperbaric treatments during the maturation period.

Evaluate the possibility of reducing the refrigeration need to preserve the

quality of fruits and vegetables while using hyperbaric storage facilities.

Develop a decompression method specially adapted to specific fruits or

vegetables.

Test hyperbaric treatment on other commodities to verify the effects in terms of

phytochemical synthesis and quality preservation.

CHAPTER 9 9. CONTRIBUTIONS TO KNOWLEDGE

The major contributions to knowledge generated by this study are:

For the first time, a hyperbaric respirometer was designed, built and tested on
tomato fruits. This novel approach allows real time measurement of the metabolism state of the tomatoes subjected to hyperbaric stress. This hyperbaric respirometer can also be used to test other commodities.

Through the investigation of hyperbaric treatment on tomatoes, lycopene was

synthesized, ripening was delayed, there was a decrease in water loss and the overall quality of the fruits was improved under a specific treatment.

Through the investigation, it was demonstrated that storage temperature and

time affect the lycopene synthesis of tomato during the maturation period.

10. REFERENCES
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APPENDIX A

163

TREATMENT 5 DAYS Treatment: 5 days, date 1; Analysis @ day 0 1 atmabs 3 atmabs 5 atmabs 7 atmabs 9 atmabs

Treatment: 5 days, date 1; Analysis @ day 5trt + 0mat 1 atmabs 3 atmabs 5 atmabs 7 atmabs 9 atmabs

Treatment: 5 days, date 1; Analysis @ day 5trt + 12mat 1 atmabs 3 atmabs 5 atmabs 7 atmabs 9 atmabs

164

Treatment: 5 days, date 2; Analysis @ day 0 1 atmabs 3 atmabs 5 atmabs 7 atmabs 9 atmabs

Treatment: 5 days, date 2; Analysis @ day 5trt + 0mat 1 atmabs 3 atmabs 5 atmabs 7 atmabs 9 atmabs

165

Treatment: 5 days, date 3; Analysis @ day 0 1 atmabs 3 atmabs 5 atmabs 7 atmabs 9 atmabs

Treatment: 5 days, date 3; Analysis @ day 5trt + 0mat 1 atmabs 3 atmabs 5 atmabs 7 atmabs 9 atmabs

166

TREATMENT 10 DAYS

Treatment: 10 days, date 1; Analysis @ day 0 1 atmabs 3 atmabs 5 atmabs 7 atmabs 9 atmabs

Treatment: 10 days, date 1; Analysis @ day 10trt + 0mat 1 atmabs 3 atmabs 5 atmabs 7 atmabs 9 atmabs

167

Treatment: 10 days, date 2; Analysis @ day 0 1 atmabs 3 atmabs 5 atmabs 7 atmabs 9 atmabs

Treatment: 10 days, date 2; Analysis @ day 10trt + 0mat 1 atmabs 3 atmabs 5 atmabs 7 atmabs 9 atmabs

168

Treatment: 10 days, date 3; Analysis @ day 0 1 atmabs 3 atmabs 5 atmabs 7 atmabs 9 atmabs

Treatment: 10 days, date 3; Analysis @ day 10trt + 0mat 1 atmabs 3 atmabs 5 atmabs 7 atmabs 9 atmabs

169

TREATMENT 15 DAYS

Treatment: 15 days, date 1; Analysis @ day 0 1 atmabs 3 atmabs 5 atmabs 7 atmabs 9 atmabs

Treatment: 15 days, date 1; Analysis @ day 15trt + 0mat 1 atmabs 3 atmabs 5 atmabs 7 atmabs 9 atmabs

170

Treatment: 15 days, date 2; Analysis @ day 0 1 atmabs 3 atmabs 5 atmabs 7 atmabs 9 atmabs

Treatment: 15 days, date 2; Analysis @ day 15trt + 0mat 1 atmabs 3 atmabs 5 atmabs 7 atmabs 9 atmabs

171

Treatment: 15 days, date 3; Analysis @ day 0 1 atmabs 3 atmabs 5 atmabs 7 atmabs 9 atmabs

Treatment: 15 days, date 3; Analysis @ day 15trt + 0mat 1 atmabs 3 atmabs 5 atmabs 7 atmabs 9 atmabs