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Effect of atmospheric pressure fluctuations on bulk gas flow and composition of flavour volatiles from… Buckley, Katherine Elaine 1995

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EFFECT OF ATMOSPHERIC PRESSURE FLUCTUATIONSON BULK GAS FLOW AND COMPOSITION OF FLAVOUR VOLATILESFROM BULKY PLANT TISSUESByKATHERINE ELAiNE BUCKLEYA THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYINTHE FACULTY OF GRADUATE STUDIES(Department of Plant Science)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIASeptember, 1995© Katherine Elaine Buckley, 1995In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. lt is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of Scctcc)The University of British ColumbiaVancouver, CanadaDate / 7)DE-6 (2188)ABSTRACTThe occurrence of total gas pressure gradients, which act as a drivingforce for the mass transport of fluids, may be common phenomena in plantorgans. A gas-exchange system was devised to characterize changes in CO2emission rates of greenhouse tomato fruit (Lycopersicon esculentum L.Dombito), greenhouse green bell pepper fruit (Capsicum annum L. Doria),slicing cucumber fruit (Cucumis sativa L. Straight Eight and Sweet Success) andjumbo yellow onion bulbs (Allium cepa L.) in response to the imposition of totalgas pressure gradients. Cyclical variations in atmospheric pressure inducedsignificantly higher rates of gas exchange in peppers and onions but not intomatoes and cucumbers. Oxygen concentration significantly affected carbondioxide efflux rates in onions subjected to variable pressures. Temperature hadno significant effect on relative efflux rates in any of the plant organs used in thisstudy. Duration of the interval between varying pressure treatments was animportant factor in CO2 emission rate in onions, tomatoes and cucumbers. Thediffering response of various commodities to varying pressure treatments wasprobably due to differences in routes of gas exchange as well as intercellularspace volumes and internal structure.To determine if variable pressure treatments had a metabolic effect ontissues, a dynamic head-space sampling technique was developed to collect andconcentrate aroma volatiles for analysis by gas chromatography/massspectrometry. Principal component analysis of pepper, onion and tomatovolatiles revealed that variable pressure storage increased levels of compoundsassociated with oxidation compared to those stored under constant pressure.Data from peppers stored under 3% oxygen and variable pressures for I weekindicated that compounds associated with off-flavours were lower than inpeppers stored in air.IITABLE OF CONTENTSABSTRACT iiTABLE OF CONTENTSLIST OF TABLES viLIST OF FIGURES viiiLIST OF ABBREVIATIONS xiiACKNOWLEDGMENTS xiiiINTRODUCTION ILITERATURE REVIEW 5A. Physiological Gases and Their Role in Tissue Function 5The Importance of Oxygen in Tissue Biochemistry and Physiology 6The Importance of Carbon Dioxide in Tissue Biochemistry andPhysiology 8The Importance of Ethylene in Tissue Biochemistry and Physiology 9The Consequences of Water Loss 11B. Diffusive Gas Flow in Bulky Plant Tissues 12Mechanism of Gas Exchange 12Application of the Principles of Gas Exchange 14C. Bulk Gas Flow in Bulky Plant Tissues 15Occurrence of Bulk Gas Flow 15Application of Mass Gas Flow 17D. Flavour Analysis in Food Research 18E. Isolation of Food Flavours 20Headspace Methods 21a. Static Headspace Sampling 21b. Dynamic Headspace Sampling 22Simultaneous Purge and Solvent Extraction 23IIISolvent Extraction Methods .23Distillation Methods 25Supercritical Fluid Extraction Method 26SECTION 1. ACCELERATION OF GAS TRANSFER IN ONION, SWEETPEPPERS, TOMATOES AND CUCUMBERS BY VARYING ATMOSPHERICPRESSURE 28INTRODUCTION 29MATERIALS and METHODS 30A. Plant Materials 30B. Variable Pressure Storage and Monitoring System 30C. Pressure Treatments 33D. Measurement of Carbon Dioxide 35E. Statistical Analysis 36RESULTS and DISCUSSION 39A. Effect of Level of Pressure Variations 39B. Effect of Temperature 45C. Effect of Interval of Constant Pressure Between Pressure Cycles(DCAP) 47D. Effect of Pressure Variation, Temperature and DCAP on Relative Rate ofCO2 Efflux 54E. Effect of Oxygen Concentration 54SECTION 2. ANALYSIS OF AROMA VOLATILES OF ONIONS, PEPPERSTOMATOES AND CUCUMBERS 68INTRODUCTION 69MATERIALS and METHODS 71A. Plant Materials 71Tomatoes 71Onions 71ivSweet Peppers .72B. Reagents and Apparatus 72C. Isolation and Concentration of Aroma Compounds 74D. Capillary GC-MS Analysis 75Analytical Conditions 75Identification of Vegetable Volatiles 76Recovery and Precision Tests 77Statistical Analysis 78RESULTS and DISCUSSION 79A. Method Development for Isolation of Volatiles 79B. Precision and Accuracy 81C. Effect of Atmospheric Pressure Treatments on Onion Volatiles 83D. Effect of Atmospheric Pressure Treatments on Tomato Volatiles 98E. Effect of Atmospheric Pressure Treatments on Sweet Pepper Volatiles.. 115F. Total Water Loss 128GENERAL SUUMARY AND CONCLUSIONS 132REFERENCES 135APPENDIX I 149APPENDIX2 150APPENDIX3 158VLIST OF TABLESTable PageSection 11.1. Varying pressure treatments applied to onions, peppers,tomatoes and cucumbers 371.2. Effect of level of pressure variation, temperature and DCAP onrelative carbon dioxide efflux rate in onion, cucumber, sweet pepperand tomato 551.3. Effect of oxygen concentration, level of pressure variation andtemperature on relative carbon dioxide efflux rate in onion, cucumber,sweet pepper and tomato 611.4. Effect of temperature, level of pressure variation and DCAP on netcarbon dioxide efflux rate of onion during constant and variablepressure treatments 1501.5. Effect of temperature, level of pressure variation and oxygenconcentration on net carbon dioxide efflux rate of onion duringconstant and variable pressure treatments 1511.6. Effect of temperature, level of pressure variation and DCAP on netcarbon dioxide efflux rate of cucumber during constant and variablepressure treatments 1521.7. Effect of temperature, level of pressure variation and oxygenconcentration on net carbon dioxide efflux rate of cucumber duringconstant and variable pressure treatments 1531.8. Effect of temperature, level of pressure variation and DCAP on netcarbon dioxide efflux rate of sweet pepper during constant andvariable pressure treatments 1541.9. Effect of temperature, level of pressure variation and oxygenconcentration on net carbon dioxide efflux rate of sweet pepperduring constant and variable pressure treatments 1551.10. Effect of temperature, level of pressure variation and DCAP on netcarbon dioxide efflux rate of tomato during constant and variablepressure treatments 156vi1.11. Effect of temperature, level of pressure variation and oxygenconcentration on net carbon dioxide efflux rate of tomato duringconstant and variable pressure treatments 157Section 22.1. Accuracy and precision determinations with a water (Model 1)and a green tomato (Model 2) matrix 822.2. Flavour constituents of onion 852.3. Means of volatile compounds identified in onion 882.4. Principal component analysis (PCA) of volatile compounds fromonions stored under constant and variable pressures 912.5. Coefficients of the first five principal components of the onionvolatiles data set 922.6. Flavour constituents of tomato 992.7. Means of volatile compounds identified in tomato 1032.8. Principal component analysis (PCA) of volatile compounds fromtomato stored under constant and variable pressure 1052.9 Coefficients of the first six principal components of the tomato volatilesdata set 1082.10. Flavour constituents of sweet pepper 1172.11. Means of volatile compounds identified in sweet pepper 1182.12. Principal component analysis (PCA) of volatile compounds fromsweet pepper stored under constant and variable pressures 1212.13. Coefficients of the first six principal components of the sweet peppervolatiles data set 1242.14. Means of volatile compounds identified in sweet pepper subjectedto variable pressures at different oxygen concentrations 129viiLIST OF FIGURESFigure PageSection 11.1. Illustration of the variable pressure storage and monitoring system 321.2. Illustration of the variable pressure cycles 341.3. Main effect of atmospheric pressure variations on net CO2 effluxrate of onions, cucumbers, sweet peppers and tomatoes 401.4. Model of bulk gas flow induced by variable pressures 441.5. Main effect of temperature on net CO2 efflux rate of onions, cucumberssweet peppers and tomatoes 461.6. Main effect of DCAP on net CO2 efflux rate of onions, cucumberssweet peppers and tomatoes 481.7. Effect of DCAP, pressure variations and temperature on net CO2efflux rate of onions 501.8. Effect of DCAP, pressure variations and temperature on net CO2efflux rate of cucumbers 511.9. Effect of DCAP, pressure variations and temperature on net CO2efflux rate of sweet peppers 521.10 Effect of DCAP, pressure variations and temperature on net CO2efflux rate of tomatoes 531.11. Main effect of oxygen concentration on net CO2 efflux rate of onions,cucumbers, sweet peppers and tomatoes 571.12. Sustainability of mass gas flow in an onion bulb during variable pressuretreatments 581.13. Effect of oxygen concentration, pressure variations and temperatureon net CO2 efflux rate of onions 591.14. Effect of oxygen concentration, pressure variations and temperatureon net CO2 efflux rate of cucumbers 63VIII1.15. Effect of oxygen concentration, pressure variations and temperatureon net CO2 efflux rate of sweet peppers 641.16. Effect of oxygen concentration, pressure variations and temperatureon net CO2 efflux rate of tomatoes 66Section 22.1 Illustration of the dynamic purge and trap apparatus 732.2 Total ion chromatogram of volatiles extracted from onion stored underconstant pressure at 15°C for 7 days 862.3. Principal component analysis of aroma compounds from onions storedunder constant atmospheric pressure and variable pressure treatments(first and second eigenvectors) 942.4. Principal component analysis of aroma compounds from onions storedunder constant atmospheric pressure and variable pressure treatments(first and third eigenvectors) 952.5. Principal component analysis of aroma compounds from onions storedunder constant atmospheric pressure and variable pressure treatments(first and fourth eigenvectors) 962.6. Principal component analysis of aroma compounds from onions storedunder constant atmospheric pressure and variable pressure treatments(first and fifth eigenvectors) 972.7. Total ion chromatogram of volatiles extracted from tomato stored underconstant pressure at 15°C for 3 days 1012.8. Principal component analysis of aroma compounds from tomatoesstored under constant atmospheric pressure and variable pressuretreatments (first and second eigenvectors) 1062.9. Principal component analysis of aroma compounds from tomatoesstored under constant atmospheric pressure and variable pressuretreatments (first and third eigenvectors) 1072.10. Principal component analysis of aroma compounds from tomatoesstored under constant atmospheric pressure and variable pressuretreatments (first and fourth eigenvectors) 112ix2.11. Principal component analysis of aroma compounds from tomatoesstored under constant atmospheric pressure and variable pressuretreatments (first and fifth eigenvectors) 1132.12. Principal component analysis of aroma compounds from tomatoesstored under constant atmospheric pressure and variable pressuretreatments (first and sixth elgenvectors) 1142.13. Total ion chromatogram of volatiles extracted from sweet pepperstored under constant pressure at 15°C for 3 days 1162.14. Principal component analysis of aroma compounds from peppersstored under constant atmospheric pressure and variable pressuretreatments (first and second eigenvectors) 1222.15. Principal component analysis of aroma compounds from peppersstored under constant atmospheric pressure and variable pressuretreatments (first and third eigenvectors) 1232.16. Principal component analysis of aroma compounds from peppersstored under constant atmospheric pressure and variable pressuretreatments (first and fourth eigenvectors) 1252.17. Principal component analysis of aroma compounds from peppersstored under constant atmospheric pressure and variable pressuretreatments (first and fifth eigenvectors) 1262.18. Principal component analysis of aroma compounds from peppersstored under constant atmospheric pressure and variable pressuretreatments (first and sixth eigenvectors) 1272.19. Comparison of water loss in pressure treated and controlvegetables 1312.20. Total ion chromatogram of volatiles extracted from onion stored underconstant pressure at 15°C for 14 days 1582.21. Total ion chromatogram of volatiles extracted from onion stored underconstant pressure at 15°C for 21 days 1592.22. Total ion chromatogram of volatiles extracted from onion stored undervariable pressure at 15°C for 7 days 1602.23. Total ion chromatogram of volatiles extracted from onion stored undervariable pressure at 15°C for 14 days 161x2.24. Total ion chromatogram of volatiles extracted from onion stored undervariable pressure at 15°C for 21 days 1622.25. Total ion chromatogram of volatiles extracted from tomato stored underconstant pressure at 15°C for 7 days 1632.26. Total ion chromatogram of volatiles extracted from tomato stored undervariable pressure at 15°C for 3 days 1642.27. Total ion chromatogram of volatiles extracted from tomato stored undervariable pressure at 15°C for 7 days 1652.28. Total ion chromatogram of volatiles extracted from sweet pepperstored under constant pressure at 15°C for 7 days 1662.29. Total ion chromatogram of volatiles extracted from sweet pepperstored under constant pressure at 15°C for 14 days 1672.30. Total ion chromatogram of volatiles extracted from sweet pepperstored under variable pressure at 15°C for 3 days 1682.31. Total ion chromatogram of volatiles extracted from sweet pepperstored under variable pressure at 15°C for 7 days 1692.32. Total ion chromatogram of volatiles extracted from sweet pepperstored under variable pressure at 15°C for 14 days 170xiLIST OF ABBREVIATIONSGC, - Gas chromatograph, gas chromatographyGC-MS - Gas chromatography coupled with mass spectrometryDCAP - Duration of the interval of constant pressure between 5 mm variablepressure treatments.HPLC - High performance liquid chromatographyNMR - Nuclear Magnetic ResonanceCA - Controlled atmosphereMA - Modified atmosphereppm - Parts per million (jiL L1)xl IACKNOWLEDGMENTSI wish to express my appreciation to Dr. Peter Jolliffe for his support andencouragement throughout the preparation of this manuscript and the studiesdescribed herein. Sincere thanks are extended to Dr. Joe Molnar for placing thefacilities of Agassiz Research Station at my disposal, and to Dr. Peter Toivonenfor his assistance during the course of this study.Assistance from Mr. Mark Gross, Mrs. Audry Nadalin and Mr. TomHelmner with greenhouse production of the sweet peppers and tomatoes isgratefully acknowledged.My gratitude to my friend, Mary-Margaret Gaye, for encouraging me tofirst undertake this program cannot be adequately expressed.I also wish to thank my husband, Wayne, for his understanding, andencouragement.Financial support was provided by a Strategic Grant to P. A. Jolliffe fromthe Natural Sciences and Engineering Research Council of Canada.XII IINTRODUCTIONShortly after the turn of the century the marketing of fresh plant producewas revolutionized by the introduction of mechanical refrigeration. Huge profitswere realized by reduction in postharvest losses, extension of the fresh produceseason, and generation of new marketing opportunities far removed from thelocation of crop production.Several decades later, refrigerated controlled atmosphere (CA) storagewas developed for commercial use in the storage of tree fruit crops in NorthAmerica and Western Europe. The primary goals of CA storage are to controlthe concentrations of oxygen, carbon dioxide, water vapour and ethylene in theatmosphere surrounding plant produce in order to slow the rates of deteriorationand decay. Conventional CA storage, with 02 levels of 2 to 5% and CO2 levelsof 2 to 5% with the remainder balanced by N2, will maintain pome fruit qualitylonger than storage in air. Since the introduction of CA storage, the system hasbenefited from a number of refinements. Storage of apples in atmospherescontaining 1.5% 02 (“Ultra-low Oxygen CA”) and the imposition of CA conditionsin less than 6 days (“Rapid CA”) have shown promise both on a research and acommercial scale (Little and Peggie, 1987).“Modified atmospheres” (MA) is a term more commonly applied to themanipulation of the atmosphere surrounding the commodity by the introductionof a specific gas mixture into a sealed wrap or the use of specially designed gaspermeable plastic films. MA differs from CA only in the precision of control overexternal gas partial pressures, CA being more precise than MA.The reasons for the beneficial effects of CA and MA on fruits andvegetables are complex. Lowering the 02 partial pressure around fruits andvegetables reduces their respiration rate in proportion to the 02 concentration.However, a shift from aerobic to anaerobic respiration will occur if the 02 levelIfalls below 1-3%, depending on the commodity (Solomos, 1982). Under suchconditions, the glycolytic pathway replaces the Krebs cycle as the main sourceof energy for plant tissues. Instead of being oxidized, pyruvic acid isdecarboxylated to form acetaldehyde, CO2 and ultimately, ethanol, resulting inoff-flavours and tissue breakdown (Kader, 1986). Although the 02 concentrationwithin a cell may be as low as 0.2% before anaerobic respiration occurs, thegradient of 02 concentration from that cell to the external atmosphere requiresthat the commodity be maintained in an atmosphere containing substantiallyhigher levels of 02 (Solomos, 1982). The required 02 concentration in thestorage environment depends on the rate of 02 consumption and the rate of gasdiffusion through dermal and subdermal tissues of the specific cultivar of eachfruit or vegetable.Elevated CO2 concentrations also reduce the respiration rate of fruits andvegetables, but above a level of about 20% (lower or higher depending on 02concentration) there is a danger of anaerobiosis in part of the plant organ due toinhibition of decarboxylation reactions of normal respiration. Storage of freshplant tissues in elevated CO2 atmospheres reportedly inhibits glycolysis andsuccinic dehydrogenase activity, reduces formation of citrate/isocitrate and cketoglutarate and may also have an uncoupling effect on oxidativephosphorylation (Kader, 1986).Ethylene has been shown to damage various fruits and vegetables byaccelerating senescence. Reduced 02 levels can decrease the rate of ethyleneproduction and reduce the sensitivity of horticultural produce to ethylene (Chenet al., 1981). Elevated CO2 may reduce plant tissue response to ethylenepresumably by competing with ethylene for binding sites on ethylene receptors(Veen, 1987) but it may also induce the production of ethylene in the presenceof light (Kao and Yang, 1982). The presence of nonethylenic volatile2compounds in the storage atmosphere can inhibit regrowth in tubers or causebleaching of green vegetables (Lougheed et al; 1987).Exchange of Go2, 02, ethylene and volatile compounds between the outeratmosphere and internal tissues occurs along a concentration gradient by:gaseous diffusion through the dermal system and the intercellular system, anexchange of gases between the intercellular atmosphere and cell sap, anddiffusion in solution within the cell to centers of metabolic activity. Diffusivemovement of gases is not always adequate to prevent excessive CO2 build-up or02 depletion in bulky plant organs or parts of the organs. Mass gas flow, on theother hand, would not be restricted to movement along concentration gradients.Although mass gas flow does not occur in bulky tissues under normal storageconditions, such gas movement may take place wherever temperature orpressure gradients exist.Many of the recent CA experiments have revealed that small changes inthe storage environment can have a significant response in terms of extendingshelf-life for days or weeks, enhancing quality, and ameliorating or controllingCA injuries. To date, little research attention has focused on mass gas transferas a means to alter internal gas compositions thus effecting an “ultra rapid CA”.Consequently there is little information characterizing the effects of pressure-driven gas transfer between fruit and vegetable commodities and theirsurroundings. Information obtained by Jolliffe and Dyck (1988) on pressure-driven bulk gas flow and Corey and Tan (1990) on temperature-driven bulk gasflow implies that internal gas compositions of large plant organs could be alteredat a much faster rate than through diffusion. In this tight, characterization offactors which may affect rate of mass gas transfer is of both theoretical andpractical interest. Identification of the more subtle metabolic effects of mass gastransfer may be useful in determining the benefit of mass gas transfer. The main3objectives of this study were: (1) to ascertain if variations in external atmosphericpressure induce bulk gas flow in onion, tomatoes, sweet peppers andcucumbers, (2) to determine the effects of factors such as temperature, oxygenconcentration, and timing of atmospheric pressure variations on the induction ofbulk flow in these plant tissues, and (3) to elucidate the short-term effects ofvariable pressure treatments on emissions of aroma compounds of onions,tomatoes, and sweet peppers. Onion, sweet pepper, tomato and cucumber werechosen for this study because these vegetables represent diverse morphologicaltypes with a variety of internal matrices and, therefore, more likely to differ intheir response to varying atmospheric pressures.4LITERATURE REVIEWA. PHYSIOLOGICAL GASES AND THEIR ROLE IN TISSUE FUNCTIONIn plant tissues there are individual gaseous spaces which may beinterconnected with one another forming a continuous air-space in the wholeplant. The air spaces contain the main components of air, i.e., nitrogen andoxygen, and also carbon dioxide, the product of respiration as well as volatileproducts of metabolism. The proportions of these gases are different fromnormal air, however. The proportion of CO2 has been found to be generally inthe range of 3 to 6%, and in many cases, as high as 20 and 30% while theconcentration of oxygen must be lower than ambient air (Phan, 1987). In anintact plant ethylene may occur at concentrations ranging from 100 to 1000 ppm.Although minute amounts of this gas may trigger an increase in respiration ratein a harvested tissue, in the attached organ such effects are suppressed,possibly due to the antagonistic action of a high internal concentration of CO2(Burg and Burg, 1965).Upon detachment of the plant organ, there is an inflow of oxygen throughthe cut end of the pedicel and an outflow of CO2 resulting in an abrupt increasein the respiration rate and an increase in all oxidative processes. Ethyleneapparently escapes through the harvest wound and the internal concentrationcan fall as low as 0.5 to I ppm. The combined effects of an increase in therespiratory process and healing of the harvest wound allows ethylene toaccumulate to preharvest levels in a short period of time (Phan, 1987).Another consequence of harvest is the interruption of the water supply tobulky organs or leafy tissues. Vaporization, or the passage of water moleculesfrom the liquid phase to the vapor phase continues after harvest creating a waterdeficit within plant tissues and a consequent loss of turgor. Postharvest5increases in respiratory activity result in a greater production of heat and anaugmented loss of moisture. Resistance to water vapour loss is derived mainlyfrom the cuticular layer (Burg and Burg, 1965; Ben-Yehoshua et al., 1985).The Importance of OxvQen in Tissue Biochemistry and PhvsiolocivGenerally, reducing the external 02 concentration will decreaserespiration rate of vegetables. Evidence indicates that the substantial decreasein rate of oxygen uptake that occurs with decreasing external oxygenconcentrations is connected with the enzymes involved in the respiratorymachinery, rather than being an indirect effect of a decrease in the generalmetabolism of the tissue (Solomos, 1982). For any given external 02 level instorage, the cells at the center of a bulky tissue will experience significantlylower 02 levels than those at the surface if the effective diffusivity is low(Rajapakse et al., 1990). Under low-02 environments, like those encountered inCA or MA storage, this gradient could result in loss of quality.Vegetable crops reacting positively to CA conditions, require a minimumof 1-3% 02 in the storage atmosphere. Asparagus and potatoes require anexternal concentration of 10% 02 otherwise inner tissues become anaerobic asinternal oxygen levels fall below the respiratory minimum which may be as lowas 0.2% 02 (Kader, 1986). This may occur not only in low 02 storage but also inpiles of warm, poorly aerated vegetable material.In an anaerobic environment pyruvic acid is no longer oxidized but isdecarboxylated to form acetaldehyde, CO2 and finally ethanol. Weichmann(1987) stated that such a change in metabolism is not the result of specificbiochemical reactions of certain crops to a low 02 environment but of theanatomy and morphology of those crops. Thus, certain varieties of applesrespond well to ultra-low 02 storage while other varieties, or even the same6variety in a different harvest year, will suffer adverse effects under suchconditions (Sharples and Johnson, 1987).The explanation for the reduction in respiratory metabolism as a reactionto low 02 concentration (10-11 %) has not yet been fully elucidated, Inhibition ofcytochrome oxidase was ruled out when it was discovered that this enzyme hasa very high affinity for oxygen, allowing it to function even when the intracellular02 concentration is only 0.01 % (Drew, 1979). Solomos (1982) theorized that thestorage atmosphere had to contain less than 2% 02 to have any influence on theactivity of cytochrome oxidase in apples. Any decrease in the rate of respirationin response to the decrease in external 02 concentration must stem, therefore,from the diminution of the activity of oxidases other than cytochrome oxidase(Solomos, 1982).An atmospheric concentration of less than 8% 02 will reduce the rate ofethylene production in fruit and vegetable tissues (Dilley et al., 1982). Oxygen isrequired for the conversion of I -aminocyclopropane-carboxylic acid to ethylene(Yang, 1985).Growth and development of crops in storage greatly reduces their retailvalue and accelerates deterioration. lsenberg (1979) suggested that 02concentration affects endogenous growth regulators either promoting orinhibiting sprouting depending on the crop.Reviews of the effects of reducing 02 levels in the storage environment onchlorophyll retention, prevention of premature softening, maintenance of nutritivevalue and flavour retention in selected crops can be found in the literature(Kader, 1986; Weichmann, 1986; Kader et al, 1989).7The lmiortance of Carbon Dioxide in Tissue Biochemistry and PhysioIoyAs previously mentioned, the internal CO2 concentration in bulky tissuesprior to harvest is much higher than that in the surrounding air and possiblysuppresses the action of endogenous ethylene. Increasing the CO2concentration in the atmosphere surrounding the harvested tissue has much thesame effect. Elevated levels of CO2 prevent or delay many responses of freshfruits and vegetables to ethylene presumably by competing with ethylene forbinding sites on receptor molecules (Veen, 1987). However, elevated CO2levels can reduce, promote or have no effect on ethylene production rates byfruits, depending on the commodity and the CO2 concentration (Kader, 1986).Raising CO2 concentration in the atmosphere to 6% can reducerespiration rate of bulky tissues possibly by preventing the oxidation of KrebsCycle intermediates (Brecht, 1980; Kader, 1985). However, CO2 concentrationsas low as 2.5% can cause tissue damage in leafy commodities (lsenberg,1979). In bulkier tissues, CO2 levels can rise as high as 15% before ethanol andacetaldehyde begin to accumulate and tissue damage is evident (Frenkel, 1977;Kader, 1985; Lougheed, 1987). Short-term exposure to 4-15% CO2 beforestorage at chilling temperatures has been shown to decrease subsequentchilling injury in okra (Ilker and Morris, 1975; Morris and Kader, 1977).The effects of reduced 02 elevated CO2 on respiration rate are additive;10% CO2 in air reduces respiratory metabolism to about the same extent aslowering the 02 concentration to 2% in the atmosphere (Kader, 1985). A mixedatmosphere of 2% 02 and 10% CO2 has approximately double the effect of eithercomponent alone (Kader, 1985).High levels of CO2 can have a beneficial or detrimental effect onappearance, flavour and nutritional qualities of fruit and vegetables. Treatmentswith 1-5% CO2 were reported to retard softening and maintain high8concentrations of sugars and acids in peaches and nectarines (Anderson andPenny, 1975). Elevated levels of CO2 have been found to reduce browning ofcut surfaces of Brussels sprouts (Weichmann, 1983) and lettuce (Singh et al.,1972). The rate of starch-to-sugar conversion in potatoes can be slowed bystorage in an atmosphere containing 5-20% CO2 (Burton, 1974). Pitting injurywas induced by 5% CO2 in asparagus kept at 6 °C for I week (Lipton, 1965).Carbon dioxide levels above 10% cause off-flavours in sweet corn (Saltveit,1985). General reviews of the effects of elevated levels of CO2 in storageenvironments on sensory and nutritional quality of various fruits and vegetablescan be found in the literature (Weichmann, 1986; Herner, 1987). In most of theresearch investigations, mixtures of high levels of CO2 and low levels of 02 wereevaluated for their effect on plant tissues.The Importance of Ethylene in Tissue Biochemistry and PhysiologyFruit have been characterized as climacteric or non-climacteric dependingon their respiratory behavior during ripening (Burg and Burg, 1967). Ripening inclimacteric fruit is associated with a large increase in respiration and ethyleneproduction. The process is irreversible once autocatalytic ethylene productionincreases to a certain level (McGlasson, 1985). A climacteric-like respiratoryincrease can be induced in non-climacteric fruit by treating them with ethylene,ethane or ethanol. However, an increase in ethylene production does notaccompany the increase in respiration which will rapidly subside once thehydrocarbon stimulus is removed (McGlasson, 1985).As a fruit develops “competence” to ripen, ethylene perception by theplant stimulates respiration and turnover of macromolecules (Grierson, 1987).An enhanced production of enzymes in the ethylene biosynthesis pathwayresults in an autocatalytic burst of ethylene which leads to synthesis of mRNAs9responsible for a host of physiological changes in the mature fruit. Some of theresponses to ethylene during ripening include: accumulation of the pigmentlycopene, accelerated degradation of chlorophyll and starch, production offlavour compounds, synthesis of cell wall degrading enzymes such aspolygalacturonases and fruit abscission from the parent plant.The accumulation of ethylene in the storage environment can have adetrimental effect on many crops, reducing shelf-life by as much as 33% in aslittle as 2 days (Kader, 1985). Schouten (1985), Knee et al. (1985) andLougheed et al. (1987) summarized the undesirable effects of ethylene uponvegetables. As little as 1-5 ppm ethylene can cause loss of chlorophyll in leafyvegetables such as celery, cabbage, broccoli, Brussels sprouts and in fruit-vegetables like cucumbers, peppers and tomatoes as well as leaf abscission insome cultivars of cabbage, Brussels sprouts and cauliflower (Kader, 1985).The effects of ethylene on textural quality of some commodities can beobserved only after cooking or processing. “Hardcore” in sweet potato can beinduced by exposure to ethylene, resulting in a hard, inedible core in the cookedproduct (Watada, 1986). Short-term exposures to ethylene have been reportedto increase spear toughness in asparagus (Haard et al., 1974) and causesoftening in pickling cucumbers (Poenicke et al., 1977).The development of bitter flavours in sweet potatoes, carrots, cabbageand Brussels sprouts has been attributed to the presence of ethylene (Chalutz etal., 1969; Kader, 1985; Hardenburg et al., 1986). Auxin-induced ethylene isthought to play a role in the production of isocoumarin, a compound responsiblefor the bitterness in carrots (Chalutz et al., 1969).10The Consequences of Water LossThe most important change induced by harvest is the loss of turgidity dueto a termination in the water supply to the harvested organ. With loss of turgor,transpiration rate is then lessened, affecting the protective cooling afforded byvaporization of water. Since biological reactions operate well only within anarrow range of temperatures, plant organs already subjected to warmtemperatures during harvest are likely to suffer biochemical damage (Phan,1987). While internal changes due to water loss affect the flavour, texture andnutrition of bulky plant tissues, the loss of the “fresh” appearance caused bywilting and withering of the outer tissues is a clear signal to the consumer thatharvest and handling was less than optimum. Transpiration has beenconsidered the major cause of postharvest losses and poor quality in leafyvegetables such as chard, lettuce, cabbage and spinach, and second inimportance only to overmaturity at harvest, to losses of fruit-type vegetablessuch as eggplant, okra, snap bean, cucumber and sweet pepper (Kader, 1983).It is important to note that the relative contributions of plant structures (lenticels,stomata, cuticle, etc.) to transpiration varies among organ types. While 02, CO2and ethylene diffuse mainly through air-filled stomata, lenticels, floral ends andstem scars, water vapour diffuses through the aqueous phase of the cuticle; theproportions may vary according to morphology (Burg and Burg, 1966; Burton,1982; Ben-Yehoshua et al., 1985).Cells that have lost their turgor are more susceptible to infection bypathogens. Storage of fruits and vegetables in a water-saturated atmospherealleviates water stress, encourages wound healing and helps maintain the skin’sresistance to pathogens (Ben-Yehoshua, 1987).Ben-Yehoshua (1987) cited numerous reviews discussing the effects ofwater stress on phytohormones. Apparently water stress promotes activities11associated with senescence, such as a drop in endogenous levels ofgibberellins and cytokinins, and a rise in the level of abscisic acid and ethylene.In most retail storage facilities, diverse commodities are stored togetherunder the same level of humidity. Since plant products vary greatly in theirresponse to water loss, different strategies must be developed to control thehumidity of the air immediately surrounding the product. Plastic films andcoatings have been used successfully to control water loss in manycommodities; however, excessive moisture in the pack atmosphere increasesrisk of pathological disorders (Geeson et al., 1985; Ben-Yehoshua, 1985).B. DIFFUSIVE GAS FLOW IN BULKY PLANT TISSUESMechanism of Gas ExchanQeThe primary mechanism for exchange of metabolic gases between theinterior and exterior of bulky plant organs is diffusion (Burg and Burg, 1965;Cameron and Yang, 1982; Ben-Yehoshua et al., 1985; Solomos, 1987) whichcan be defined as the net movement of gas molecules from one point to anotherbecause of the random kinetic activities or thermal motions of molecules.Metabolic rates and skin resistance to gas diffusion, as well as effective avenuesfor gas exchange such as gaseous pores, lenticels or stomata, calyx or pedicelopenings, result in gas concentration gradients between the externalatmosphere and the atmosphere just beneath the fruit skin (Burg and Burg,1965). In most plant tissues, especially those containing parenchymatoustissues, a network of intercellular spaces forms a continuous air-space. Gasconcentration gradients can also exist between these intercellular gas spacesand internal cells, the magnitude of which is influenced by apparent diffusivity ofinternal tissues, fruit size and rate of gas production or consumption (Burg and12Burg, 1965; Solomos, 1987; Rajapakse et al., 1989). Kader (1987) outlined thesteps for gas exchange between a plant organ and its environment which are: 1)diffusion in the gas phase through the dermal system, 2) diffusion in the gasphase through the intercellular system, 3) exchange of gases between theintercellular atmosphere and the cellular solution, and 4) diffusion in solutionwithin the cell to centers of 02 consumption, or away from centers of CO2production.The rate of gas exchange in bulky storage organs can be approximated byFicks first law of diffusion (Burg and Burg, 1965; Cameron and Yang, 1982;Solomos, 1987). This law states that the flux of a gas in or out of a plant tissuedepends on the concentration gradient across the barrier involved, the surfacearea of the barrier and the resistance of the barrier to diffusion. A simplifiedversion of Fick’s Law (Cameron and Yang, 1982) can be written as:cls = (C— CL)Adt Rwhere ds/dt is the rate of efflux (nLs1), R is the resistance coefficient (s cm-I), Ais the surface area of the tissue (cm2), and Ct0 and Ct1 are the concentrations(nLIcm3) outside and inside the tissue, respectively, at time t. Once theproduction (or consumption) rate of the gas by the organ and the concentrationsof the gas in the internal and external atmospheres are determined, thenresistance can be calculated from:P.concentration gradientproduction rateAlthough partial pressure gradients between the interior and exterior ofplant organs may occur for individual gases, normally the total internal pressureof gases approximates atmospheric pressure for organs in a gaseousenvironment (Solomos, 1987).13Part of the difficulty in developing a model to study gas exchange in bulkytissues is the limited availability of information on resistances of dermal andparadermal tissues, gas transport pathways, tissue porosity and internal gasconcentrations. The sheer diversity of cultivars and types of edible vegetableand fruit tissues makes the collection of such detailed gas exchange informationa formidable task.Application of the Principles of Gas ExchaneDetermination of diffusivity of gases in bulky plant tissues is important forthe development of controlled-atmosphere (CA) and modified (MA) atmospherestorage treatments which extend the shelf-life of many plant products bymodifying respiration rate and metabolic changes. The terms ‘controlledatmosphere’ and ‘modified atmosphere’ mean that the atmospheric gascomposition surrounding a perishable product is different from that of normal air.Both commonly involve manipulation of C02, 02 and N2 levels; however, othergases such as CO, C2H4,C2H and C3H6 are sometimes included. MA isusually developed in a package, either by passive diffusion or purging thepackage with a premixed gas, after which there is no precise control over thegases surrounding the product. Surface coatings and plastic films are also usedto generate MA within the tissue. Controlled atmosphere conditions involveconstant control and monitoring of atmospheric gases.The effects of CA and MA on respiration are dependent on the plantmaterial itself and on the concentration gradient that develops between thecenters of metabolic action and the outer integument of the plant material(Burton, 1978). lsenberg (1979) suggested that the effects of CA on therespiratory process depended more on the anatomy and morphology of the plantorgan than on its biochemical system. According to Lougheed (1987),14identifying CA disorders in vegetables is complicated by the constant changes incultivars and variations among vegetables in anatomy, morphology andphysiology. In fruit, porosity may be important in the successful outcome of CAstorage. Values for 02 diffusivity in apples have been found to vary with cultivar,being broadly consistent with intercellular space volume (Rajapakse et aL,1990). The commercial success of CA storage of pome fruits, and more recentlywith MA packaging of cut vegetables, provides continued incentive to continuethe investigation of gas diffusion characteristics of diverse plant species todevelop strategies to increase effectiveness of CA and MA treatments, and todetermine the changes required in a storage atmosphere to ameliorate CA-induced disorders (Ladeinde and Hicks, 1988; Banks and Kays, 1988; Andrich etal., 1989; Bertola et al., 1990; Lee et al., 1991; Solomos, 1989). These types ofinvestigations are conducted with the knowledge that small changes in thestorage environment such as lowering 02 concentrations to 1.5% from 2-5%(Ultra Low 02 Storage) or rapid establishment of CA conditions (Rapid CA) haveresulted in superior quality and reduced disorder levels of stored apples (Littleand Peggie, 1987).C. BULK GAS FLOW IN BULKY PLANT TISSUESOccurrence of Bulk Gas FlowIn addition to diffusive transfer of gases, bulk flow of gases, in which gasesmove collectively along a gradient of total pressure, may also occur in largeplant organs. At the present time, evidence of bulk gas exchange in planttissues comes almost exclusively from studies with wet-land plants and leaves oftrees. In one such study, small pressure increases within the intercellular airspaces of water lily (Dacey, 1981) directed bulk flow of gases down petioles to15the rhizomes and out the older leaves. Bulk gas flow has also been reported inlotus leaves (Dacey, 1987), although the direction of gas flow in the petioles wasnot determined. Day and Parkinson (1979) calculated that the contribution ofmass flow to gas exchange in leaves exposed to wind should increase rapidlywith leaf oscillation frequency and a concomitant reduction in the boundary layerthickness. Although Dacey (1981) considered bulk gas flow in wet-land plants toresult from gradients in temperature and water vapour concentration, factorssuch as flow-limiting ‘pores’ in palisade parenchyma, stomatal function andphotosynthetic capability may be equally important (Dacey, 1987, Armstrong andArmstrong, 1991).Although reports of mass gas flow in large plant organs are limited, thereare indications that supplementary gas flow or pressurization of internal tissuesmay occur during postharvest activities. Reduced gas permeabilities oradditional diffusion barriers to gas exchange may permit development of totalgas pressure gradients between a bulky plant organ and its externalenvironment (Corey and Tan; 1990). Research by Corey and Tan (1990)revealed that sudden temperature changes may cause mass gas flow to occur,resulting in internal gas pressure changes. Employing an apparatus designed toproduce oscillating atmospheric pressure cycles, Calbo (1985) determined thatmass gas flow under ambient gas concentrations had no effect on onset of theclimacteric and tissue softening in “Gravenstein” apples compared to applesstored at constant pressure. Oscillating pressure treatments combined with anatmospheric concentration of 8% CO2 had a beneficial effect on colour retentionand tissue firmness compared to apples stored at ambient pressure and 8%CO2. This effect was thought to be due to inhibition of ethylene synthesis andaction by elevated internal concentrations of CO2 although oscillating pressuresin a flow-through system reduced internal and external concentrations of16ethylene (Calbo, 1985). On the other hand, experimental enhancement of massgas flow in cabbage stored in air, by means of variable low-pressure storage,improved shelf-life and reduced trim loss (Onoda et al. 1989).The effect of variable atmospheric pressure treatments on postharvestmetabolism is largely unknown, although there is evidence that a pronouncedreduction in ethylene production rate may occur in apples during storage inatmospheres with elevated C021 ratios (Calbo, 1985).Application of Mass Gas FlowIt has been known for some time that by reducing the normal atmosphericpressure in the environment around plant tissue, the effective partial pressuresof individual ambient gases are also lowered. A 115th reduction in the totalpressure of normal air would result in an effective oxygen partial pressureequivalent to 4% oxygen. Therefore, unlike CA or MA, no gas other than airneed be supplied in a hypobaric system (Brecht, 1980; Jamieson, 1980). Inaddition to lowering the partial pressures of gases in air, including water vapour,low-pressure systems allow gases to escape more rapidly (Burg, 1975).According to Burg (1975), this is due to the fact that the diffusion coefficients ofvarious gases, including ethylene and other volatiles are inversely proportionalto atmospheric pressures. Although there are reports of the successful use ofhypobaric storage for extending the storage life of tomatoes (Burg and Burg,1966; Wu et aL, 1972; Mermelstein, 1979), avocados, mangos, sweet cherries,limes, guava (Burg and Burg, 1966; Mermelstein, 1979), apricots (Wu andSalunkhe, 1972), sweet peppers, lettuce, mushrooms, floral products(Mermelstein, 1979), and tropical and subtropical fruits (Lougheed et al., 1978),this form of storage never became popular. Grumman Allied Industriesdeveloped the Dormavac System for hypobaric transportation of perishables17(Mermelstein, 1979; Jamieson, 1980), but this met with only limited commercialsuccess and was discontinued (Sherman, 1985). Interest in hypobaric storagemay have diminished due, perhaps, to the cost of constructing a storage facilitycapable of withstanding enormous negative pressures and evidence thatindicates that flavour volatiles could be lost during storage at low pressures (Wuand Salunkhe, 1972, Lougheed et al., 1978). Geeson et al. (1986) observedthat ethylene concentrations of less than 0.1 ppm in the hypobaric storageatmosphere resulted in poor and uneven ripening of tomatoes following storage,hampering flavour development.Most of the information describing gas exchange in plant tissues hasbeen amassed through studies with fruit rather than vegetables. The majority ofthose investigations indicate that the superficial tissues represent the mainsignificant barrier to gas diffusion and are thus the primary factor regulating theinternal concentrations of gases within a commodity (Burg and Burg, 1965; BenYehoshua et al., 1985; Andrich et al, 1990; Rajapakse et al., 1989, 1990; Bertolaet al., 1990). There is an obvious need for quantitative determination of theresistances through the various pathways for exchange of 02, GO2, ethylene andwater vapour in vegetables in order to model and develop storage systemswhich are capable of creating and maintaining internal atmospheres beneficial toplant tissues.18D. FLAVOUR ANALYSIS IN FOOD RESEARCHThe ability to monitor volatile components of flavour adds a newdimension to expressions of quality often used by horticulturists such as yield,size, texture, appearance or percentage of waste and sensory assessments.However, the task of isolating and identifying volatile flavour components isformidable. Biologically generated aromas are present in low concentrations ina complex matrix and comprise a large number of compounds representingnumerous chemical classes. Ideally, once the flavour compounds have beenextracted, concentrated, separated and detected, the contribution of eachchemical to the perception of flavour should be established. This latter task isreceiving increasing attention by flavour chemists and sensory analysts in effortsto identify important components in various foods (Maarse, 1991; Grosch, 1993).Wide availability of a technology with the capability to isolate and identifynumerous flavour compounds has resulted in the publication of copious lists ofconstituents from many common and exotic food species. While it is true thatmuch of the information on flavour analysis has been reported in the form ofrelative peak areas or height and cannot be related to the actual concentrationsin the products, such data are still important when the intent of the research isconsidered.Mazza and Pietrzak (1990) used GC/MS analysis and sensory analysis ofheadspace volatiles to identify a commercial postharvest treatment for sproutsuppression as the origin of an undesirable musty, earthy aroma in potatoes.Statistical comparisons of the peak areas and peak arealtotal area ratiosrevealed that six major components were more concentrated in off-ifavourpotatoes than in the good quality potatoes. Three of these compounds werealso isolated from an adjuvant in a commercial formulation of sprouting inhibitorused on the potatoes (Mazza and Pietrzak, 1990). This resewh led to the19successful resolution of a problem that threatened the continuing operation ofthe largest frozen French fry production plant in Canada.Qualitative or semi-quantitative methods for determining volatileconstituents in foods have been applied in many creative ways including:evaluation of exotic fruits as potential sources of novel flavours and flavourconstituents (Potter and Fagerson, 1990; Wyllie and Leach, 1990; Peppard,1992; Nisperos-Carriedo et al., 1992; Farka et al., 1992), identification ofpreviously unknown compounds in common crops (Tang et al., 1990; Takeoka etal., 1991; Kuo and Ho, 1992a; Kuo and Ho, 1992b), appraisal of preservation,processing and storage methods (Chung et al., 1983; Crouzet et al., 1985; ElNemr et al., 1988; Nisperos-Carriedo et al., 1992; St. Angelo et al., 1992;Hansen et al., 1992; Yen et al., 1992; Narain and Bora, 1992; Shamaila et aL,1992; Moshonas et al., 1993; Piggott and Othman, 1993), evaluation of cultivars(Wyllie and Leach, 1990; Kallio and Salorinne, 1990; Horvat et al., 1992;Takeoka et al., 1992; Shamaila et al., 1993), monitoring the ripening process(Chyau et al., 1992; Hansen et al., 1992; Perez, et al., 1992; Mattheis et al.,1992), the study of processes leading to the generation of off-flavours (Seitz andSauer, 1992; Singh, 1992; Rouseff et al., 1992), and the determination of theeffect of growing conditions on flavour development (Van Wassenhove et al.,1990; Fischer, 1992).Because of the complexity of typical fruit and vegetable volatiles, nosingle method of analysis will provide a flavour profile truly representative of thefood. Quantitative and qualitative data on volatile compounds in many edibleproducts, obtained using various methods, were compiled by a Dutch researchorganization and published recently in a three volume edition (Maarse andVisscher, 1989).20E. ISOLATION OF FOOD FLAVOURSTechniques employed in flavour analysis of diverse food products havebeen reviewed elsewhere (Bemelmans and Schafer, 1981; Cronin, 1982; loffeand Vitenberg, 1982; Heath and Reineccius, 1986; Parliment, 1986; Burgard andKuznicki, 1990; Maarse, 1991). A brief description of methods commonly usedfor isolation of aroma compounds in vegetables and fruits used as vegetables,together with examples of recent applications, are presented here.HeadsDace MethodsHeadspace analysis is considered the preferred technique for isolationand identification of important odour compounds in foods and beverages. Theheadspace techniques described in the following text are limited in theircapability to isolate a complete representation of flavour components due tovariation in concentration, stability, volatility and solubility of the components.a. Static Headspace SamplinciThe procedure referred to as static headspace (equilibrium) sampling,can be manual or automated. The manual technique involves use of a hand-held syringe to sample volatiles in the headspace above a sample contained in asealed system. Automated headspace analyzers are commercially availablewhich produce better chromatographic reproducibility than the manual method.Unfortunately, except in liquid products, it is very difficult to relate theconcentration of flavour volatiles in the vapour phase to that in the product(Maarse, 1991). In addition, the normally low concentrations of aroma volatilesreleased by most vegetables (especially when they are intact) precludes the useof this technique, since identification is difficult if not impossible.21Methanethiol, ethyl alcohol and ethyl acetate have been identified inheadspace gases of broccoli florets by Hansen et al. (1992), who used themanual method to determine the effect of low-oxygen atmosphere storage on lowboiling volatiles in broccoli. Kallio and Salorinne (1990) identified onion aromacomponents by direct injection of headspace gas of chopped onion and on-column high-resolution capillary GC and GC-MS. Volatile flavour components intomato homogenate (2 mL of liquefied tomato) were quantified by Baldwin et al.(1991) using an automated headspace sampler to introduce the sample onto aGC column.b. Dynamic Headspace SamølinQDynamic headspace (non-equilibrium) sampling involves the entrainmentof odourous compounds on a solid adsorbent by purging the headspace of theholding vessel with nitrogen or some other gas. This procedure can beperformed manually (in which case the volatile components are eluted withsolvent), semi-automatically or automatically, with any of at least fourcommercially available instruments. Desorption of volatiles obtained by thesemi-automated or automated procedure is effected by thermal desorption,which may cause degradation of thermally labile compounds. Commonly usedadsorbents are activated coconut charcoal and synthetic polymers such as:chromasorb 105, porapack Q, Tenax TA and Tenax GC, either used alone or incombinations. Resins such as XAD-4, XAD-7 and XAD-9 are less popularadsorbents. Although Tenax GC and TA have become the most frequently usedporous polymers for trapping volatiles from headspace vapours, their adsorptioncapacity is much less than that of activated carbon (Heath and Reineccius,1986). Excellent recoveries of a range of organic compounds trapped on 2 mgof charcoal have been reported (Clark and Cronin, 1975). Despite the22availability of high quality carbon in convenient forms for trapping volatiles, useof this material seems to be mainly directed toward the identification andquantitation of pollutants.Van Langenhove et al. (1991) used Tenax GC to capture and concentratevolatiles from brussels sprouts and cauliflower during the blanching processboth on a lab scale and an industrial scale. Volatiles were thermally desorbed ina heating block and analyzed by GC-MS.Tokitomo and Kobayashi (1992) entrained fresh onion volatiles on TenaxTA. Following the collection, they used a thermal desorption cold trap injector(Chrompack, The Netherlands) to desorb the volatiles, which were thencryofocused in the injector and injected onto a GC capillary column.Kohlrabi volatiles, from the liquid fraction of a homogenate of kohirabi andphosphate buffer, were collected on Tenax TA by Fischer (1992) and eluted withpentane/diethylether (1:1 v) for subsequent analysis by GC.Tenax GC (10 g contained in a glass trap) was used by Hansen et al.(1992) to trap volatiles from whole broccoli florets by purging a flask containingthe broccoli with purified air. The isolation was carried out for 3 h at 25°C withan airflow rate of 3 L/min. Volatiles were eluted with 100 mL of diethyl ether andconcentrated by careful distillation prior to analysis by GC and GC-MS.Simultaneous PurQe and Solvent ExtractionA device developed by Umano and Shibamoto (1986), and later used byMacku and Shibamoto (1991) to extract headspace volatiles of chopped celeryinto solvent, has not been included in review literature. The apparatus consistsof a large containment vessel with an inlet and outlet for purge gas. The outletof the vessel is connected to a gas-washing bottle and a liquid-liquid continuousextractor joined in tandem. The extraction solvent (50 mL) was condensed to I23mL in vacuo using a Vigreux distillation column and the volatiles were analyzedby GC and GC-MS. The main advantage of this system, compared to trappingon porous polymers, is the very high capacity of the trap.Solvent Extraction MethodsThe solvent extraction method takes advantage of the solubility of flavourcompounds in organic solvents such as pentane, diethyl ether, dichloromethaneand carbon disulfide. Following steam distillation of homogenates or juices,aqueous distillates can be shaken together with a solvent that is immiscible inwater. As the water and solvent separate to form two layers the volatiles willremain in the solvent. Once contained in the organic solvent, aroma compoundscan be concentrated allowing easier detection and identification by GC analysis.Nonfat-containing foods can be directly extracted with solvent by using aSoxhlet apparatus (and heat) or blending the tissue or juice with the solventfollowed by filtration and/or centrifugation. Often solvent extraction requires afurther ‘cPeanup method to eliminate nonvolatile constituents.Kuo and Ho (1 992b) prepared solvent extracts of onions and scallions byblending the samples with distilled water, adding dichloromethane and stirringfor 12 hours. The samples were then filtered, dried over sodium sulfate andpassed through a silica gel column to remove the chlorophyll. Followingconcentration of the extract, methanol was added to precipitate the waxes andthe samples were filtered and analyzed by GLC and GC-MS. Block et al.(1 992a, I 992b) used this method to extract flavour compounds from a number ofAllium species except that after blending the samples with water and filtering,they saturated the filtrate with sodium chloride and extracted twice withdichloromethane. The solvent extracts were dried, concentrated, filtered andanalyzed by GC-MS, HPLC and NMR spectroscopy. Weinberg et al. (1993)24blended celery and carrot juices with water and dichloromethane to extractflavour compounds. The extract was condensed on Kuderna-Danish apparatusbefore analysis by GC-MS.Volatile compounds from celery have been isolated by refluxing choppedcelery with diethyl ether in a Soxhiet apparatus for 4 h (Van Wassenhove et al.,1990). The extracts were dried over sodium sulfate, concentrated underreduced pressure and analyzed by two-dimensional GLC and GC-MS. Inanother report of volatile compounds in celery (Tang et al; 1990), the juiceobtained by macerating celery in a blender was introduced onto an AmberliteXAD-2 column which was then rinsed with distilled water to eliminate sugars,acids and other substances. Pentane was passed through the column to elutethe adsorbed aroma compounds (free volatiles) and methanol was used to elutearoma compounds bound to glycosides. Bound volatiles were hydrolyzed with f3-glucosidase, taken up in dichloromethane and the extracts were concentratedunder a stream of nitrogen and analyzed by GC and GC-MS.Ohta and Osajima (1992) devised a cold trap apparatus to overcome theproblem of emulsion formation in solvent extracts. In their procedure, theyimmersed fresh onion (1 kg) in ethyl ether for 5 days at 5°C, dried the etherextract (4 L) over sodium sulfate, and reduced the solvent to 100 mL in vacuo.The volatile fraction was recovered in liquid N2 under vacuum using the cold trapapparatus and analyzed by GC-MS.Distillation MethodsThis technique includes steam distillation, and distillation under vacuumwhich allows lower temperatures to be used. Distillation methods takeadvantage of the volatility of flavour components and non-volatility of the major25food constituents, and is one of the oldest methods for flavour isolation fromfoods.A number of newly identified volatile components of onions (Alilum cepaL.) were reported by Farka et al. (1992) who used a steam-distillation techniqueemploying a water-immiscible volatile oil separator trap. After distillation,centrifugation at high speed separated the onion oil from the water condensate.The oil was analyzed by GLC and GC-MS. This method was also used by Kuoand Ho (1992a, 1992b) to determine volatile constituents of Welsh onions andscallions (both Allium fistulosum L.).Block et al. (1992a, 1992b) subjected chopped Allium species to highvacuum at room temperature, using an oil bath to prevent the contents of thedistillation flask from freezing, and collected the aqueous extract at -196°C.They found that subsequent analysis of the methylene dichloride extract of theaqueous sample by HPLC and NMR spectroscopy, gave good qualitativethiosulfinate composition profiles. The advantages of this method over solventextraction were reduced emulsion formation and absence of interference frompigments, waxes and other non-volatile plant components.A popular method of isolating flavours by distillation is to usesimultaneous distillation/extraction (SDE). Various modifications of thedistillation head designed by Likens and Nickerson (1964) have evolved but theprinciple of operation remains unchanged. The design permits the mingling ofvapours of the product with vapours of the extracting solvent allowing veryefficient flavour extraction. The apparatus was modified by Schultz et al. (1977)for use under vacuum. Van Wassenhove and his associates (1990) used theunmodified apparatus to isolate volatiles from celery plants grown with differentlevels of organic and/or inorganic fertilizers. A comparison of Soxhlet andLikens-Nickerson extraction methods indicated that Soxhiet extraction was less26efficent than the Likens-Nickerson apparatus for isolation of terpenes (VanWassenhove et al., 1990).Supercritical Fluid Extraction MethodIsolation of flavour compounds by supercritical fluid extraction (SFE)shows promise as a sample preparation technique because of minimal losses oflow boiling compounds, preservation of thermolabile constituents and rapidity ofextraction. In addition, SFE grade carbon dioxide is nonflammable, inexpensive,chemically inert and leaves no toxic residue (Maarse, 1991). The techniqueinvolves pumping liquid CO2 at a controlled pressure through a samplecontained in a heated extraction vessel. Extract recovery is accomplished byusing either solvent recovery or direct interfacing to a capillary column throughthe injector of a GC. Thus far, however, the technique has been applied mainlyto spices (Moyler, 1986; Hawthorne et al.,1989; Gopalakrishnan et al., 1990;Huston and Ji, 1991), but as techniques and equipment improve the method willlikely be applied to a wider range of food materials.The progress in methods of flavour analysis offers exciting opportunitiesto improve the quality of fruits and vegetables at every step in the marketingchain between the producer and the consumer. Monitoring changes in flavourvolatiles may be useful as an early indicator of the development of a harmfulstorage evironment or an unsuitable cultivar for storage. Chemical analyses arealso objective, fast and easier to perform than formal sensory analyses.27SECTION 1ACCELERATION OF GAS TRANSFER INONION, SWEET PEPPERS, TOMATOES AND CUCUMBERSBY VARYING ATMOSPHERIC PRESSURE28INTRODUCTIONModification of internal gas concentrations by manipulating environmentalgases, applying surface treatments or using special packaging materials canprolong storage life of a number of fruit and vegetable commodities. Refinementof CA storage has produced a number of specialized storage environments, oneof these being rapid CA, where the desired temperature is achieved 1 day afterharvesting and the level of low 02 is achieved in less than 6 days afterharvesting (Little and Peggie, 1987). Minimizing the time to obtain CA in pomefruit storage, results in superior quality and reduced disorder levels compared tothe passive development of CA over a period of 2 to 3 weeks (Little and Peggie,1987).The rate and direction of diffusive exchange of gases between planttissues and their environment depends on partial pressure gradients of each gasand is limited by available pathways for gas movement. Partial pressuregradients are a function of resistances along pathways of diffusion and the rateof production or consumption of the component gases. Jolliffe and Dyck (1988)proposed that interchange of gases between tissues and the surroundingatmosphere could be improved by supplementing diffusion with bulk gas flowinduced by rapid variations in external gas pressure. Presently, however, thereis little information on pressure driven gas exchange in bulky tissues.The main purpose of this portion of my research was to study the effect ofcyclic atmospheric pressure variations on CO2 efflux rates in onion, tomatoes,sweet pepper and cucumbers. A secondary objective was to characterize theinfluences of temperature, oxygen concentration, and interval between pressurecycles on gas transfer in bulky plant tissues subjected to variable pressuretreatments.29MATERIALS and METHODSA. PLANT MATERIALSJumbo yellow onions (Allium cepa L.) were purchased from a localsupermarket, selecting only those onions (average weight ca. 460 g) which werefree of apparent botrytis neck rot or soft rot. Sweet bell peppers (Capsicumannuum L. c.v. Doria) and tomatoes (Lycoperison esculentum L. c.v. Dombito)were reared in the greenhouse at Agriculture and Agri-Food Canada’s PacificResearch Center, Agassiz, B.C. The peppers, each weighting ca. 200g, wereharvested at the immature green stage, and tomatoes, each weighing ca. 260 gwere harvested at the red ripe stage. Slicing cucumber (Cucumis sativa)varieties were produced in field plots (c.v. Straight Eight) and in the greenhouse(c.v. Sweet Success) on the Research Station. Fruits 15 cm in length andweighing ca. 170 g were selected for experimentation. Immediately afterharvest, tomato, pepper and cucumber fruit surfaces were sanitized with a 100ppm hypochlorite solution. The plant material was placed in microperforatedpolyethylene bags and held overnight in the dark at temperatures approximatingthose to which the material would be subjected the following day. Experimentswere repeated on 3 individual plant organs over 3 periods in the growing (orstorage, as in the case of onions) season. Rates of gas exchange weremeasured on a fresh mass basis.B. VARIABLE PRESSURE STORAGE AND MONITORING SYSTEMA closed gas-exchange system was constructed to control environmentalconditions while generating cycles of pressure changes in the atmospheresurrounding bulky tissues. The materials in the gas circuit comprised aluminum,stainless steel, copper and glass. The circuit had a fixed internal volume of 5.630L (Fig. 1.1). The treatment vessel and a secondary chamber, each with avolume of 2.6L, were constructed from aluminum. They had 1.3 cm thick wallsand their rims were mounted with 6 protruding screw threads. The vessels werecapped by 1.1 cm thick aluminum plates with 6 smaller holes to allow the screwthreads to pass through the lid. The caps had 5 holes drilled to accommodate 6mm Swagelok bulkhead unions. A rubber 0-ring was installed in the rims of thevessels to ensure a gas-tight seal between vessel and lid when wing-nuts weretightened down on the lid. A temperature sensor (Model 47 Scanning Telethermometer, Yellow Springs Instrument Co., Inc., TX), as well as an electricalsource for fans, were passed through the bulkhead unions which were sealedair-tight with epoxy sealant. Pressure was measured by a gauge (0-18 kPa)mounted on the top of the vessel using compression fittings. A constanttemperature was maintained in the insulated treatment chamber by immersing itin a Haake recirculating bath (Haake, West-Germany). Muffin fans, rated at 600L min1 at zero static pressure, were placed in the treatment chamber and thesecondary chamber in order to mix gases and reduce boundary layer effects.Circulating gases were bubbled through distilled water in a glass WheatonPurge and Trap unit to saturate the gases entering the treatment chamber.Oxygen concentration in the system was monitored with an in-line Servomex570A Portable Oxygen Analyser. Carbon dioxide was sampled in a computerprogrammed sequence by means of a gas-sampling bulb installed in the gascircuit.Room air, or commercially prepared premixed gases composed of 1% or3% 02, balance N2 (Linde Specialty Gases Ltd., Edmonton, AB), were circulatedaround the system at a flow rate of 1.5 L min1 by a pump (Metal Bellows Corp.,Sharon, MA). Atmospheric pressure inside the treatment chamber was31BUFFERFig. 1.1, Iiiustration of the variable pressure storage andmonitoring system.GAB SAMPLINGBULB SOLENOIDSHUT-OFFWLV E02TR EAr U E NAN ALYB ERCHAMBERGASOHROMATOQRAPH32varied by directing the gas flow either through a bypass or against a flowrestrictor. This simple design caused air pressure to increase in the downstreamvessel when gas flow was directed against the restrictor, then to subside toatmospheric pressure as gas was redirected through the bypass. Aprogrammable timer (771 Programmable Sequencer, Cole-Parmer InstrumentCo.) controlled switching of a 3-way universal stainless steel solenoid valve(Ascoelectric Lt., Brantford, ON) to obtain a fluctuating pressure cycle of thedesired period. This type of system allowed the redistribution of gases withoutvarying system volume as was done in an apparatus constructed by Calbo(1985) or introducing external gases in a manner similar to the variable lowpressure storage apparatus of Onoda et al. (1989). The design of this systemalso circumvented difficulties reportedly associated with using a paramagneticgas measuring device in conjunction with pressure treatments, requiring that theCO2 analyser be isolated from the gas circuit (Jolliffe and Moloney,unpublished).Leaks were minimized in the gas exchange circuit by the use ofcompression fittings and valves capable of withstanding 670 kPa. To test forleaks the system was pressurized to 10 kPa above atmospheric pressure usingcompressed N2, sealed and allowed to stand for 4 hours. No noticeable drop inpressure occurred over this period.C. PRESSURE TREATMENTSThe design of the gas exchange system permitted only positive increasesin total pressure to occur with a minimum value of 101 kPa (atmosphericpressure) and a maximum value of 111 kPa. Increases of 3.5 and 6.9 kPaabove atmospheric pressure were chosen for the purposes of this study.Phases of the pressure cycles are illustrated in Fig. I .2. Starting at atmospheric337wDCI)Cl)wa:::aC-)LUa-Cl)0LU>0(15a65432IDCAPgasmeasurementgasmeasurement0o ioo 200 300 400 500 600 700SECONDSFig. 1.2. Variable pressure cycles. A programmable timer regulating timingof administration of pressure variations at 3.5 and 6.9 kPa wasco-ordinated with a computer-controlled gas sampling valveto automate gas measurements.34pressure, pressure increased rapidly to the maximum value, remained at theacrophase for about 16 s, and rapidly returned to the minimum value. Openingor closing the needle valve of the restrictor in the gas circuit controlled the levelof pressure variations. The time required to complete the maximum andminimum phases of one cycle, i.e. the period of the cycle, was 40 seconds.To begin a pressure treatment, the different plant materials were weighedand placed in the treatment chamber. The gas exchange circuit was thenflushed with room air or a premixed gas using a metal bellows pump, the fanswere started, the system closed and the humidifier included in the flow. Eachsample of plant material was allowed to equilibrate for 2 hours before startingtreatments, at which time the humidifier was excluded from the circuit. Thevolume of the system was sufficient to prevent the buildup of more than 1800ppm of CO2 before completion of the trial that day. Following treatment, theplant materials were reweighed and their volumes were determined by waterdisplacement.D. MEASUREMENT OF CARBON DIOXIDEGas measurements were performed automatically by programmed streamsampling with a 6-port air-actuated Valco valve (Chromatographic Specialties,Brockville, Ont). Sample volumes of 0.25 mL were injected onto a 6 ft, 1/8 inchID stainless steel column packed with Porapak Q, 1001120 mesh mounted in aShimadzu GC9A gas chromatograph. Carbon dioxide was catalyticallyconverted to methane by means of a Shimadzu MTN-1 methanizer for detectionby a Flame ionization detector. Peak integration and data calculations wereperformed by a Shimadzu C-R3A data processing unit. Helium (Ultra-highPurity) was used as the carrier gas at a flow rate of 40 mL/min. The flow rate ofthe detector gases were 340 mL/min and 50 mL/min for air (Extra Dry) and35hydrogen (Ultra-high Purity), respectively. A moisture and hydrocarbon trap wasplaced in the carrier gas line and each gas line was fitted with a 5 micron particlefilter. GC analytical runs were isothermal with temperatures as follows: columntemperature 500 C, methanizer 3500 C, methanizer transfer line 800 C anddetector 2500 C.Carbon dioxide efflux rates (pL.kgl•s1) of all tissue samples weremeasured several times at constant atmospheric pressure prior to initiation ofpressure treatment to confirm that equilibration was complete. Data werecollected as paired observations i.e. net CO2 emission rate at constant pressurewas paired with the following CO2 emission measurement taken after a 5 mmvariable pressure treatment. Relative response to pressure variations wasdetermined by the ratio (variable pressure/constant pressure) of these two ratemeasurements. Five consecutive paired constant and variable pressuremeasurements were collected for each of the different bulky tissues.Experimental treatments are outlined in Table 1.1. In addition to determining theinfluence of pressure level, temperature and oxygen concentration on theresponse to pressure variations in onions, sweet peppers, tomatoes andcucumbers, effects due to the duration of the interval of constant pressurebetween 5 mm pressure treatments (DCAP), either 5 or 15 mm, were examined.E. STATISTICAL ANALYSESData for the onion, tomato, sweet pepper and cucumber trials wereanalyzed as a 3-way factorial design using the General Linear Model procedureof the SAS/STAT (SAS Institute Inc., 1987) procedures. A factorial model withinteraction was specified. For onion data, CONTRAST statements were used inorder to partition the interaction effects between temperature and pressure level,36Table 1.1. Varying pressure treatments applied to onions, peppers,tomatoes and cucumbers.Treatments Onion Pepper Tomato CucumberPressure Level6.9kPa x x x x3.5kPa x x x xDCAP15mm x x x x15mm x x x xTemperature0°C x10°C x x x x20°C x x x x% OxygenI x3 x x x21 x x x xI Duration of the interval of constant atmospheric pressure between5 mm variable pressure treatments.2 DCAP=1 5 mm for trials testing the effect of different oxygen concentrations.37temperature and DCAP, and, temperature and 02 concentration (see Appendix Ifor contrast statements). Multiple comparisons of temperature means for oniontreatments were performed by the Student-Newman-Keuls multiple range test (P0.05).Because of the exploratory nature of this work, and the necessity to limitthe number of temperature and pressure treatment levels to those that could bemanaged accurately within the gas circuit system, much of the statisticalanalysis involved paired comparison of means using the PROC MEANSstatement of the SAS procedures. A new variable was created for each of themain treatments containing the difference between the value for CO2 efflux rateat constant pressure and the value for CO2 efflux rate as a result of variablepressure treatments. Using statements to obtain a t statistic and probabilityvalue, the mean differences were tested to determine whether they weresignificantly different from zero (PO.05).38RESULTS and DISCUSSIONA. EFFECT OF LEVEL OF PRESSURE VARIATIONSBoth levels of pressure variation had a significant effect on CO2 emissionrate of onions (P<O.0001) and peppers (P<O.OO1) but no significant (P>O.05)effect on cucumbers and tomatoes (Fig 1.3). After each series of pressurefluctuations, carbon dioxide emission rates returned to levels close to the initialconstant pressure values. The level of pressure variations, either 3.5 or 6.9kPa, did not affect carbon dioxide emission rates for each organ type duringintervals of constant pressure. Carbon dioxide efflux rates in onions increasedfour-fold and seven-fold, and two-fold and three-fold in peppers, during pressurevariations at pressure levels of 3.5 kPa and 6.9 kPa, respectively.Increases in bulk gas movement with increasing pressure is consistentwith Darcy’s law for fluid flow through porous media (Nobel, 1974). Bulk flowalong a pressure gradient will be effected only if sufficient time is allowed forwhole gas movement before reversal of the gradient. This response is welldemonstrated in research by Jolliffe and Moloney (unpublished) which showedthat increases in convective gas flow rates were proportional to the amplitude ofpressure variation and that a short period of pressure oscillation preventedmaximum bulk gas flow from being achieved. Lack of a response by cucumbersand tomatoes to pressure variations indicates that where there are limitedavenues for gas exchange, opportunities for mass gas transfer are minimal.In onion bulbs, it is expected that gas exchange occurred primarilythrough the root plate, which has been determined to be the most importantavenue for gas exchange (Ladeinde and Hicks, 1988). Onions are organs3910(I) h- -J uJ F >< D4-J U U--woc’j 0 C-)23.56.93.56.9ONIONTOMATOAMPLITUDEOFPRESSUREVARIATIONS(kPa)Fig.1.3.MaineffectofatmosphericpressurevariationsonnetCO2effluxrateofonions,cucumbers,sweetpeppersandtomatoes.A,BBarsassociatedwitheachplanttissue,withdifferentletters,aresignificantly(P<O.05,Ftest)different.a,bBarsassociatedwitheachpressurelevel,withdifferentletters,aresignificantly(P<0.05,pairedt test)different.AaIConstantpressure____VariablepressureAaBa3.56.93.56.9CUCUMBERPEPPERcomposed of individual leaves, each of which is covered on both sides by anepidermis. It has been noted that air bubbles coming from onion bulbs subjectedto a moderate vacuum under water came mainly from the base plate, withrelatively few from the neck and none through the dry scales (Ladeinde andHicks, 1988). Based on this observation, Ladeinde and Hicks (1988) theorizedthat gas exchange between adjacent leaves of mesophyll tissue may beinsignificant and gas exchange takes place mainly via the root plate rather thanby a lateral, shorter path of greater resistance. This observation is alsoconsistent with the pattern of gas exchange noted in this experiment. Movementof gases between leaf scales rather than laterally from the center of the onionwould result in the relatively large flux in CO2 observed under conditions ofvarying atmospheric pressure.In contrast, the major avenue for gas exchange in isolated sweet pepperfruit is at the pedicel (Burg and Burg, 1965). Burg and Burg (1965) found thatCO2 emanation was retarded by about 60% when lanolin was applied to thepedicel end. Evidence that the outer tissue layer of sweet pepper fruit is highlyresistant to gas exchange was obtained by Corey and Tan (1990) who found thatpartial vacuums occurred in pepper fruits following their exposure to cool air andcool water. The implication of their findings is that the pressure gradient causedby exposure of warm fruit to cool water (as can occur during postharvesthydrocooling) would result in water uptake with possible bacterial contamination.On one hand, the pressure gradient caused by moving warm fruit into cool airwould be beneficial in cooling internal tissues, but, moving cool fruit to warmenvironmental temperatures may have undesirable consequences such assignificant water loss. Evidence in this study that small pressure changes resultin substantial movement of internal gases could have important implications forpostharvest transportation and handling practices where pressure gradients may41occur. For example, depressurization during air transportation, pressurizationduring CA storage, temperature gradients caused by storage room defrost cyclesor movement of cold produce to a warm retail shelf, and even opening andclosing cold storage room doors may contribute significantly to mass gas flow.Diffusion of gases in tomatoes occurs through the stem scar and theepidermis. The permeability of gases through the latter depends on thesolubility and diffusivity of gases in the waxy layer that normally covers theepidermis. Cameron and Yang (1982) estimated that the stem scar region is thesite for 97% of the gas exchange taking place in tomato fruit. The liquid matrixof the fruit presents an obstacle in the evaluation of the stem scar as an area formass gas transfer. Using peeled tomato fruit, Bertola et al. (1990) resolved thatdiffusive resistance in tomato tissue was a significant factor in the rate of internalmass gas transfer. By determining the mass transfer coefficient of intact tomatofruit and fruit with the blocked stem scar they found that the specific resistanceof the peel is approximately 200 times greater than that of the stem scar, andthat two-thirds of the gas diffusing in the fruit passes through the scar. Thisfinding agrees more closely with that of Burg and Burg (1965) who determinedthat 60% of gas exchange takes place through the stem scar. The substantialdiffusion resistance of the liquid matrix, low gas permeability of the peel and thesmall surface area of the stem scar, often representing no more than I % of thetomato surface area, probably accounts for the lack of response to varyingatmospheric pressure.Part of the response to pressure variations exhibited by cucumbers andtomatoes, aside from limited opportunities for gas exchange, could be due to theperiod of the pressure cycle. A single period of 40 seconds was chosen for thisstudy based on information by Jolliffe and Moloney (unpublished). They42speculated that short period cycles may be too brief to allow maximum bulk gasflow along a pressure gradient before the gradient was reversed.Where bulk gas flow occurs, a model can be used to describe themovement of CO2 out of internal tissues by artificial means (Fig. 1.4) where V isthe tissue volume, V, is the internal gas volume, C and C0are the internal andexternal CO2 concentrations (masslL), Fd is the CO2 efflux rate at a constantpressure (massC02/fresh wtltime) and FD is CO2 efflux rate caused by diffusiveflow. During the first cycle of pressure variations, CO2 efflux driven by diffusiveflow can be expressed as:FDt = Fd[ ]t (mass/fresh wt)where Ca equals C1,., only under constant pressure and t is the period per cycle(time). If external gases flow back into internal gas spaces during a pressurecycle (as may happen when pressure drops back to the ambient level) thenduring one variable pressure cycle, CO2 efflux driven by bulk flow will be:çVA - COUVA = (Cm—C0)V1A (mass/fresh wt)where A is the amplitude of the pressure cycle. When it is assumed that Fd isunaffected by atmospheric pressure cycles then CO2 influx from internalproduction during one pressure cycle will equal Fd.If the initial internal concentration of CO2 could be established, modelingof the effect of external variations in pressure on internal concentrations of CO2may be possible.43GoutFig. I .4. A model of bulk gas flow.44B. EFFECT OF TEMPERATURERaising the storage temperature resulted in increased net rates of CO2emission from produce stored under both constant and varying pressures (Fig.1.5). This increase was significant (P<O.0001) for all plant organs at alltemperatures, except in onions where a temperature increase from 0 to 10 °Chad no significant (P>O.05) effect at constant pressure (see Table 1.4 ofAppendix 2 for more information). There was no indication that temperatureaffected the response to changes in atmospheric pressure nor that anysignificant interaction between temperature and level of pressure variationsoccurred for any of the plant organs. It was expected that temperature mightaffect the response to variable pressures particularly in bulky tissues with a moreliquid matrix like tomato. The temperature coefficient of CO2 diffusion in air ismuch smaller than that of its solubility in water. The differences in the diffusivityof CO2 at different temperatures, therefore, are expected to be quite large whenCO2 is diffusing in aqueous media (Solomos, 1987). Tomatoes, however, didnot show any response to pressure changes at either temperature.The synthesis of volatile productsand ethylene are among the metabolicprocesses not strongly suppressed by cold temperature. Therefore, even ifother metabolic processes are slowed down through imposition of lowtemperatures, autocatalytic ethylene synthesis goes on, increasing internalconcentrations of the phytohormone. When produce is removed from coldstorage to the warm retail shelf, supraoptimal concentrations of internal ethylenemay have a negative effect on shelf-life. Fruits and vegetables stored for longperiods have a shorter shelf life upon rewarming and there are indications that abuild-up of ethylene during cold storage may be an important factor in qualitydecline (Phan, 1987). Low ethylene storage has been found to be highlyeffective in reducing storage disorders and maintaining quality of many4512:1OFig.1.5.MaineffectoftemperatureonnetCO2effluxrateofonions,cucumbers,sweetpeppersandtomatoes.A,B,c,A’,BBarsassociatedwitheachplanttissue,withdifferentletters,aresignificantly(P<O.05,Ftest)different.a,bBarsassociatedwitheachtemperature,withdifferentletters,aresignificantly(P<O.05,pairedttest)different.AaIIConstantpressureVariablepressureAaBaA’A01020102010201020ONIONCUCUMBERPEPPERTOMATOTEMPERATURE(°C)horticultural crops but, according to Sherman (1985), the technology for ethylenecontrol during commercial storage and handling requires improvement.Research by Jolliffe and Moloney (unpublished) supplies evidence that variablepressure treatments cause bulk flow of ethylene gas in apple fruit. Since lowtemperatures do not appear to interfere with pathways of convective flow it ispossible that external pressure variations could improve the control of internalconcentrations of ethylene in fruits and vegetables. The present studies did notinvestigate ethylene flow in onions, cucumbers, sweet peppers and tomatoesbecause of the low sensitivity of the instrumentation to ethylene.C. EFFECT OF INTERVAL OF CONSTANT PRESSURE BETWEENPRESSURE CYCLES (DCAP)Increasing the length of the interval of constant atmospheric pressure(DCAP) following the 5 minute variable pressure treatment from 5 to 15 minutesincreased (P<O.OO1) the measured response to cycling atmospheric pressures inonions, cucumbers and tomatoes but had no effect on net CO2 emission rate ofsweet pepper (Fig. 1.6). Net CO2 emission rate at constant pressure wassignificantly (P<O.005) reduced in onions when DCAP was increased from 5 to15 mm but significantly (P<O.005) increased in tomatoes and unchanged incucumbers and sweet peppers. It is expected that the response to DCAP issimilar to the response to period noted by Jolliffe and Moloney (unpublished)where short period cycles may be too brief to allow maximum bulk gas flow alongthe pressure gradient before the gradient is reversed. It may be useful toinvestigate period effects in cucumber more fully since there is some indicationthat lengthening DCAP results in a higher CO2 emission rate during variablepressure treatments (Fig 1.6). The lack of a response by peppers to lengthening4712____10if) -8-JDCAP(mm)Fig1.6.MaineffectofDCAPonnetCO2effluxrateofonions,cucumbers,sweetpeppersandtomatoes.A,BA’,B’Barsassociatedwitheachplanttissue,withdifferentletters,aresignificantly(P<O.05,Ftest)different.a,bBarsassociatedwitheachDCAPlevel,withdifferentletters,aresignificantly(P<O.05,pairedt test)different.ConstantpressureVariablepressureABaabAABB515515515515ONIONCUCUMBERPEPPERTOMATODCAP indicates that lack of internal resistance allows rapid bulk gas flow in thedirection of the pressure gradient.Interaction between temperature and DCAP, and level of pressurevariations and DCAP was significant (P<O.0001) only for onions undergoingvariable pressure treatment. The F values for temperature x DCAP (linear) andtemperature x DCAP (quadratic) indicated a significant interaction for linear netCO2 emission rates and no quadratic interaction. The effects of temperatureand DCAP on net CO2 efflux rates of onions are illustrated in Fig. 1.7 showingthat the magnitude of the response to variable pressures appeared to be muchgreater when DCAP was 15 mm rather than 5 mm (see Table 1.4 in Appendix Ifor further information). In cucumber, sweet pepper and tomato fruit there wereno significant interactions between DCAP and any of the other treatments asdetermined by measuring net CO2 efflux rate. However, lengthening DCAPappeared to allow a fuller expression of the response to variable pressuretreatment in cucumber as well as reducing the variability between cucumbersamples (Fig 1.8). Although DCAP appeared to have no significant effect onresponse to changes in atmospheric pressure surrounding pepper fruit,variability in response among individual fruit was reduced by lengthening DCAP(Fig 1.9) suggesting that timing of the pressure variations should be examinedfurther. In tomato fruit, lengthening DCAP resulted in higher rates of CO2 effluxbut there was no visible reduction in sample variability (Fig. 1.10). Means andstandard deviations for experimental results for cucumbers, peppers andtomatoes are given in Appendix 2, Tables 1.6, 1.8 and 1.10, respectively.49‘D)-wI—><D-JLLUw(N0C-)I I oristaiit ressi..Ire I 3.5 II’a — 6.9 Ic1’aFig. 1.7. Effect of DCAP, pressure variations and temperature on CO2 efflux rateof onions. (A) DCAP = 5 mm, (B) DCAP = 15 mm. Bars representtreatment means and standard deviations for observations at constantatmospheric pressure (n=6) and variable pressure at levels of 3.5(n=3) and 6.9 (n=3) kPa above atmospheric pressure.A0 10 2043210108-6-4-2-0-TEMPERATURE °CB‘U)1!0 10TEMPERATURE OC205010::.u-II><D-JUUwc’J0C-)wI><D-JUUU-IC’.’0C-)I I or—stait f:ressi.Ire 1 3.5 II’a 6.9 1l:aFig. 1.8. Effect of DCAP, pressure variations and temperature on CO2 efflux rateof cucumbers. (A) DCAP = 5 mm, (B) DCAP = 15 mm. Bars representtreatment means and standard deviations for observations at constantatmospheric pressure (n=6) and variable pressure at levels of 3.5(n=3) and 6.9 (n=3) kPa above atmospheric pressure.A86420108642010 20TEMPERATURE °CB10 20TEMPERATURE °C51z.wF><D-JUUw(N0C)‘U)‘cwFxD-JUUw(N0C)I I Constant pressure I I 3.5 kPa____6.9 kPaFig. 1.9. Effect of DCAP, pressure variations and temperature on CO2 efflux rateof sweet peppers. (A) DCAP = 5 mm, (B) DCAP = 15 mm. Bars representtreatment means and standard deviations for observations at constantatmospheric pressure (n=6) and variable pressure at levels of 3.5(n=3) and 6.9 (n=3) kPa above atmospheric pressure.A201816141210864201412108642010 20TEMPERATURE °CB10 20TEMPERATURE °C5251‘Co‘0)w><D-JLLLiwc’J0C-)‘CowF><D-JLLLiLu(N0C-)I I iistart ressL.Ire 1 3.5 lla 6.9 lF’aFig. 1.10. Effect of DCAP, pressure variations and temperature on CO2 efflux rateof tomatoes. (A) DCAP = 5 mm, (B) DCAP = 15 mm. Bars representtreatment means and standard deviations for observations at constantatmospheric pressure (n=6) and variable pressure at levels of 3.5(n=3) and 6.9 (n=3) kPa above atmospheric pressure.A432I05432I010 20TEMPERATURE °CB10 20TEMPERATURE °C53D. EFFECT OF PRESSURE VARIATION, TEMPERATURE AND DCAP ONRELATIVE RATE OF CO2 EFFLUXAnalysis of ratios of net gas flow during variable pressure treatments andintervals of constant pressure (Table 1.2) indicated that temperature had nosignificant (P>O.05) effect on relative rate of CO2 emission rate of onion,cucumbers, peppers or tomatoes. Although some of the differences in meanrelative CO2 efflux rates of onions at various temperatures were quite large, theywere not significant due to sample variation. These results indicate that theresponse to variations in external pressures will be of the same magnituderegardless of temperature as long as the external and internal temperatures areequal.In onions, main effects for level of pressure variation and DCAP weresignificant (P<O.05) for relative rate of CO2 emission with the higher pressurelevel and longer DCAP resulting in higher relative rates. Although interactionbetween pressure level and DCAP was significant (P<O.05) this appeared tooccur in the mean comparison of 15 mm DCAP at 3.5 kPa and 5 mm OCAP at6.9 kPa which is not a comparison of interest. Pressure level and DCAP hadlittle effect on relative rate of CO2 emission in cucumbers and tomatoes whileincreasing the level of pressure variation from 3.5 to 6.9 kPa resulted in anincreased relative CO2 efflux rate in sweet peppers.E. EFFECT OF OXYGEN CONCENTRATIONTo determine if oxygen concentration would affect the CO2 efflux rate ofonions, cucumbers, sweet peppers and tomatoes subjected to cyclic pressurevariations, pressure treatments were carried out in a low oxygen atmosphere.54Table 1.2. Effect of level of pressure variation, temperature and DCAP onrelative carbon dioxide efflux rate1 in onion, cucumber, sweet pepperand tomato.Pressure Level (kPa)3.5 6.9DCAP2 (mm)Tissue °C 5 15 5 15Onion 0 1.5 (±0.7) 7.6 (±3.5) 2.9 (±0.8) 14.9 (±4.9)10 1.8 (±0.2) 9.2 (±1.8) 4.6 (±1.1) 20.1 (±7.9)20 1.9(±0.1) 6.9(±2.0) 2.4(±0.3) 12.7(±2.6)Cuc 10 0.9(±0.1) 1.2(±0.3) 0.9(±0.1) 1.2 (±0.2)20 0.8 (±0.2) 1.1 (±0.1) 0.9 (±0.1) 1.0 (±0.1)Pepper 10 2.1 (±0.7) 2.7 (±0.6) 2.9 (±0.4) 5.4 (±1.5)20 2.5(±0.1) 2.3(±0.3) 3.3(±0.8) 2.9(±0.6)Tomato 10 0.8 (±0.3) 1.0 (±0.2) 1.1 (±0.2) 1.4 (±0.2)20 1.5(±0.3) 1.0(±0.2) 0.9(±0.1) 1.0(±0.1)1 Measurement of relative rate of CO2 efflux determined by (CO2 efflux rateduring variable pressure)/(C02efflux rate during constant pressure), Means ±SD.2 Duration of the interval of constant atmospheric pressure between 5 mmvariable pressure treatments.55Recommended postharvest levels of 1% 02 for onions and 3% 02 forcucumbers, sweet pepper and tomatoes (Saltveit, 1985) were established byflushing the gas exchange apparatus with premixed gases. Since DCAPappeared to have an effect on CO2 emission rate, an interval of 15 mm ofconstant pressure between pressure treatments was chosen for the low oxygenstudies.At 1% 02, net CO2 efflux rate of onions during variable atmosphericpressure changes was significantly (P<O.0001) lower than that in onionsundergoing pressure treatments in air even though diffusive efflux at a constantpressure was unaffected by 02 concentration (Fig. 1.11). However, changes inatmospheric pressure still resulted in an increase (P<O.0001) in net CO2 effluxrate over constant pressure at low atmospheric 02. In cucumbers, low oxygenconcentrations reduced (P<O.05) net CO2 emission rates during constantpressure and variable pressure treatments but the overall response to variablepressure treatments was the same for both 02 levels. Low oxygen storage didnot appear to affect the response of tomatoes or sweet pepper to variablepressure treatment.It is possible that longer term storage of onions under low-oxygen mayresult in a damping of response to variable atmospheric pressures. Carbondioxide measurements taken over a period of 7 hours indicate that generally theresponse was not diminished over this period (Fig. 1.12). Similar results wereobtained with onions stored under fluctuating atmospheric pressures at 10 and20°C (not shown). Level of pressure variation at 3 different temperatures hadlittle effect on net CO2 efflux rate of onions held under I % 02 compared to theresponse at ambient 02 concentrations (Fig 1.13). As well, there were nosignificant (P>’0.05) differences between efflux rates at constant or variablepressure for all pressure treatments at I % oxygen and 0° C, and at5681J)1)-6 I: 021321ONIONTOMATOATMOSPHERICOXYGENCONCENTRATION(%)Fig.1.11.MaineffectofatmosphericoxygenconcentrationonnetCO2effluxrateofonions,cucumbers,sweetpeppersandtomatoes.,B,A,BBarsassociatedwitheachplanttissue,withdifferentletters,aresignificantly(P<O.05,Ftest)different.a,bBarsassociatedwitheachoxygenlevel,withdifferentletters,aresignificantly(P<O.05,pairedttest)different.IIConstantpressureIVariablepressureaaBBaI321CUCUMBER321PEPPER0.8 -0.7-U) 0.6-0.5-:::30.2-0.10.0- I I I0 100 200 300 400 500TIME (mm)Fig. 1.12. Sustainability of mass gas flow in an onion bulb duringvariable pressure treatments. Net CO2 emission rateduring variable pressure treatments is represented byclosed symbols and during constant pressure by opensymbols. Variable pressure cycles of 6.9 kPa amplitude witha 40 s period were applied for 5 mm at 0°C.58U)c,):1wFa::><D-JLLULuc.’100l)‘0)LuIa::><D-JLIULuC400I Constant pressure 3.5 kPa zzi 6.9 kPaFig. 1.13. Effect of oxygen concentration, pressure variations and temperature onnet Co efflux rate of jumbo onions. (A) 3% oxygen, (B) 21% oxygen.Treatmnt means and standard deviations for observations at constantatmospheric pressure (n=6) and variable pressure at levels of 3.5 (n=3)and 6.9 (n=3) kPa above atmospheric pressure.A1.51.00.50.010864200 10 20TEMPERATURE °CB0 10 20TEMPERATURE °C59constant pressure and a pressure level of 3.5 kPa at 10° C (see Table 1.5 ofAppendix 2 for details). In exploring the significant interaction between °2concentration and temperature (P<0.0001), contrast statements revealed thatthe relationship between temperature and oxygen is linear (P<0.0001). Thecombination of low oxygen and temperature must have reduced respiration rateto such an extent that mass gas transfer was restrained. This resulted in largedifferences in relative rate of gas transfer between onions held at I % oxygencompared to those held at 21% (Table 1.3).Ladeinde and Hicks (1988) reported that 24 h was required toestablish equilibrium level of internal 02 in onions held in atmospherescontaining different an 02 concentrations. At 0°C internal 02 concentrationsequaled 75.7, 77.9 and 93.7% of external atmospheres containing 4.5, 15 and21% 02, respectively; at 15°C the internal 02 concentrations equaled 55.3, 57.4and 78.9% of external concentrations, respectively; and at 30°C, theseconcentrations were 6.7, 31.5 and 48.6% of external concentrations,respectively. By extrapolating these results, the level of 1% 02 used in thisstudy should have resulted in internal 02 concentrations of about 0.75% at 0°Cand 0.4% at 20°C. According to Kader (1985), onions can continue to respireaerobically at very low external 02 concentrations which would explain whyonion respiration rate at constant pressure under both atmospheres wasunchanged. It is thought to be unlikely that changing atmospheric pressuresaffected respiration rate at a cellular level by increasing the internal partialpressure of 02 during application of the pressure treatments thereby increasingCO2 emission rate. If this were true then one might expect an increase inrelative CO2 emission rate with an increase in temperature. According to thedata in Table 1.3 relative rate decreased slightly at higher temperatures as itwould if 02 became more limited in internal tissues. In other words, the fact that60Table 1.3. Effect of oxygen concentration, level of pressure variation andtemperature on relative carbon dioxide efflux rate1 in onion,cucumber, sweet pepper and tomato.3.5Pressure Level (kPa)6.9Cuc 10 1.1 (±0.1)20 1.2 (±0.2)1.2 (±0.3)1.1 (±0.1)1.2 (±0.1)1.1 (±0.2)1.2 (±0.2)1.0 (±0. 1)Pepper 10 2.9 (±0.5)20 2.2 (±0.2)2.7 (±0.6)2.3 (±0.3)4.0 (±0.5)3.3 (±0.3)5.5 (±1 .5)2.9 (±0.6)Tomato 10 1.0 (±0.1)20 1.0(±0.1)1.0 (±0.2)0.9 (±0.2)1.2 (±0.2)1.3 (±0.2)1.3 (±0.2)1.0 (±0.1)I Measurement of relative rate of CO2 efflux determined by (CO2 efflux rateduring variable pressure)/(C02efflux rate during constant pressure), Means ±SD.Tissue °COnion 01020Oxygen Concentration (%)1 21 1 212.1 (±0.7) 7.6 (±3.5) 2.5 (±1.2) 14.9 (±4.9)1.6 (±0.4) 9.2 (±1.8) 2.4 (±0.5) 20.1 (±7.9)1.5 (±0.1) 6.9(±2.0) 2.3(±0.5) 12.7(±2.6)3 21 3 2161at 20°C, under an atmosphere containing I % 02, net CO2 efflux rate in onionswas not that much greater than at 0°C may indicate that varying atmosphericpressure only affected the movement of gases in the intercellular air spaces ofporous tissues. On the other hand, cycling pressures with a period longer than40 s may allow for diffusive transfer of 02 to centers of respiration resultingingreater respiratory activity.Lowering external 02 concentration to 3% reduced CO2 emission rate incucumbers during constant pressure and variable pressure cycles, but thereduction in response to low 02 was not as remarkable as that of onion (Fig.1.14, see Table 1.7 of Appendix 2 for details). It may be possible thatcucumbers must be stored for a longer period than the few hours required tocarry out the treatments in this study before respiration rate stabilizes inresponse to a lower concentration of 02. A study by Solomos (1982) indicatesthat for dense tissues like sweet potato, 48 hours is required for respiration todecline and stabilize after external 02 concentration is lowered. The data inTable 1.3 indicates that external 02 concentrations or levels of pressuretreatments at 10 and 20°C had little effect on the relative rate of CO2 emission.A 20.8% reduction in respiration rate of Chili peppers held at 10°C wasobtained in one published study by reducing external 02 concentration fromnormal ambient levels to 5% (Kader et al, 1989). In the present study, comparedto air, external concentrations of 3% 02 resulted in a 13.5% reduction (P>0.05)in CO2 emission rate in sweet pepper at 20°C (constant pressure), but there wasno reduction at 10°C (Fig 1.15). Although there was a tendency for pepperssubjected to pressure treatments at a low 02 concentration and 10°C to havehigher CO2 efflux rates than the peppers pressure-treated in air, this trend wasreversed at 20°C (see Table 1.9 of Appendix 2 for details).6210wIxD-JLLLiLU00‘I))UixD-JLLULUC.”00I I Constant pressure 1 3.5 kPa 6.9kPaFig. 1.14. Effect of oxygen concentration, pressure variations and temperature onnet CO2 efflux rate of cucumbers. (A) 3% oxygen, (8)21 % oxygen.Treatment means and standard deviations for observations at constantatmospheric pressure (n=6) and variable pressure at levels of 3.5 (n=3)and 6.9 (n=3) kPa above atmospheric pressure.A86420108642010 20TEMPERATURE °CB10 20TEMPERATURE °C63‘Cl)‘o.LUI><D-JL1LLUi0C-)‘Cl)-:1UiI><D-JLILLLiiC”0C)______Constant pressure I 3.5 kPa______6.9 kPaFig. 1.15. Effect of oxygen concentration, pressure variations and temperature onnet CO2 efflux rate of sweet peppers. (A) 3% oxygen, (B) 21% oxygen.Treatment means and standard deviations for observations at constantatmospheric pressure (n=6) and variable pressure at levels of 3.5 (n=3)and 6.9 (n=3) kPa above atmospheric pressure.A141210864201412108642010 20TEMPERATURE °CB10 20TEMPERATURE °C64There is no information, to the author’s knowledge, about the effect ofreduced atmospheric 02 concentrations on red ripe tomatoes since storageresearch is generally done with mature-green tomatoes, which have a less fluidinternal matrix and respire at a much higher rate. However, there is a greatinterest in bringing a ripe tomato to the retail shelf to assure the consumer ofbetter flavour quality. There is some value therefore, in investigating techniquesthat could maintain high quality in tomatoes harvested at a later stage ofripeness. It is currently unknown if altering internal gas composition of ripetomatoes has a noticeable effect on quality attributes.External oxygen concentration and level of pressure treatment had nosignificant effect (P>0.05) on net CO2 emission rate of tomatoes, however therewas a significant temperature effect (P<O.0001) on tomatoes held at both oxygenconcentrations. All interactions between main effects were insignificant(P>0.05). It was interesting to note that like peppers, the lower 02 concentrationat 10°C tended to result in a slightly higher net CO2 efflux rate in tomatoes butthis trend was reversed at 20°C (Fig 1.16, see Table 1.11 of Appendix 2 fordata). Relative CO2 emission rates for all treatments were similar (see Table1.3).The lack of a response to the lower 02 concentration is likely due to theshort term of the treatment. Because the internal matrix of tomato, especiallyripe tomato, is very liquid and the area where most gas exchange occurs isrelatively small more time was required for internal gases to equilibrate in a lowoxygen atmosphere. However, it was in the interests of this study to determine ifvariable pressure treatments affected the rapidity of a response to low levels ofatmospheric 02.In conclusion, this study shows that bulky fruits and vegetables with largemorphological differences vary greatly in their response to changes in651)IiiI—><D-JLIUw0C)I I I1iess.iie 3 5 I(I 6 9 Ic1:)aFig. 1.16. Effect of oxygen concentration, pressure variations and temperature onnet CO2 efflux rate of tomatoes. (A) 3% oxygen, (B) 21% oxygen.Treatment means and standard deviations for observations at constantatmospheric pressure (n=6) and variable pressure at levels of 3.5 (n=3)and 6.9 (n=3) kPa above atmospheric pressure.A5432I05432I010 20TEMPERATURE °CB‘0).I10 20TEMPERATURE °C66atmospheric pressure. Onions and sweet peppers both showed large relativeresponses to variable pressure treatments, but by changing temperatures, thetime interval between series of pressure fluctuations and external 02concentration, the effect of differing pathways for gas movement became moreapparent. Changing the time interval between the 5 mm pressure treatmentsfrom 5 to 15 mm more than doubled the relative rate of CO2 efflux in onions whileevoking little or no response in sweet peppers. This observation is probably dueto the fact that there was no long time lag in movement of CO2 from centers ofproduction to the cavity of the pepper fruit. Although cucumbers and tomatoeswere unresponsive to variable pressure treatments regardless of temperature,level of pressure treatment or 02 concentration, there was some indication thatincreasing the interval between series of pressure variations had an effect onCO2 efflux rate in cucumbers.Researchers have focused their attention on the effects of externalatmospheric concentrations of gases on fruits and vegetables while there is stillmuch to discover about the mechanisms and pathways of gas exchange in bulkytissues. These studies suggest that pressure-driven gas exchange mayaccelerate movement of internal and external gases, offering the potential forbetter control of gas concentrations in plant tissues.Other issues that need to be addressed are: assessment of effectivenessof positive and/or negative pressure pulses in promoting mass gas flow, whethersystematically varying the atmospheric pressure surrounding bulky tissues alterstotal gas concentrations in core tissues or only in intercellular spaces of thesuperficial tissues, and determination of the effect of long term applications ofvariable pressures on shelf-life and rate of loss of cellular constituents.67SECTION 2ANALYSIS OF AROMA VOLATILES OF ONIONS,PEPPERS, AND TOMATOESSTORED UNDER VARYING PRESSURES68INTRODUCTIONChanges in carbohydrates, organic acids, proteins, amino acids, lipidsand phenolic compounds can influence the flavour of fresh fruits andvegetables, either enhancing flavour quality or resulting in the production ofundesirable off-flavours. Controlled atmosphere and MA storage have beenused successfully to prolong shelf-life and maintain flavour quality in pome fruits,broccoli and root crops by suppressing senescence; to prevent enzymaticdegradation of structural carbohydrates, storage starch, amino acids and lipids;and to reduce the rate of formation of harmful phenolics (Kader, 1986).Improper control of CO2 and 02 concentrations can result in anaerobicrespiration resulting in the formation of acetaldehyde and ethanol, impartingundesirable flavour characteristics (Carlin et al., 1990). Controlled atmospherestorage for extended periods can decrease the production rate of volatiles byapples, pears and other fruits presumably by reducing the rate of alcoholsynthesis (Kader, 1986). Minor disturbances in the balance of variouscompounds constituting flavour may render a poor sensory quality rather than aserious off-flavour, nevertheless, changes of this nature can lead to reducedconsumer confidence in the product, resulting in economic loss.Sensory panels made up of trained testers are currently utilized fordetermining differences in flavour characteristics among food products.Considerable effort has been expended in the development of instrumentalanalysis to supplement or replace sensory analysis which is considered to beexpensive, difficult, labour intensive and inherently inaccurate and impreciseespecially when large numbers of samples or variables are involved in aninvestigation (Zervos et al., 1992).Salunke and Do (1976) asserted that the aroma of fruits and vegetables isthe key factor for assessing their flavour quality. Headspace analysis is69considered to be the appropriate method for application to flavour researchsince it reveals the identity and the concentration in the vapour phase of thosecompounds that are directly responsible for the odour of the product (Maarse,1991). Trapping headspace volatiles on a solid sorbent concentrates the traceanalytes sufficiently for identification by GC/MS. The use of a solvent to desorbthe volatile compounds from the solid support produces a liquid sample whichcan be stored or used for multiple gas chromatographic analyses.Early in the development of experiments to test the effect of variableatmospheric pressure on mass gas flow in bulky tissues, a suggestion was madethat variable pressures may enhance enzymatic degradation of vegetables dueto an influx of 02 into internal tissues (Solomos, T., private communication).Since flavour is an important factor in assessing changes in quality, this studywas undertaken to determine if cyclic variations in total external atmosphericpressure would alter chromatographic profiles of aroma volatiles. The dynamicheadspace sampling method of Buttery et al. (1987) was modified by theinnovative use of disposable charcoal traps rather than Tenax traps which havea much lower loading capacity and are negatively affected by the presence ofwater vapour.70MATERIALS AND METHODSA. PLANT MATERIALSTomatoesTomato plants (LycopersIcon esculentum L. cv Dombito) were grown in agreenhouse at Agriculture and Agri-Food Canada’s Pacific Research Center atAgassiz. The fruit were harvested at the “turning” stage, randomly assigned totwo groups and weighed. One group was placed in the treatment chamber of thegas exchange circuit described in Section 1 and the other group was placed in aforced-air, walk-in cooler.Both containment systems were maintained at 15 °C and 95% RH. Afterloading, the gas-exchange circuit was closed and variable pressure treatments(see Section 1) were initiated. The 5 mm treatments were applied at a level of6.9 kPa with a 15 mm interval of constant pressure between treatments. Thetreatments were carried out in triplicate for 3 and 7 days. At the end of each ofthese time periods, the tomatoes were removed from the treatment chamber andthe walk-in cooler, weighed and stored in the laboratory at room temperature(average 24 °C) under normal laboratory lighting and allowed to ripen to a fullred coloring. This ripening period was 13 and 11 days for the 3 and 7 daytreatments, respectively. After ripening, the fruits were prepared for isolation ofaroma volatiles.OnionsOnions (Allium cepa L.) were purchased from a commercial source asrequired for the experiments, resulting in a sample selection from variousorigins, maintained under unknown conditions. Care was taken to select onions71free from defects and disease. The onions were divided into two groups andsubjected to the same conditions as the tomatoes except the treatment periodswere 7, 14, and 21 days, replicated 3 times. Following treatment the onionswere immediately prepared for isolation of aroma compounds.Sweet PeppersSweet pepper plants (Capsicum annuum L. cv Doria) were grown in agreenhouse on the Research Station at Agassiz. Immature pepper fruitsweighing approximately 200 g each were harvested as required for eachexperimental replicate and divided into two treatment groups. The sweetpeppers were subjected to the same treatments as tomato and onions overperiods of 3, 7 and 14 days. In addition, a preliminary trial was performed todetermine if pepper volatiles were affected by the concentration of oxygen whilestored under variable atmospheric pressures. Sweet peppers were stored for Iweek, surrounded by either 21 or 3% oxygen, under a variable atmosphericpressure of 6.9 kPa. Following treatment the peppers were prepared forisolation of aroma volatiles.B. REAGENTS and APPARATUSJacketed jars from Wheaton (Millville, New Jersey 08332, U.S.A.) fittedwith teflon and glass connectors (Fig. 2.1) served as extraction vessels. A glasschromatography column containing a plug of silanized glass wool allowed mostof the water to condense before reaching the activated charcoal tube. Thetemperature of the vessels was controlled by a recirculating water bath (Haake)and jars could be joined in tandem for multiple analyses. High-grade purified N2was delivered at a rate of 30 mLlmin by four tube rotameters (Matheson, 740072C)N2activatedcharcoalglasswoolwater4dCjacketedjarFig.2.1.Illustrationofthedynamicpurgeandtrapsystemforthecollectionofvegetablevolatilecompounds.Series) fitted with model 610 flowmeter tubes. The activated charcoal adsorbenttubes (Orbo-32TM, 6 mm OD x 4 mm ID x 10 cm, 20/40 mesh, single bed) usedas volatile traps, were obtained from Supelco (Oakville, ON). Authenticreference chemical compounds of high purity were obtained from commercialsources (Sigma Chemical Company, St. Louis, MO and Aldrich Chemical Co,Milwakee, Ml). Methylene chloride, J.T. Baker “capillary analyzed”, wasobtained from Caledon Laboratories Ltd. (Edmonton, AB) and used as thedesorbing solvent without further purification. Ethyl antioxidant 330[1,3, 5-trimethyl-2,4,6-tris(3, 5-di-tert-butyl-4-hydroxybenzyl)benzene; Ethyl Corp.,Baton Rouge, LA] was added to the methylene chloride (0.001%) as apreservative, just prior to desorption of volatile compounds. Internal standardsolutions were prepared separately by the addition of accurately measuredamounts of 2-octanone (175 pL), ethylbenzene (200 pL) and 3-pentanone (250pL) to 5 ml methanol. A saturated CaCl2 (ICN Biomedicals Canada Ltd.,Mississaugha, ON) solution was prepared as previously described (Buttery etal., 1987) by adding an excess of CaCI2 to grade I water and boiling the solutionin an open Erlenmeyer flask for I h to remove volatile impurities.C. ISOLATION AND CONCENTRATION OF AROMA COMPOUNDSA tomato sample (400 g at 24 °C) of Ca. equal pieces cut from fourdifferent tomatoes from each of the storage treatments, was blended for 45 secin a Braun food processor. Saturated CaCl2 solution (400 mL at 24 °C) wasadded all at once and the mixture blended for 15 seconds. The mixture wasplaced in a preheated Wheaton purge and trap jar. The jar was sealed and thehomogenate was purged briefly with nitrogen gas flowing in through one of theside arms of the jar. Five microliters of the 3-pentanone solution was addedthrough the other side arm on the vessel, the charcoal trap was attached and the74jar resealed. Tomato volatiles were captured in the trap by bubbling nitrogenthrough the vigorously stirred mixture for 4 h. Following the collection period,the charcoal trap was removed and volatile compounds were extracted with 3 mLof methylene chloride. The extract was concentrated to ca. 30 pL on ice under agentle flow of purified nitrogen.Volatile concentrates were obtained from onion and sweet pepper inessentially the same way except that 300 g of onion or sweet pepper tissue wasblended with 400 mL of saturated CaCI2. Onion homogenates were allowed tostand for 100 sec before the addition of CaCl2 since the characteristic flavour ofthe onions develops only after rupture of the cells. Control over the time periodbefore addition of CaCI2 was important in replication of the sample collection.Five microliters of 2-octanone were added to sweet pepper homogenates as theinternal standard and for onion, 5 pL of the 3-pentanone standard solution wasused.The area count ratio between peaks of interest and that of the internalstandard was calculated to determine relative quantities of volatile compounds.D. CAPILLARY GC-MS ANALYSISAnalytical ConditionsThe analytical column used in this study was a 30 m x 0.32 mm (i.d.)bonded phase DB-1 fused-silica capillary column (J&W Scientific, Folsom, CA)with a I pm phase thickness. Helium (ultra high purity) carrier gas, set at a flowvelocity of 30 cm/sec, was further purified by placing a 5 pm screen, an oxygenand a hydrocarbon trap in line. The GLC oven was held at 35 °C for 10 mm afterinjection, programmed at 5 °C/min to 180 °C, then to 240 °C at 10 °C/min and75held at the final temperature for 10 mm. The injector temperature was 220 °C. Asample size of 2 pL was injected in splitless mode.A Hewlett-Packard 5890 gas chromatograph, mounted with a HP 7673Aautoinjector and interfaced to an HP 5970B quadrupole Mass Selective (MS)Detector was used for all volatile compound analyses. The MS detector wasoperated in the electron impact mode at 70 eV, taking scans from 30 to 350 mlzin a 1-s cycle. The temperature of the open-split interface was 245 °C.Identification of Vegetable Volati lesChromatographic peaks were identified by using a software driven librarysearch routine which automatically compared user-specified sample spectra to auser-generated or NBS (National Bureau of Standards) library. Authenticstandards with a purity of at least 95% were chromatographed under the sameconditions as the samples. Mass spectra from these standards were compiledon hard disk in a custom-made library and, together with the retention times,were used to confirm the identity of unknown peaks in the total ionchromatogram.In this study, n-paraffinsC6-C24 were chromatographed under the sameconditions as the samples to develop retention indices for unknown peaks forcomparison with published indices. A comparison of linear retention indices isoften useful to tentatively identify unknown compounds. The most widely usedindex is the Kovats retention index, I, which utilizes the linear relationshipbetween log retention time, t , and the carbon number of an n-alkane standard(Lee et al., 1984). The retention time of compound x[t] is interpolated betweenthe values of t for two adjacent n-alkanes with carbon numbers z and z + I[retention times t(z) and t(z +1) ]:76I(x)=lOOz +100logt(x)—logt(z)log t (z +1)—log t (z)In the case of temperature-programmed gas chromatography, log t isreplaced by the elution temperature, TR. The temperature programmed retentionindex, l(x), for a compound eluted between two n-alkanes with carbon numbersz and z + I is given by the following equation provided that the program beginsat a low enough temperature.I (x)=lOOz+100 TR(x)—TR(z)p 7?(z+1)—2(z)Recovery and Precision TestsTwo model systems are described for testing the recovery rate andprecision of the methodology for capturing aroma volatiles. Two mixtures ofcompounds representative of vegetable aromas were prepared (see Table 2.1 inResults and Discussion on p. 81). For each mixture, 20 pL of each compound(weight determined from density) was accurately measured into a container and0.1 % of Ethyl Antioxidant 330 was added. The mixtures were stored at -4 °C.Two microliters of the first model mixture were added to 400 mL of aCaCl2 solution (near saturation) together with the 2-octanone standard solutionin each of 6 Wheaton jars. The volatile compounds were collected in the samemanner as vegetable volatile samples. The volatiles were desorbed with 3 mL ofdichloromethane which was reduced to a volume of Ca. 30 pL. Three microlitersof a 50 ppm standard of ethylbenzene were added to each sample which wasthen taken up in a 100 pL syringe and the volume brought to 50 pL. Onemicroliter of the extract was injected automatically and analyzed by GC-MS.Similarly, 2 pL of the second model mixture were added to a homogenate77of 300 g of green tomato and 400 mL of saturated CaCI2 solution. A largehomogenate of green tomato was first prepared and divided into 6 jars for evenreplication. Five microliters of 3-pentanone internal standard solution wereadded to the homogenate instead of 2-octanone. Sample collection, elution andanalysis for the second model mixture were the same as for the first mixture.Response factors (MS) were determined relative to the internal standards3-pentanone, 2-octanone and ethylbenzene by making known solutions indichloromethane.Statistical AnalysisData for the different components were analyzed by analysis of varianceusing the General Linear Model (GLM) procedure, a program of the SASInstitute Inc. (Gary, NC). The experiment was analyzed as a one-way analysis ofvariance with 3 replications. Treatment comparisons were carried out usingFisher’s (protected) least significant difference (LSD) test (Steel and Torrie,1980) at a 5% level of significance. Principal component analysis (PCA) wasapplied to the GC data to determine if, by condensing the variability into a limitednumber of variables, the effect of variable pressure storage of tomatoes, onionsand peppers could be distinguished from a constant pressure treatment (SAS,1987; Zervos and Albert, 1992).78RESULTS and DISCUSSIONA. Method Develooment for Isolation of VolatilesThere were many considerations in selecting a technique to isolatevolatile compounds such as: the cost of the more commonly used specializedglassware and instrumentation such as the Likens-Nickerson apparatus, HewlettPackard headspace analyzer or Teckmar purge and trap unit, the limitedavailability of fume hood space, the low concentrations and thermolability of thecompounds of interest and the need for easy replication. Another importantconsideration was the need for a liquid rather than a gaseous sample soanalysis by GC-MS could be automated.The dynamic headspace sampling technique using a solid sorbantdeveloped by Buttery et al. (1987), was modified for use in this study. Inemploying this technique, Buttery and his associates used 10 g of Tenax in eachcollection tube and a very fast flow of purified air (3 L/min) for entrainment oftomato volatiles. Attempts were made in this lab to use smaller amounts ofTenax GC and TA (150 mg) and a lower gas flow rate (30 mL/min), in a mannersimilar to Shamaila et al. (1992, 1993). The amount of volatile compounds fromtomatoes and sweet peppers that could be captured on this adsorbent, however,was insufficient for analysis by GC-MS. Although Tenax can be purified andreused, a less expensive sorbant which did not require time-consumingregeneration was desired.Tubes containing activated coconut charcoal (custom ordered Orbo32TM,6 mm O.D. x 10 cm long with a single 7.5 cm bed of charcoal) of acceptablepurity, were inexpensive, simple to use and elute, and had a greater capacity toadsorb volatile compounds than the same weight of Tenax. Since this studyutilizing activated charcoal was initiated, there have been two reports of the use79of the same product for isolation of strawberry (Perez et al. 1992) and apple(Ollas et al. 1992) volatiles.In order to deactivate enzyme systems, plant tissues were homogenizedwith a saturated solution of CaCl2 as described by Buttery et al. (1987). Enzymedeactivation was necessary due to the long collection period (4 h) and thepresence of an anaerobic atmosphere in the sample jar, both of which wouldalter the volatile composition. The use of CaCI2 rather than heavy metal saltswas more environmentally sound and also increased the air/water partitioncoefficient of many volatile compounds, causing a “salting out” effect noted byButtery and his associates (1987). In other words, CaCl2 in the homogenatedecreased the solubility of aroma compounds in the aqueous mixture.Unlike Buttery et al. (1987), surrogate internal standards (2-octanone,ethylbenzene and 3-pentanone) were prepared in methanol rather than water toimprove reproducibility of the standard addition. Due to its low molecular weightmethanol is not retained well on charcoal. Nevertheless, amounts of methanolwere kept to a minimum to avoid binding some of the active sites on thecharcoal.Air cannot be used as the purge gas with activated charcoal because ofits reactivity, but the charcoal sorbant can be eluted with a wide range ofsolvents and its activity is unaffected by water in headspace vapours.Dichioromethane was chosen as the eluting solvent because fresh batchescould be quickly obtained from the manufacturer allowing maximum shelf-life ofthe solvent. The use of carbon disulfide was attempted but a supply ofacceptable purity could not be found and redistillation on an efficientfractionating column proved unsatisfactory. It was observed during the course ofthis work that Tenax can only be eluted with non-polar solvents. At low initial80temperatures in the GC oven, pentane or diethyl ether both cause excessivepeak tailing on a non-polar capillary column, obscuring early eluting peaks.B. Precision and AccuracyPrecision is a measure of the reproducibility of an analytical procedureaccounting for individual variabilities from the sampling, extraction andinstrumental analysis procedures. Overall precision is estimated by calculationof the standard deviation of a sample population which is normalized to themean value to yield the relative standard deviation (RSD) or coefficient ofvariation (CV) as in the following equation:RSD1=s1/XxlOOTo reduce variability due to instrumental precision, a calibration mixture was rundaily and the Mass Selective Detector was calibrated by a software controlledprogram.Accuracy refers to how close the reported value is to the true value. Theaccuracy of the calculated result depends on the precision of the procedure,interferences in the determination, effects of the matrix, instrumental calibrationand losses that occur during sample preparation. Attempts were not made inthis study to discover the true bias in the found vs. known results since theheadspace capture methodology will not extract total volatiles. By reporting theamounts of identified compounds in relation to an internal standard some of thesources of error involved in extraction and analysis of minute amounts ofvolatiles are defined.The results of the accuracy and precision tests are given in Table 2.1.Measurement of extraction recoveries was not performed since, in applying thepurge and trap method to fresh vegetables, it would have been impossible toachieve complete recovery of aroma compounds. Complete validation of the81Table 2.1. Accuracy and precision determinations with a water(Model 1) and a green tomato (Model 2) matrix.Model I Accuracy (%)8 CVLL-terpineol 5.8 4.6(S)-(-)-Iimonene 21.5 6.91-heptanol 11.3 4.92-ethylfuran 15.0 3.22-furaldehyde 3.2 9.82-methyl-I -butanol 13.6 5.82-octanone 49.6 3.2eugenol trace -geranyl tiglate trace -hexenal 31.1 8.3terpinen-4-ol 11.9 10.7trans-2-heptenal 24.0 11.0trans-2-hexenal 34.2 5.2Model 2 Accuracy (%) CVx-terpineol 1.5 6.5f3-ionone trace -I -penten-3-one 5.6 8.92-isobutylthiazole 41.2 6.33-methyl-I -butanol 10.4 4.83-pentenone 17.7 5.46-methyl-5-hepten-2-one 11.3 9.5geraniol 25.4 8.6geranyl acetone trace -hexenal 24.4 8.2linalool 32.5 10.5terpinen-4-ol 8.7 7.4trans-2-heptenal 1 8.1 4.2a Accuracy % = observed amount/known amount x 100.n=682method would have required the availability of all of the chemical compoundsidentified in this work.Buttery et al. (1987, 1988) observed that percent recovery of 3-pentanone, 2-octanone, I -penten-3-one, geraniol, 6-methyl-5-hepten-2-one,hexenal, trans-2-hexenal, 3-methyl-1-butanol, 2-isobutyithiazole and linalool onTenax was approximately twice as great as the amounts trapped on activatedcharcoal in the present study. Since the volume of the sweep gas used byButtery et al. (1987, 1988) was 2.5 times greater than in the present study thisresult was not totally unexpected. The poor rate of capture of geranylacetonewas unexpected, however, since Buttery et al. (1988) observed an absoluterecovery of 36% of this compound from water solutions. It appears thereforethat geranylacetone must have been tightly adsorbed to the activated charcoal.Eugenol, 3-ionone and geranyl tiglate could have been strongly bound to theadsorbent as well since only traces were found in the extracts (Table 2.1).Recovery of some of the volatiles could be related to a their affinity for water andhence their volatility [air to water partition coefficient] (Buttery et al., 1988).The green tomato matrix appeared to affect the amount of cx-terpineol,hexenal, trans-2-heptenal and terpene-4-ol captured on activated charcoal.Green tomatoes were chosen as an organic matrix for the precision testsbecause, other than trans-2-hexenal, the amounts of volatiles are very low.Petro-Turza (1987) noted that the acidity of the medium or the conditions ofsample preparation (enzyme activity, the amount of oxygen in the sample or theextent of comminution) affect the quality and quantity of aldehydes and alcoholsrecovered from tomato.C. Effect of Atmospheric Pressure Treatments on Onion Volatiles83The characteristic aroma of onions is attributed to the numerous sulfur-containing volatiles in these plants. One hundred and forty compounds, most ofwhich contain sulfur, have been isolated from fresh and cooked onions byvarious extraction methods. The composition and formation of volatiles in onionhave been recently reviewed (Carson, 1987; Whiffield and Last, 1991).The twenty-three organic compounds listed in Table 2.2 were recoveredfrom the headspace of onion homogenates and are numbered according to theirorder of elution from the gas chromatographic column. The aroma compoundswere identified by comparing GC peak retention indices with those in theliterature (RI) or matching sample spectra with spectra from authenticcompounds (CS) as well as spectra contained in the NBS library (MS). Total ionchromatograms of volatile components from onions subjected to variablepressure treatment for I to 3 weeks were compared to those from onions storedfor the same length of time under constant pressure (Fig. 2.2, see Fig. 2.20 to2.24 in Appendix 3). Peak numbers in the Table 2.2 correspond to numberedpeaks in Fig. 2.2.Pure, authentic chromatographic standards for comparison with onionvolatiles are not easily obtained from commercial sources, due the extremetoxicity of such compounds. As a result, much of the identification of onionaroma compounds by a number of researchers (Brodnitz and Pascale, 1971;Boelens et al., 1971; Galetto and Hoffman, I 976a, I 976b; Kallio and Salorinne,1990; Farka et al., 1992; Block et al., 1992a, 1992b; Kuo and Ho, 1992a,1992b; Tokitomo and Kobyagashi, 1992; Ohta and Osajima, 1992) has beenaccomplished through in-lab or custom synthesis of the desired compounds,interpretation of MS fragmentation patterns, comparison of spectra and retentionindices with previous reports, or the use of other instrumentation such asNuclear Magnetic Resonance (NMR) to assist in confirming the identity of a84Table 2.2. Flavour constituents of onion.PeakNumbera Component Ip (DB-1 )b IDI I ,3,5-cycloheptatriene 749 MS2 5-hexyn-3-ol 769 MS3 2-methyl-2-pentenal123 816 MS RI CS4 S-propyl thioacetate 851 MS RT5 1 ,4-dimethyl benzene 853 MS CS6 2,4-dimethyl thiophene123 855 MS RI7 3,4-dimethyl thiophene123 882 MS RI8 unknown 8969 unknown 90310 methylpropyl disuIfide123 905 MS RI11 methyl 1-propenyl disuIfide123 915 MS RI12 propanthioic acid 940 MS13 unknown 96014 2-pentylfuran3& unknown 978 MS CS RI15 decane 1000 MSCS16 dipropyl disulfide123 1095 MS RI17 propyl 1-propenyl disulfide12. 1097 MS RI18 unknown 114619 unknown 122420 unknown 122621 dipropyl trisuIfide123 1270 MS RI22 3, 5-diethyl-1 ,2,4-trithio-Iane 2,3 1280 MS RI23 3,5-diethyl-1,2,4-trithiolane (isomer)l’23 1283 MS RIMS, mass spectra of unknown tentatively matched with spectra in the massspectral library of the National Bureau of Standards (Probability BasedMatching); RT, retention time of unknown peak matched with retention time ofpeak chromatographed under similar conditions in the published literature; RI,retention index of unknown peak matched against retention index in publishedliterature; CS, mass spectra of unknown matched with spectra and retentiontimes of authentic compounds.a Peak number matches peaks numbered in Fig. 2.2b KovaVs linear retention indices with temperature programming (Lee et al.,1984).I Farka et al. (1992), 2 Kuo and Ho (1992a), Kuo and Ho (1992b)85TICofIJATA:ONIOR1A.IJB.E6-1617SB.E65.EB3C18iti-4ØEBC30E689141921_______Time(mm.)______Fig.2.2.Totalionchromatogramofvolatilesextractedfromonionstoredunderconstantpressureat15Cfor7days.NumbersrefertocompoundslistedinTable2.2.compound.Some relatively large peaks in the onion samples were unidentifiableeither by comparing spectra or relative retention index (Table 2.2). These peakscould be overlapping compounds (which makes spectral identification verydifficult), degradation products of thermolabile sulfur-containing constituents andnot identifiable from the literature or true unknowns. The thiosulfinates found inonions are unstable and break down nonenzymatically to yield alkyl and alkenylmono-, di- and trisulfides or may be oxidized to the corresponding sulfonates(Whitfield and Last, 1991).The main purpose of this study was not to study onion volatilecomponents, but to determine if storage under variable atmospheric pressurehad an effect on the chemical composition of aroma extracts. A visualcomparison of the total ion chromatograms reveals very little about changes involatile composition due to storage treatment (Fig. 2.2 and Fig. 2.20 to 2.24 inAppendix 3). Moreover, the complexity of the data obtained from GCIMSanalysis (Table 2.3) does not allow the observer to readily distinguish majortreatment effects although individual peak differences are apparent.Variable pressure treatments increased relative amounts of 2-methyl-2-pentenal recovered from onions compared to onions held under constantpressure (Table 2.3). This increase, however, was significant (P<0.05) only forthe 7 and 14 day treatment periods. Total relative concentration of volatiles washigher in the pressure-treated onions than in the onions stored at constantpressure although this effect was significant (P<0.05) only for the comparisonbetween onions stored I week under variable pressure and onions stored 1-3weeks under constant pressure. By week 3, volatile composition of variablepressure-treated onions was very much like that of the constant pressuretreatments.87Table2.3.Means1ofvolatilecompoundsidentifiedinonion.Relativeamountsofcompounds3PeakNumberComponentCON(7)2CON(14)CON(21)VAR(7)VAR(14)VAR(21)I1,3,5-cycloheptatriene0.380.540.390.410.420.4025-hexyn-3-ol0.190.250.401.271.420.2532-methyl-2-pentenal555Cd3•jjd8.28Cd20.91a17.Ogab11.994S-propylthioacetate0.080.110.080.100.090.1151,4-dimethylbenzene0.02c0.O4bc0.O7abc0.O7abc0.lla0.O8ab62,4-dimethylthiophene0.050.050.160.180.140.1973,4-dimethylthiopheneO.382b0.31b0.32b0.75aQ74aO.422b8unidentified2.471.237.1913.9910.657.449unidentified2.94ab1.17b5.712b9.48a797ab5.66ab10methylpropyldisulfide0.140.220.170.140.120.1811methyl1-propenyldisulfide0.070.110.050.180.170.0912propanthioicacid0.14b0.15b0.18bQ49a022b0.17b13unidentified2.18b1.18b2.95ab6.41a313ab457ab142-pentylfuran&unidentified2.83abI.49b3•73ab74Qa435ab5.57ab15decane0.080.100.060.080.080.0616dipropyldisulfide3.O9ab5.4222.64b3.64ab4.25ab3•93ab17propyl1-propenyldisulfide4.86ab7.80a3•95b7.O3ab6.382b4.9lab18unidentified2.24bC2.84abc1.52c4.1033jab1.78bc19unidentified0.720.380.842.061.331.6020unidentified1.19b0.43b1.05b3.85a1.27b1.70b21dipropyltrisulfide0.89bcI.4Oabc0.57cI9321.76abI.O2abc223,5-diethyl-1,2,4-trithio-lane0.801.621.312.091.931.65233,5-diethyl-1,2,4-trithiolane(isomer)1.37”°I.88abC0.67c435a3•79abI97abcTable2.3continuedRelativeamountsofcompounds3CON(7)2CON(14)CON(21)VAR(7)VAR(14)VAR(21)Totalamountsrelativetointernal32.67b3181b42.27b9J9a70.78ab5589abstandardImeansofthreeobservations.2CON-constantpressure,VAR-variablepressure,numberofdaysoftreatmentinparenthesis.Amountsarerelativeto3-pentanone(internalstandard)peak.abcMeansinthesamerowwithdifferentlettersaresignificantly(P<O.05)different.Because of the complexity of flavour analysis, statistical treatment of onevariable (i.e. one compound) at a time is often not sufficient to determinerelationships among treatments. Principal component analysis (PCA)condenses the variability into a limited number of variables and extracts theeigenvectors and eigenvalues used to perform the calculation of sets ofcoordinates. Two-dimensional plots of the points indicated by these coordinatesmay suggest some natural groupings of sample materials. According to Zervosand Albert (1992), one of the simplest analyses of flavour data is a point cloudon a plane formed by a pair of principal components. Zervos and Albert (1992),using data from the flavour analyses of orange juice, illustrate how samplesassociated with a treatment sometimes cluster in different regions of a system ofnormal orthogonal axes, presenting an identifiable pattern.Principal component analysis was used to extract the eigenvectors andeigenvalues from the onion volatile data (Table 2.4). According to Afifi and Clark(1984), a rule of thumb adopted by many investigators is to select only theprincipal components explaining 5% or more of the total variance. In this case,the first 5 principal components (eigenvector numbers I to 5) account for 85.2%of the variability in the onion data (Table 2.4). Examination of the coefficientsdefining each of the principal components reveals the correlation between agiven variable and the principal component (Table 2.5). For each principalcomponent, the variables with a correlation greater than 0.5 with thatcomponent, are underlined. The value of 0.5 was chosen for illustrationbecause it was used by Afifi and Clark (1984) in their discussion oninterpretation of multivariate analysis. The correlation between the ith principalcomponent C and the j’ variable x is = a,/VarC1 therefore, a coefficient ais underlined if it exceeds O.5/1]VarC.90Table 2.4. Principal component analysis (PCA) of volatile compoundsfrom onions stored under constant and variable pressures.Eigenvector Variance preservednumberEigenvalue Each Total1 10.741 46.7 46.72 3.883 16.9 63.63 2.058 8.9 72.54 1.650 7.2 79.75 1.277 5.5 85.26 1.067 4.6 89.97 0.626 2.7 92.68 0.503 2.2 94.89 0.310 1.3 96.110 0.259 1.1 97.211 0.224 1.0 98.212 0.164 0.7 98.913 0.104 0.5 99.414 0.069 0.3 99.715 0.043 0.2 99.916 0.017 0.1 100.017 0.004 0.0 100.091Table 2.5. Coefficients of the first five principal components of the onionvolatiles data set.Principal ComponentPeakNumber 1 2 3 4 5PK1 0.058632 0.409061 -.108862 0.360228 0.054402PK2 0.175463 0.007802 0.275465 0.044524 -.510911PK3 0.280391 -.162314 0.034219 -.058251 0.027632PK4 0.096838 0.242732 -.338864 0.153860 0.142915PK5 0.213376 -.080227 0.176382 0.363961 -.008061PK6 0.147642 0.029712 0.412833 0.362022 0.269231PK7 0.290305 -.004466 0.035958 -.021501 -.188106PK8 0.261106 -.123413 -.188010 0.098871 0.159776PK9 0.263306 -.186953 -.132618 0.049956 0.034413PKIO -.081276 0.237002 0.438254 0.125573 0.086326PK11 0.222694 0.193197 -.028676 0.238520 -.260267PKI2 0.159617 0.091409 0.325464 0.035036 0.137405PKI3 0.252381 -.224238 -.041702 -.036783 0.163831PK14 0.253247 -.200429 0.009109 -.014892 0.143866PKI5 0.081509 0.323757 -.416617 0.200462 -.062269PKI6 0.020687 0.346202 0.222071 -.331318 0.242101PKI7 0.143371 0.331550 -.029688 -.389036 0.142632PK18 0.219496 0.207498 0.105198 -.300986 -.211122PKI9 0.223856 -.202076 -.014757 -.022174 -.153123PK2O 0.222727 -.110101 -.032914 -.159171 0.444817PK2I 0.260163 0.133881 -.007829 -.244089 -.214168PK22 0.232647 0.209034 -.034991 0.064651 0.182379PK23 0.286055 0.072699 -.049405 -.089328 -.103541Var C1 10.741CPEb 46.7O.5/.jVarC 0.1533.883 2.058 1.650 1.27763.6 72.5 79.7 85.20.254 0.348 0.389 0.442a Eigenvaluesb Cumulative proportion explainedFor each principal component, the variables with a correlation greater than 0.5with that component, are underlined.92Table 2.5 shows that many peaks were highly correlated (greater than0.5) with the first component, with all of the correlations being positive. Peaks 1,15, 16, and 17 were highly correlated with the second component and peaks 6,10 and 15 (a strong negative correlation) with the third component. The thirdcomponent is a contrast of peaks 6 and 10 against 15.The factor pattern calculated by the statistical procedure can be plottedfor each onion treatment. The arrangment of points (each representing atreatment rep) in a cloud plane (Fig. 2.3 and Fig. 2.4) indicate that variablesassociated with the first principal component were important in separatingtreatment effects. The first three eigenvectors extracted from the onion volatiledata accounted for 72.5% of the variability and were, therefore, important inidentifying differences in aroma compounds between variable and constantpressure treatments. It appears from the plots in Fig. 2.3 and 2.4 that variablepressure treatments had the greatest effect on onion volatile constituents inweek I and week 2 of storage. Due to extensive variation in onion samples,there was no clear separation of the treatments according to duration of storageperiod in the principal component plots.Since early eigenvectors may not always best show the existing classseparation in the sample space it is worthwhile plotting later values against thefirst component (Zervos and Albert, 1992). The plots in Fig. 2.5 and Fig. 2.6show a tighter grouping of onion volatiles from onions stored under constantpressure, although onion volatile constituents from the variable pressuretreatments were very similar.Onion aroma contains of a number of strong chemical irritants. Sensoryproperties of a fresh vegetable such as this are very difficult to assess bysensory evaluation. Although the onions in this study were not evaluatedformally by a trained panel, it was noted that the presence of the9380)CDCNCG)00E0C-)CDC-)C0• constant pressure (1 week)• constant pressure (2 weeks)A constant pressure (3 weeks)o variable pressure (1 week)D variable pressure (2 weeks)variable pressure (3 weeks)Fig. 2.3. Principal component analysis of aroma compounds from onionsstored under constant atmospheric pressure and variablepressure treatments (n=3). A two-dimensional plot of thefirst and second eigenvectors.6-4-2-0—-2--4- I I I I I I-6 -4 -2 60 2 4Principal component 1 (46.7%)89480)CC1)a.)C00.E0000Fig. 2.4. Principal component analysis of aroma compounds from onionsstored under constant atmospheric pressure and variable pressuretreatments (n=3). A two-dimensional plot of the first and thirdeigenvectors.6-4-2-0--2--4--6 -4 8Principal component 1 (46.7%)• constant pressure (1 week) 0 variable pressure (1 week)• constant pressure (2 weeks) ci variable pressure (2 weeks)A constant pressure (3 weeks) variable pressure (3 weeks)-2 0 2 4 695C4.1-0G)00E0()00C• constant pressure (1 week)I constant pressure (2 weeks)A constant pressure (3 weeks)o variable pressure (1 week)D variable pressure (2 weeks)variable pressure (3 weeks)Fig. 2.5. Principal component analysis of aroma compounds from onionsstored under constant atmospheric pressure and variable pressuretreatments (n=3). A two-dimensional plot of the first and fourtheigenvectors.8-6-4-2-0--2 --4-A.-----6I I I I-4 -2 20 4Principal component 1 (46.7%)6 89680’1’,4-’Ca)C00E0C.)00• constant pressure (1 week)• constant pressure (2 weeks)A constant pressure (3 weeks)o variable pressure (1 week)D variable pressure (2 weeks)variable pressure (3 weeks)Fig. 2.6. Principal component analysis of aroma compounds from onionsstored under constant atmospheric pressure and variable pressuretreatments (n=3). A two-dimensional plot of the first and fiftheigenvectors.6-4-2-0—-2--4- T—---6 -4 -2 0 2 4Principal component 1 (46.7%)6 897lachrymatory factor, thiopropanal S-oxide (not found in GC-MS analysis due todegradation in the hot GC oven), did not seem as intense in onions stored undervariable pressure compared to onions stored under constant pressure.D. Effect of Atmospheric Pressure Treatments on Tomato VolatilesVolatile substances, imparting the characteristic flavour and aroma oftomatoes, develop during ripening and during cellular disruption as a result ofenzyme action. Flavour constituents are derived mainly from fatty acids andamino acids (PetrO-Turza, 1987). Of the approximately 400 volatile compoundsso far identified, compounds considered to be most important in the aroma ofraw tomato are Z-3-hexenal, 3-methylbutanal, J3-ionone, I -penten-3-one,hexanal, Z-3-hexen-1 -ol, E-2-hexenal, 2- and 3-methylbutanol, 2-isobutyl-th iazole, eugenol, 6-methyl-5-hepten-2-one and dimethyl trisulfide (PetrO-Turza,1987; Whiffield and Last, 1991). Aroma volatiles identified in tomatoes storedunder constant and variable pressure treatments for 3 and 7 days are listed inTable 2.6 and total ion chromatograms showing typical volatile profiles for eachtreatment are shown in Fig. 2.7 and Fig. 2.25 to 2.27 in Appendix 3. Eugenoland J3-ionone were not captured well by the head-space methodology used inthis study so the concentration of these compounds was insufficient foridentification by GC-MS.Volatiles are principally derived from two different classes of precursors,the straight-chain compounds from unsaturated fatty acids and the branchedchain compounds from free amino acids (PetrO-Turza, 1987). Alanine, leucineand valine are considered to be the most important free amino acids in tomatofor formation of volatile compounds such as 3-methylbutanal and 3-methylbutanol (Yu and Spencer, 1970). Linoleic and linolenic acids are oxidizedto hydroperoxides which yield mainly hexanal and Z-3-hexenal (Whiffield and98Table 2.6. Flavour constituents of tomato.PeakNumbera Component Ip (DBI)b IDI 2-methylfuran 601 MS CS2 2-methyl-I -propanol 613 MS CS3 3-methylbutanal 633 MS CS4 2-methylbutanal 641 MS CS5 I -penten-3-one 660 MS CS6 1 -penten-3-ol 664 MS CS7 unidentified 6988 E-2-methyl-2-butenal 715 MS CS9 Dimethyl disulfide and 3-methyl-I -butanol 721 MS CS10 2-methyl-I-butanol and E-2-pentenal 724 MS CSII 3-methylpentanal 743 MS RT12 Z-3-hexenal 769 MS RT13 hexanal 780 MSCS14 2-methylpropanoic acid2 786 MS15 octane 800 MSCS16 E-2-hexenal 818 MSCS17 3-methyl-I -pentanol1 822 MS RT18 Z-3-hexen-1 -ol 840 MS CS19 ethylbenzene 845 MS CS20 p- or m-xylene 851 MS CS21 2,4-hexadienal 871 MSCS22 5-ethyl-2(5H)-furanone 903 MS23 E-2-heptenal 920 MS CS24 dimethyl trisulfide1 940 MS25 6-methyl-5-hepten-2-one 960 MS CS26 2-octanone 965 MS CS27 2-pentylfuran 978 MS CS28 2-isobutylthiazole and unknown 1005 MS CS29 linalool 1073 MS CS30 undecane 1100 MSCS31 Dimethyl tetrasulfide 1200 MSMS, mass spectra of unknown tentatively matched with spectra in the massspectral library of the National Bureau of Standards (Probability BasedMatching); RT, retention time of unknown peak matched with retention time ofpeak chromatographed under similar conditions in the published literature; CS,mass spectra of unknown matched with spectra and retention times of authenticcompounds.a Peak numbers match peaks in Fig. 2.799Table 26 continuedbKovatls linear retention indices with temperature programming (Lee et al.,1984).McGlasson et al., 19872Petró-Turza, 1987 3Maarse and Visscher, 1989100TICofIJRTA:TOMAR1A.IJ5.OEB-3S164.OES-13U) o3.0E6C28o-o9-C :2.0E6-2510 /710E6 0_____________15191221413010203040Time(mm.)Fig.2.7.TotalionchromatogramofvolatilesextractedfromtomatoesstoredunderconstantpressureatI5Cfor3days.PeaknumberscorrespondtopeaknamesinTable2.6.Last, 1991). In an acidic environment, Z-3-hexenal is quickly converted to E-2-hexenal (PetrO-Turza, 1987). E-2-hexenal is considered to be an importantaroma component and is associated with ‘green tones’ of tomato flavour.However, too high a concentration of this component is associated with anunpleasant, rancid aroma (PetrO-Turza, 1987).The abundances of dimethyl disulfide + 3-methyl-I -butanol, E-2-hexenaland Z-3-hexen-I -01, and were greater in the tomatoes stored 7 days comparedto tomatoes stored 3 days regardless of the atmospheric pressure treatmentalthough these compounds tended to be significantly (P<O.05) moreconcentrated in extracts from tomatoes subjected to varying pressure treatmentfor 7 days (Table 2.7). Total relative amounts of volatiles were not significantly(P>O.05) affected by treatment although there was a tendency for the tomatoesstored for 7 days to have greater amounts of volatiles.Principal Component Analysis of the chromatographic data revealed that11 components are required to account for the total variability with the first threecomponents explaining 61.2% of the variance (Table 2.8). Plots of principalcomponents I and 2 (Fig. 2.8) and components I and 3 (Fig. 2.9) were closelyoverlapped for tomatoes stored 3 days regardless of atmospheric pressuretreatment. Nevertheless, variable pressure treatment appeared to have someeffect on tomato volatile profiles after 7 days of storage although variability inconcentration of aroma compounds associated with the second and thirdcomponents indicated by the spread of points on the Y axis may have obscuredthe differences. The data seem to indicate that a delay in ripening caused bycold storage affected the subsequent development of volatile compounds. Theconstruction of a table of coefficients defining each of the principal components(Table 2.9) is helpful in determining how variable atmospheric pressures mayhave affected the development of flavour compounds, based on chemical102Table2.7.Means1ofvolatilecompoundsidentifiedintomato.Relativeamountsofcompounds3PeakNumberComponentCON(3)2CON(7)VAR(3)VAR(7)I2-methylfuran0.540.7Ô0.479122-methyl-i-propanol0.28b0.42ab0.39ab0.58833-methylbutanal4.045.495.176.9042-methylbutanal0.180.180.200.2951-penten-3-one0.140.110.060.0461-penten-3-ol0.180.140.130.087unidentified0.97bI35b1.32bIgga8E-2-methyl-2-butenal0.560.600.830.929Dimethyldisulfideand3-methyl-i -butanol3•7306.384.96bC11.218102-methyl-I-butanolandE-2-pentenal2.102.242.733.02113-methylpentanal0.460.290.840.3912Z-3-hexenal0.280.210.250.2213hexanal4.123.163.443.00142-methylpropanoicacid0.450.510.811.2015octane0.040.060.070.0516E-2-hexenal8.66b12.4688.47b13.85a173-methyl-1-pentanol0.62a0.16b0.61a0.278b18Z-3-hexen-1-ol0.16b0.46a0.05b0.36a19ethylbenzene0.470.840.440.5820p-orm-xylene0.340.420.390.30212,4-hexadienal0.370.490.300.51225-ethyl-2(5H)-furanone0.460.310.480.4223E-2-heptenal0.180.190.250.20Table2.7continued- 0 .Imeansofthreeobservations.Relativeamountsofcompounds32CON-constantpressure,VAR-variablepressure,numberofdaysoftreatmentinparenthesis.3arerelativeto3-pentanone(internalstandard)peak.abcMeansinthesamerowwithdifferentlettersaresignificantly(P<0.05)different.PeakNumberComponentCON(3)2CON(7)VAR(3)VAR(7)24dimethyltrisulfide0.230.220.180.23256-methyl-5-hepten-2-one1.52ab1.64ab1.16b2.21a262-octanone0.150.120.660.07272-pentylfuran0.120.080.100.09282-isobutylthiazoleandunknown6.335.476.113.0529linalool0.53b0.88ab0.57b1.4130undecane0.070.120.060.1131Dimethyltetrasulfide0.250.280.340.39Totalamountsrelativetointernalstandard38.5045.9741.9254.79Table 2.8. Principal component analysis (PCA) of volatile compoundsfrom tomato stored under constant and variable pressure.Eigenvector Variance preservednumberEigenvalue Each TotalPRINI 7.193 23.2 23.2PRIN2 6.451 20.8 44.0PRIN3 5.329 17.2 61.2PRIN4 4.294 13.9 75.1PRIN5 2.705 8.7 83.8PRIN6 1.984 6.4 90.2PRIN7 1.231 3.9 94.1PRIN8 0.836 2.7 96.8PRIN9 0.701 2.3 99.1PRINIO 0.151 0.5 99.6PRINII 0.125 0.4 100.0105Co0CNC.J•1-’C0E0C)Cu00C0• constant pressure (3 days)I constant pressure (7 days)o variable pressure (3 days)D variable pressure (7 days)Fig. 2.8. Principal component analysis of aroma compounds from tomatoesstored under constant atmospheric pressure and variable pressuretreatments (n=3). A two-dimensional plot of the first and secondeigenvectors./II//I/I4-3-2-1—0-—1 —-2 --3 --4--5 -//I/I1/1/1/1/.I I I I-6 -4 -2 2 40Principal component 1 (23.2%)61066C1)00E00CuC.)C0• constant pressure (3 days)I constant pressure (7 days)o variable pressure (3 days)D variable pressure (7 days)Fig. 2.9. Principal component analysis of aroma compounds from tomatoesstored under constant atmospheric pressure and variable pressuretreatments (n=3). A two-dimensional plot of the first and thirdeigenvectors.1075-4-3-2-1—0-—1 —-2--.3 -///N //-6 -4 -2 0 2 4Principal component 1 (23.2%)6Table 2.9. Coefficients of the first six principal components of the tomatovolatiles data set.a Eigenvaluesb Cumulative proportion explainedPrincipal ComponentPeakNumber 1 2 3 4 5 6PKI 0.252032 0.157853 -.098856 -.009496 -.262972 0.094547PK2 0.349089 0.026923 -.008700 0.066610 0.065681 -.187940PK3 -.037968 0.124601 0.376815 -.157032 -.060335 0.035525PK4 -.157412 0.237867 0.180744 -.155755 0.199438 0.017477PK5 -.088230 -.263780 0.166253 0.103648 -.044905 0.053191PK6 0.068281 0.023008 0.015759 0.408863 0.247513 0.154121PK7 0.251459 0.156650 0.157450 -.134568 -.142740 -.168451PK8 0.059803 0.286327 0.199294 -.103065 0.149525 -.157223PK9 0.328465 0.050693 0.148247 -.086978 -.035705 -.124941PKIO 0.097800 0.327133 0.127607 -.065856 0.073625 -.086879PKII -.062941 0.287549 -.164758 0.116210 0.241082 -.137224PK12 -.083164 0.327261 -.068987 0.135887 -.091068 0.223969PKI3 -.051688 0.255068 0.142042 0.244018 0.063609 0.250140PK14 0.218469 0.161268 -.260067 -.034317 -.031964 -.223456PKI5 0.057688 -.051890 0.039888 0.029413 0.492967 -.088616PKI6 0.246465 -.197375 0.163415 0.169055 0.054763 -.043240PKI7 -.287818 0.038309 0.180820 0.061190 0.008431 -.176823PKI8 0.189719 0.026330 -.028933 -.170745 -.298354 0.326941PK19 0.123062 -.243536 0.143572 0.133327 -.080313 0.170681PK2O 0.028776 -.133787 0.155281 0.181516 -.058526 0.063506PK2I 0.236399 0.165580 -.196637 0.128836 -.082803 0.242290PK22 -.075907 0.321134 0.002560 0.184894 -.158270 -.062825PK23 0.082163 0.020385 0.166854 0.386052 -.183155 -.177331PK24 0.037510 -.074906 0.278474 -.176020 0.178984 0.300664PK25 0.252065 0.154381 -.063406 -.041746 0.240462 0.263189PK26 -.074397 0.029598 0.129079 0.275780 -.31 8249 -.343365PK27 0.111087 -.059665 -.036927 0.369364 0.277530 -.025560PK28 -.209085 0.169795 0.062553 0.250435 -.121010 0.244396PK29 0.330287 -.085813 0.119169 0.049650 0.045549 -.114948PK30 0.159990 0.017667 0.347329 0.055881 -.058634 0.193321PK31 -.043743 0.113218 0.392060 -.094411 0.013422 -.078511Var CCPEbO.5IjVarC37.193 6.451 5.329 4.294 2.705 1.9840.232 0.440 0.612 0.751 0.838 0.9020.186 0.197 0.217 0.241 0.304 0.355108Table 2.9 continuedFor each principal component, the variables with a correlation greater than 0.5with that component, are underned.109analysis. In tomato, variables highly correlated (more than 0.5 absolutecorrelation) with the first principal component were 2-methylfuran, 2-methy-I -propanol, an unidentified peak, dimethyl disulfide + 3-methyl-I -butanol, 2-methylpropanoic acid, E-2-hexenal, Z-3-hexen-I -ol, 2, 4-hexad ienal, 6-methyl-5-hepten-2-one and linalool. Of these compounds, E-2-hexenal, Z-3-hexen-I -oland 6-methyl-5-hepten-2-one are important in determining tomato flavour andthe concentrations of two of these compounds were found to be significantlyhigher in tomatoes stored for seven days at low temperatures (Table 2.7).Coefficients for the second component indicate that 2-methylbutanal, Ipenten-3-one, Z-3-hexenal, hexanal, ethylbenzene and 5-ethyl-2(5H)-furanonewere the variables which correlate well th this component (Table 2.9) and twoof these compounds, namely 2-methylbutanal and hexanal, have been identifiedin the literature as important flavour constituents of tomatoes. Although loadingsfor this component appeared to be quite variable among tomatoes used in thisstudy, there is some indication that the second component captured enough ofthe variance structure of the data matrix to group the treatments into 3 maingroups (Fig. 2.8). An explanation of a negative correlation between I -penten-2-one and ethylbenzene and the second component may be found in the datacontained in Table 2.7. Concentrations of these chemical compounds werehigher for tomatoes stored for 7 days under constant pressure compared totomatoes stored 7 days under variable pressure and may in part, have led to theseparation between the two treatments in the plot. Variables having more than0.5 absolute correlation with the third component were 3-methylbutanal, 2-methyipropanoic acid, dimethyl trisulfide, undecane and dimethyl tetrasulfide(Table 2.9). Of these compounds 3-methylbutanal and dimethyl trisulfide havebeen previously identified as important odor compounds of tomato. It appearsfrom the plot of the first and third principal components (Fig. 2.9) that these110variables not only confirm the class difference between number of days instorage but also determine the commonality of the treatment for 7 days atconstant pressure and the treatment for 7 days at variable pressure since theplots for these treatments overlap.Again it is worthwhile plotting later values against the first component toidentify variables responsible for differentiation between treatments. Referringto the plot of the first and fourth eigenvectors in Fig. 2.10 and the table ofcoefficients in Table 2.9, it appears that 1-penten-3-ol, E-2-heptenal, 2-octanone, 2-pentylfuran and 2-isobutylthiazole + unknown were importantvariables in the separation of treatment classes, namely, variable pressuretreatment for 7 days. Plots of the first and fifth eigenvectors (Fig. 2.11) and firstand sixth eigenvectors (Fig. 2.12) also show that distinct differences in volatileproduction occur in tomatoes stored for 3 days compared to tomatoes stored for7 days. There is some indication in Fig. 2.12 that differences in volatilecomposition exist between tomatoes stored for 3 days at constant and variablepressures. However, although the sixth principal component accounted for 6.4%of the variablility, none of the coefficients listed in Table 2.9 had a significantassociation with that component.Informal tasting of the various treatments found that all tomatoespossessed a good flavour, although it was noted upon removal from 3 or 7 daysstorage, that tomatoes subjected to atmospheric pressure variations were moreevenly colored than tomatoes stored at constant pressure. The deleteriouseffect of refrigerated storage on certain tomato volatiles is well supported in theliterature although this is thought to occur only at chilling temperatures, i.e.below 12°C (Whitfield and Last, 1991).11140)C’.)40CG)C0E000C)C0• constant pressure (3 days)I constant pressure (7 days)o variable pressure (3 days)D variable pressure (7 days)Fig. 2.10. Principal component analysis of aroma compounds from tomatoesstored under constant atmospheric pressure and variable pressuretreatments (n=3). A two-dimensional plot of the first and fourtheigenvectors.3—2-1—0——1 —-2--3 -—IVVVV /VVI\\\\\\\\\-6I I I-4 -2 0 42Principal component 1 (23.2%)61124CIf)•1-G)0E0C.)CuC)0• constant pressure (3 days)• constant pressure (7 days)o variable pressure (3 days)D variable pressure (7 days)Fig. 2.11. Principal component analysis of aroma compounds from tomatoesstored under constant atmospheric pressure and variable pressuretreatments (n=3). A two-dimensional plot of the first and fiftheigenvectors.113INNNNNNNN3-2-1—0-—1 —-2--3 --4-//-6 -4 -2 2 40Principal component 1 (23.2%)63CDCDCCL)C00.E0C)CuC.)CI0• constant pressure (3 days)• constant pressure (7 days)o variable pressure (3 days)D variable pressure (7 days)Fig. 2.12. Principal component analysis of aroma compounds from tomatoesstored under constant atmospheric pressure and variable pressuretreatments (n=3). A two-dimensional plot of the first and sixtheigenvectors.1142-1—0-—1-2--3 --6I I I-4 -2 0 2 4Principal component 1 (23.2%)6E. Effect of Atmospheric Pressure Treatments on Sweet Pepper VolatilesAccording to Whiffield and Last (1991) a total of 23 compounds havebeen identified in the volatile oil of uncooked peppers with the most importantcompounds being; (E)-f3-ocimene, methyl salicylate, limonene and 2-(2-methylpropyl)-3-methoxypyrazine. Other pyrazi nes such as 2-isopropyl-3-methoxypyrazine and 2--butyI-3-methoxypyrazine have been captured inappreciable quantities by a headspace concentration technique (Murray andWhitfield, 1975).Total ion chromatograms of sweet peppers stored for 3, 7 and 14 days atconstant atmospheric pressure or varying atmospheric pressure are shown inFig. 2.13 and Fig. 2.28 to 2.32 in Appendix 3. The compounds identified in thechromatograms are listed in Table 2.10. Much of the published literature onsweet pepper volatiles appeared before 1975, and contains little in the way ofcapillary ‘aromagrams’ [other than that of Buttery et al. (1969)] and no retentionindices. Consequently, it was not possible to confirm the identity of some of thelater eluting compounds in the absence of authentic standards, and as a resultmany of the unknown peaks were identified by matching spectra with thosecontained in the computer mass spectral library. Means as shown in Table 2.11,indicate that variable pressure treatment may have had an effect on C6compounds. Means for relative amounts of hexanal, E-2-hexenal and Z-3-hexen-1 -01 were higher for peppers stored under variable pressure treatmentscompared to peppers stored at constant pressure, but this increase was notsignificant (P>0.05) for all storage periods. Although 2-isobutyl-3-methoxy-pyrazine and total volatiles were more abundant in the variable pressure-treatedpeppers compared to peppers stored under constant pressure, this differencewas not significant (P>0.05).115TICc-FIJRTA:PEPPROBA.D8.OES-s?.ØES-6S.E6-5.0E6C4.ØE6C3.0E62ØE67215161__10.1?11::22.25:.•Time(mm.)Fig.2.13.Totalionchromatogramofvolatilesextractedfromsweetpepperstoredunderconstantpressureat15Cfor3days.PeaknumberscorrespondtopeaknamesinTable2.10.Table 2.10. Flavour constituents of sweet pepper.PeakNumbera Component I (DB-1 )b IDI I -penten-3-one 660 MS CS2 unidentified 6713 3-pentanone 673 MS CS4 E-2-pentenal 723 MS CS5 hexanal12 780 MS CS6 E-2-hexenal3 820 MS CS7 Z-3-hexen-1-o1 841 MSCS8 1-hexanol 851 MS CS9 1,3-dimethyIbenzene2 853 MS CS10 2-heptanone3 861 MS CS11 2,4-hexadienal 871 MS CS12 E-2-heptenaI13 920 MS CS13 dimethyl trisulfide 940 MS14 5-ethyl-2-(5H)-furanone 980 MS15 cyclobutanone oxime 1016 MS16 D-Iimonene123 1017 MS CS17 E-3,7-dimethyl-1,3,6-octatriene(Ocimene) 1021 MS18 E-3,7-dimethyl-1,3,6-octatriene2(3-ocimene) 1029 MS19 3,7,7-trimethyl-bicyclo[4. I .0]hept-2-ene 1074 MS20 3,7-dimethyl-1 ,6-octadien-3-oI 1076 MS21 2-isobutyl-3-methoxypyrazine12 1164 MS22 methylsalicylate 1168 MS CS23 dimethyl tetrasulfide 1200 MS24 2-methyltridecane 1290 MS25 unidentified 1400 MS26 copaene1 1405 MS27 unidentified 1430MS, mass spectra of unknown tentatively matched with spectra in the massspectral library of the National Bureau of Standards (Probability BasedMatching); CS, mass spectra of unknown matched with spectra and retentiontimes of authentic compounds.a Peak numbers match peaks in Fig. 2.13b Kovat’s linear retention indices with temperature programming (Lee et al.,1984).1 Maarse and Visscher (1989) 2 Buttery et al. (1969) Whitfield and Last(1991)117Table2.11.Means1ofvolatilecompoundsidentifiedinsweetpepperRelativeamountsofcompounds3PeakNumberComponentCON(3)2CON(7)CON(14)VAR(3)VAR(7)VAR(14)Il-penten-3-one0.310.3lab0.09b0.31O.400.29ab2unidentified0.100.120.210.200.150.1833-pentanone0.loab0.llab0.Olb0.140.160.194E-2-pentenal0.l9ab0.22ab0.08bO.28aQ•37a0.315hexanal2.883.673.384.904.065.276E-2-hexenal17.1lb18.45ab25.7Oab22.39ab24.5lab26.77a7Z-3-hexen-l-olI.2Oabc1.21abc0.78cI.38abcj.85I.62ab81-hexanol0.200.250.200.210.280.2591,3-dimethylbenzene0.25ab0.1Db0.39a0.25ab0.l6ab0.188b102-heptanone0.23c0.22c0.34ab0.25bc0.32abc0.372112,4-hexadienal0.3Db0.20b0.08b1.19aQ.38ab0.32b12Z-2-heptenal0.180.160.140.230.190.1913dimethyltrisulfide0.180.150.250.180.220.17145-ethyl-2-(5H)-furanone0.l4abo.l2ab0.08b0.180.l80.1715cyclobutanoneoxime3.272.832.183.843.563.5816D-limonene0.130.O8ab0.lOab004bQQ9abQOSab17E-3,7-dimethyl-1,3,6-octatriene0.390.260.340.270.280.2518E-3,7-dimethyl-1,3,6-octatriene1.881.510.761.481.541.62193,7,7-trimethyl-bicyclo[4.1.0]hept-2-ene0.120.160.150.810.300.27203,7-dimethyl-1,6-octadien-3-ol0.37b0.36b0.6lab0.78a0.442bQ45ab212-isobutyl-3-methoxypyrazine1.001.041.031.081.251.2722methylsalicylate0.430.220.190.340.300.1923dimethyltetrasulfide0.19b0.17b0.4020.2Db0.23ab0.20bTable2.11continuedRelativeamountsofcompounds3PeakNumberComponentCON(3)2CON(7)CON(14)VAR(3)VAR(7)VAR(14)242-methyltridecane0.170.120.140.170.180.1625unidentified0.280.190.160.280.250.1726copaene0.150.030.060.240.070.0727unidentified1.81bci74bc0.5703.32a2.87ab3.22abTotalamountsrelativetointernalstandard33.5533.9838.4344.9544.5247.84_1meansofthreeobservations.2-constantpressure,VAR-variablepressure,numberofdaysoftreatmentinparenthesis.3Amountsarerelativeto2-octanone(internalstandard)peak.abcMeansinthesamerowwithdifferentlettersaresignificantly(P<0.05)different.Principal component analysis indicated that 84.7% of the samplevariability could be accounted for by the first six principal components (Table2.12). To ascertain if a recognizable treatment pattern could be discerned, thelatter 5 components were plotted against the first component. The first 2 plots(Fig. 2.14 and Fig. 2.15) illustrate the close relationship of all the variablepressure treatments. Calculation of the coefficients of the first six principalcomponents indicates that 16 of the 27 compounds were important variables inthe first component (Table 2.13). An unidentified peak, hexanal, E-2-hexenal,I ,3-dimethylbenzene, dimethyl trisulfide and dimethyl tetrasulfide were thevariables associated with the second component. Smaller differences betweenconcentrations of these compounds among the variable pressure treatmentswould account for close overlapping of these treatment classes in Fig. 2.14.Clear separation of the treatment class representing constant atmosphericpressure treatment for 14 days from other pressure treatment classes may beevidence that over time, metabolic events taking place in peppers stored atconstant pressure were different than peppers stored under variableatmospheric pressure (Fig. 2.15). Plotting later eigenvalues against the firstcomponent did not assist in further treatment class separations (Fig. 2.16 to Fig.2.18).The gas chromatographic data indicate that extensive oxidation of cellularconstituents of sweet peppers did not occur as a result of variable atmosphericpressure storage. If oxidation occurred, significant lipid changes would havebeen detected by the observation of much higher concentrations of E-2-hexenaland Z-3-hexen-1 -ol in peppers stored under variable pressures compared tothose stored under constant pressure.Data from preliminary experiments to determine if pepper volatiles wereaffected by 02 concentration during storage under variable atmospheric120Table 2.12. Principal component analysis (PCA) of volatile compoundsfrom sweet pepper stored under constant and variablepressures.Eigenvector Variance preservednumberEigenvalue Each Total1 8.889 32.9 32.92 4.388 16.3 49.23 3.550 13.1 62.34 2.750 10.2 72.55 1.909 7.1 79.66 1.389 5.1 84.77 1.189 4.4 89.18 1.130 4.2 93.39 0.626 2.3 95.610 0.418 1.6 97.211 0.279 1.0 98.212 0.173 0.6 98.913 0.111 0.4 99.314 0.081 0.3 99.615 0.046 0.2 99.816 0.043 0.2 99.917 0.021 0.1 100.0121CoCNCciC00E0C-)Cu0C-)C0• constant pressure (3 days)constant pressure (7 days)A constant pressure (14 days)o variable pressure (3 days)ci variable pressure (7 days)variable pressure (14 days)Fig. 2.14. Principal component analysis of aroma compounds from peppersstored under constant atmospheric pressure and variable pressuretreatments (n=3). A two-dimensional plot of the first and secondeigenvectors.\\\\\\\\\\\8-6-4-2-0--2 --4--6 -\\-6I I-4 -2 0 2Principal component 1 (32.9%)4 61226Cl)C1)ci0E00C’,00Ca-• constant pressure (3 days)• constant pressure (7 days)A constant pressure (14 days)o variable pressure (3 days)D variable pressure (7 days)variable pressure (14 days)Fig. 2.15. Principal component analysis of aroma compounds from peppersstored under constant atmospheric pressure and variable pressuretreatments (n=3). A two-dimensional plot of the first and thirdeigenvectors.4-2-0--2--4--6--------s•-4 -2 0 2 4Principal component 1 (32.9%)6123Table 2.13. Coefficients of the first six principal components of the peppera Eigenvaluesvolatiles data set.b Cumulative proportion explainedFor each principal component, the variables with a correlation greater than 0.5with that component, are underlined.Principal ComponentsPeakNumber 1 2 3 4 5 6PK1 0.286491 -.051463 -.030297 -.057584 0.062031 0.275943PK2 0.044831 0.391753 -.048385 0.121284 -.192524 -.149661PK3 0.295805 0.102107 0.070661 -.069663 -.092605 0.225534PK4 0.297260 0.049597 0.129180 -.120951 -.053125 -.066998PK5 0.218747 0.252239 -.015331 0.042901 -.286418 -.193381PK6 0.128078 0.353740 0.109818 -.123300 0.162832 -.237491PK7 0.231918 0.021079 0.226030 -.126472 0.115434 0.379242PK8 0.123794 0.213074 0.164193 -.249695 -.181762 0.250550PK9 -.182691 0.309925 -.130620 0.213528 -.013021 0.001360PKIO -.037210 0.237325 0.255213 -.168507 0.230813 -.316406PK11 0.050870 -.126713 0.337249 0.418142 -.007870 -.016764PK12 0.250579 0.031291 0.007334 0.008172 -.107308 0.019841PK13 -.107026 0.277162 -.099847 0.081339 0.406171 0.262334PKI4 0.273671 -.152741 0.134559 0.072374 0.216445 -.034324PKI5 0.220148 -.196614 -.050694 0.064611 0.233225 -.209830PKI6 -.210767 -.093251 0.040428 0.003087 0.186615 0.423850PKI7 0.168635 -.005485 -.356483 0.027254 0.234116 -.095602PK18 0.235876 0.041436 -.251467 -.003370 0.044716 0.036915PK19 0.029491 -.113212 0.351315 0.396945 0.049591 -.066591PK2O -.025922 0.139842 0.255089 0.41 8095 0.168514 0.0581 72PK21 0.218292 0.225512 0.055490 -.047581 0.276006 0.138810PK22 0.198119 -.116106 -.351135 0.139805 0.122347 0.014449PK23 -.138218 0.374425 -.074671 0.084030 0.255930 0.014536PK24 0.227363 -.108670 -.192253 0.137787 0.226308 -.212262PK25 0.079881 0.080971 -.175292 0.334763 -.136691 0.167931PK26 0.011705 0.131575 -.256993 0.283360 -.267427 0.199368PK27 0.278338 0.071777 0.109348 0.169802 -.202438 0.015097Var c1a 8.889 4.388 3.550 2.750 1.909 1.389CPEb 0.329 0.492 0.623 0.725 0.796 0.847O.5/.jVarC1 0.168 0.239 0.265 0.302 0.362 0.4241245CN0-I-’ci)C0E00Cu0C)C0• constant pressure (3 days)• constant pressure (7 days)A constant pressure (14 days)o variable pressure (3 days)D variable pressure (7 days)variable pressure (14 days)Fig. 2.16. Principal component analysis of aroma compounds from peppersstored under constant atmospheric pressure and variable pressuretreatments (n=3). A two-dimensional plot of the first and fourtheigenvectors.4-3-2-1—0-—1-2--3-6 -4 -2 0 2 4Principal component 1 (32.9%)61254I3210—1-2-3Principal component 1 (32.9%)6• constant pressure (3 days)• constant pressure (7 days)A constant pressure (14 days)o variable pressure (3 days)EJ variable pressure (7 days)variable pressure (14 days)Fig. 2.17. Principal component analysis of aroma compounds from peppersstored under constant atmospheric pressure and variable pressuretreatments (n=3). A two-dimensional plot of the first and fiftheigenvectors.126-6 -4 -2 0 2 442-• constant pressure (3 days)• constant pressure (7 days)A constant pressure (14 days)o variable pressure (3 days)EJ variable pressure (7 days)variable pressure (14 days)Fig. 2.18. Principal component analysis of aroma compounds from peppersstored under constant atmospheric pressure and variable pressuretreatments (n=3). A two-dimensional plot of the first and sixtheigenvectors.3-////1—0-—1 —-2A:,.—-6 -4 -2 0 2 4Principal component I (32.9%)6127pressure, indicated that oxygen concentration may affect the production of E-2-hexenal and Z-3-hexen-1-ol (Table 2.14). Presumably, reducing theconcentration of 02 in the atmosphere surrounding the peppers resulted in lowerrates of lipid oxidation hence a lower production of E-2-hexenal. Furtherinvestigations are required to confirm this observation since the trial lackedadequate control samples, which were not included due to time constraints.F. Total Water LossWater loss from the plant material in the treatment vessel of the gasexchange circuit was a concern because of the high flow rate of the circulatinggases (1.5 Umin) and the presence of the fan in the treatment vessel. Studieswith leaves (Nobel, 1974) showed that, in still air, relative humidity near theevaporating surface was higher than that of the ambient air and thus there islittle water vapour movement from the leaf to the air. Air currents greater than84 cm s1 disturb this boundary layer and decrease its relative humidity so thattranspiration increases (Shive and Brown, 1978). Shive and Brown (1978)suggested that mass flows of gases (including water vapour) may be driven byexternal air movement. There is little information in the literature about theimportance of air flow rate in transpiration rate of fruit and vegetables. Onestudy revealed that, at a constant air flow rate and relative humidity, air currentsaccounted for less than a 5% increase in transpiration rate in apples (Pantastico,1975). Other research indicated that stored apples showed a 30-100%acceleration of transpiration rates when air currents of increasing velocities wereused (Sastry et al. 1978).The benefit of high humidity storage (98-100% RH) of vegetables hasbeen recognized for nearly 20 years (van den Berg and Lentz, 1977). Acomprehensive mathematical analysis was performed by van den Berg and128Table 2.14. Means1 of volatile compounds identified in sweet pepper subjectedto variable pressures at different oxygen concentrations.Relative amounts ofcompounds3PeakNumber Component VAR(3%)2 VAR(21 %)I 1-penten-3-one 0.05 0.112 unidentified 0.12 0.073 3-pentanone 0.08 0.084 E-2-pentenal 0.06 0.155 hexanal 3.92 4.626 E-2-hexenal 1O.17b 24.42a7 Z-3-hexen-1 -ol 0.882 Q73b8 1-hexanol 0.41 0.219 1,3-dimethylbenzene 0.23 0.1810 2-heptanone 0.21 0.1211 2,4-hexadienal 0.10 0.0912 Cis-2-heptenal 0.14 0.1413 dimethyl-trisulfide 0.28 0.1514 5-ethyl-2-(5H)-furanone 0.04 0.1115 cyclobutanone oxime 1.59 2.3616 D-limonene 0.04 0.0817 E-3,7-dimethyl-1 ,3,6-octatriene 0.63 0.5018 E-3,7-dimethyl-1 ,3,6-octatriene 3.29 2.6519 3,7,7-trimethyl-bicyclo[4. I .0]hept-2-ene 0.13 0.0820 3,7-dimethyl-1 ,6-octadien-3-ol 0.30 0.1521 2-isobutyl-3-methoxypyrazine 0.92 0.7822 methylsalicylate 0.23 0.2923 dimethyl tetrasulfide 0.28 0.1924 2-methyltridecane 0.01 trace25 3-hydroxy-2,4,4-trimethylpentyl ester 0.03 0.0526 copaene 0.03 0.0127 unidentified 1.24 2.00I means of two observations.2 VAR = variable pressure treatment only, oxygen concentration (%) inparenthesis.3 are relative to 2-octanone (internal standard) peak.a,b Means within rows with different letters are significantly (P<0.05) different.129Lentz (1978) in which they noted that an increase in air velocity lowered the rateof moisture loss in stored produce. However, this effect was only apparent atdistances of at least I m between the air intake and the produce.In the present study, the distance between the air intake and the producewas only 2-7 cm, allowing gases to flow over the produce. The results of totalweight loss measurements in Figure 2.19. suggest that variable pressuretreatments did not accelerate water loss in tomatoes and peppers compared tothose fruits stored at constant pressure. Moisture loss in onions was unaffectedby treatment probably due to the low transpiration rate and protective dry scales.In conclusion, data collected from GC/MS analyses indicate that exposingvegetables to variable atmospheric pressure changes may subtly alter the profileof aroma compounds. Application of multicomponent analysis assisted indiscriminating among constant and variable pressure treatments and identifiedthe compounds which were important in making that distinction. Variablepressure treatments did not promote water loss in onions, tomatoes or sweetpeppers.1302-0C,)C,)0-JI—03 1.0CI)Cl) Cl)0 0•_JF- I-Ii I0.0Fig. 2.19. Comparison of water loss in pressure treated and control vegetables.Treatments were: variable pressure treatment at 6.9 kPaand the control treatment at constant pressureTOMATO1—0-I I I I I I I I012345678STORAGE PERIOD (DAYS)SWEET PEPPER ONION00 3 6 9 12 15STORAGE PERIOD (DAYS)0 4 8 12 16 20STORAGE PERIOD (DAYS)131GENERAL SUMMARY AND CONCLUSIONSAlthough gas exchange in bulky tissues has been studied for many years,most of the research effort has concentrated on fruits, rather than vegetables,and on diffusive gas flow, rather than bulk gas flow. Bulk gas flow has beenreported to occur in aquatic plants (Dacey, 1981, 1987; Grosse et al, 1991;Armstrong and Armstrong, 1991) and in leaves (Day and Parkinson, 1979).There is also some indication that temperature-driven (Corey and Tan, 1990)and pressure-driven (Jolliffe and Dyck, 1988) bulk gas flow can be induced infruit tissues. The main objective of this study was to determine whether flow ofCO2 could be enhanced by varying the atmospheric pressure surrounding bulkytissues. Since temperature and 02 concentration affect respiration rate, asecondary objective was to determine the effect of other environmental factorson the expression of the response to atmospheric pressure. Longer termexposures to varying atmospheric pressures (3 days to 3 weeks) were used inan attempt to determine the effect of pressure-driven gas flow on water loss andchanges in volatile aroma compounds. The vegetables used in this study weredeliberately chosen because of their widely diverse morphological types andinternal matrices. The major findings and accomplishments of the current studywere:1. The acquisition of new information regarding mass gas flow in bulky tissues inan environment of fluctuating external gas pressures. Small changes inatmospheric gas pressure induced large increases in CO2 emissions in thoseplant organs possessing relatively low resistance to gas exchange (onions andsweet peppers). Following pressure treatments, net CO2 efflux rates returned tothe same rate, or slightly lower than pretreatment values, thus there was noevidence of enhanced metabolic rate due to treatment. Increases in the level of132pressure variations resulted in a corresponding increase in CO2 efflux rate whichis in agreement with Darcy’s law for fluid flow through a porous media (Nobel,1983). Gas pressure variations had the greatest effect on net CO2 efflux ratewhen sufficient time was allowed between treatments for maximum gas flowalong the gas transport pathways. It was evident that due to their anatomicalstructure, sweet peppers could exchange gases at a very rapid rate and wereunaffected by time lag between the series of gas pressure pulses (Fig 1.6).In an oxygen limiting environment the magnitude of the response tovariable gas pressure was diminished for onions, but net CO2 efflux rates ofpressure-treated organs still exceeded that of onions held at constant pressure.The lack of response to variable pressure treatments by cucumbers andtomatoes is consistent with available information on the barriers to diffusion inthese organs.Fluctuating gas pressure storage may have an application as apretreatment or intermittent treatment for bulky organs in which diffusive gas flowdoes not satisfy the potential of those tissues to emit respiration gases. Smallexternal pressure variations may potentially be used to establish optimuminternal gas concentrations in produce placed in CA storage or to removeharmful buildup of CO2 during rewarming of plant tissue.2. The development of a purge and trap sampling method for the collection andconcentration of volatile vegetable aroma compounds. This method wasinexpensive, rapid and easy to perform. Activated charcoal captures a broadrange of organic compounds which, when eluted with a polar solvent such asdichloromethane, yield definitive chromatograms with little peak tailing. At thetime of developing the method, use of a charcoal trap technique had not beenreported in studies of vegetable aroma compounds. The main advantage of the133new technique, compared to steam distillation and solvent extraction, is thecollection of trace volatiles with a minimum of degradation and interference fromother compounds.3. The novel use of multicomponent analysis to determine differences in aromaprofiles of onions, sweet pepper and tomatoes subjected to variable pressuretreatments. Using a trained panel to discriminate between experimentaltreatments may not always be practical or yield results that are easilyinterpreted. Compared to other statistical methods, multicomponent analysis ofchromatographic results provided an improved technique for segregatingvegetable responses to storage treatments.4. The design and construction of a device to vary the atmospheric pressuresurrounding the test material while maintaining a constant temperature.Pressure was cycled inside a vessel for a predetermined length of time beforereturning to constant conditions. The period of the pressure cycle could bevaried as desired. 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Jadhav and D. K. Salunkhe (1972) Effects of sub-atmosphericpressure storage on ripening of tomato fruits. J. Food Sci. 37:952-956.Wyllie, S. G. and D. N. Leach (1990) Aroma volatiles of Cucumis melo cv.Golden Crispy. J. Agric. Food Chem. 38:2042-2044.Yang, S. F. (1985) Biosynthesis and action of ethylene. HortScience 20:41 -45.147Yen, G., H. Lin and P. Yang (1992) Changes in volatile flavor components ofguava puree during processing and frozen storage. J. Food Sci. 57:679-685.Yu, M. H. and M. Spencer (1970) cc-Alanine aminotransferase from tomato fruit.Phytochem. 9:341-343.Zervos, C. and R. H. Albert. (1992) Chemometrics: the use of multivariatemethods for the determination and characterization of off-flavors. In: Off-Flavorsin Foods and Beverages. G. Charalambous (Ed.). Elsevier Science Pub., B. V.pp. 669-742.148Appendix 1. Contrast statements used in analysis of variance.contrast ‘temp(Iiny temperature -1 0 1contrast ‘temp (quad)’ temperature 1 -2 1contrast IP*T(Iin) temperature*pressure -1 0 1 1 0 -1contrast P*T(quad)I temperature*pressure -1 -2 1 -1 2 -1contrast D*T(Iin) temperature*duration -1 0 11 0 -1contrast D*T(quad)I temperature*duration 1 -2 1 -1 2 -1contrast IP*D*T(Iin)I temperature*pressure*duration -1 0 11 0 -II 0 -1 -1 0 1contrast iP*D*T(quad) temperature*pressure*duration 1 -2 1 -1 2 -1 -1 2 -11 -2I149Appendix 2. Mean net carbon dioxide emission rates in onions, cucumbers,sweet peppers and tomatoes stored under variable and constant atmosphericpressure.Table 1.4. Effect of temperature, level of pressure variation and DCAP on netcarbon dioxide efflux rate (tL kg1 s1) of onion during constant andvariable pressure treatments.Variable Constant°C kPa1 DCAP2 Meana SD Mean SD0 3.5 5 0.43±0.10 0.33±0.090 3.5 15 1.69±0.85 0.22±0.010 6.9 5 0.68 ± 0.16 0.26 ± 0.090 6.9 15 3.56±0.75 0.25±0.0510 3.5 5 0.88±0.28 0.50±0.1810 3.5 15 3.18±0.69 0.36±0.0610 6.9 5 1.82±0.23 0.44±0.1510 6.9 15 5.49±1.10 0.29±0.0620 3.5 5 1.80± 1.19 0.94±0.5920 3.5 15 5.57±1.09 0.75±0.1220 6.9 5 3.74±0.73 1.45±0.3120 6.9 15 8.91 ± 1.61 0.78 ±0.06aI Increase in pressure above ambient atmospheric pressure.2 Duration of the interval of constant atmospheric pressure between 5 mmvariable pressure treatments.150Table 1.5. Effect of level of pressure variation, temperature and oxygenconcentration on net carbon dioxide efflux rate (iiL kg1 s1) of onionduring constant and variable pressure treatments.Variable Constant°C kPa1 Meana SD Mean SD0 3.5 1 0.76±0.29 0.35±0.110 3.5 21 1.70±0.85 0.22±0.010 6.9 1 0.78 ± 0.36 0.32 ± 0.040 6.9 21 3.56±0.75 0.25±0.0510 3.5 1 0.64±0.22 0.41 ± 0.1110 3.5 21 3.18±0.69 0.36±0.0610 6.9 1 0.83±0.14 0.37±0.1110 6.9 21 5.49±1.10 0.29±0.0620 3.5 1 0.80±0.22 0.54±0.1520 3.5 21 5.57 ± 1.09 0.75 ±0.1220 6.9 1 1.12 ± 0.23 0.52 ± 0.0720 6.9 21 8.91 ± 1.61 0.78±0.06a1 Increase in pressure above ambient atmospheric pressure.151Table 1.6. Effect of temperature, level of pressure variation and DCAP on netcarbon dioxide efflux rate (iL kg1 1) of cucumber during constantand variable pressure treatments.Variable Constant°C kPa1 DCAP2 Meana SD Mean SD10 3.5 5 3.67 ± 0.82 3.89 ± 1.2110 3.5 15 3.84±0.08 3.43±0.5110 6.9 5 3.65±1.82 3.86±1.9810 6.9 15 4.30±0.13 3.69±0.5820 3.5 5 5.75±2.18 7.00 ± 1.2420 3.5 15 8.58±0.10 8.13±0.2520 6.9 5 7.00±2.52 7.50±2.0520 6.9 15 8.76±0.69 8.48±0.97aI Increase in pressure above ambient atmospheric pressure.2 Duration of the interval of constant atmospheric pressure between 5 mmvariable pressure treatments.152Table 1.7. Effect of temperature, level of presssure variation and oxygenconcentration on net carbon dioxide efflux rate (giL kg1 s1) ofcucumber during constant and variable pressure treatments.Variable Constant°C kPa1 Meana SD Mean SD10 3.5 3 3.25±1.45 2.93±1.3710 3.5 21 3.84±0.08 3.43±0.5110 6.9 3 3.09±1.33 2.73±1.2310 6.9 21 4.30±0.13 3.69±0.5820 3.5 3 5.31 ±2.18 4.55 ±2.0320 3.5 21 8.58±0.10 8.13±0.2520 6.9 3 6.77±1.95 6.11±1.4420 6.9 21 8.76 ± 0.69 8.48 ± 0.97a n=3I Increase in pressure above ambient atmospheric pressure.153Table 1.8. Effect of temperature, level of pressure variation and DCAP on netcarbon dioxide efflux rate (jiL kg1 s1) of sweet pepper during constantand variable pressure treatments.Variable Constant°C kPa1 DCAP2 Meana SD Mean SD10 3.5 5 3.16 ± 1.54 1.59 ± 0.8910 3.5 15 2.69±0.73 1.05±0.2610 6.9 5 5.48 ± 1.35 1.91 ± 0.4610 6.9 15 4.97±0.94 0.97±0.3820 3.5 5 8.97±1.85 3.64±1.0120 3.5 15 10.36±2.15 4.63±1.0920 6.9 5 14.25±5.75 4.13±0.9920 6.9 15 13.04± 1.89 4.64 ±0.95a1 Increase in pressure above ambient atmospheric pressure.2 Duration of the interval of constant atmospheric pressure between 5 mmvariable pressure treatments.154Table 1.9. Effect of temperature, level of pressure variation and oxygenconcentration on net carbon dioxide efflux rate (jiL kg1 s1) of sweetpepper during constant and variable pressure treatments.Variable Constant°C kPa1 Meana SD Mean SD10 3.5 3 3.36±0.62 1.30±0.4510 3.5 21 2.69±0.74 1.05±0.2610 6.9 3 5.34±3.17 1.34±0.8510 6.9 21 4.97 ± 0.94 0.97 ± 0.3820 3.5 3 8.68±2.06 4.06±1.1620 3.5 21 10.36±2.15 4.63±1.0920 6.9 3 12.78± 1.75 3.91 ±0.9020 6.9 21 13.04±1.19 4.64±0.95aI Increase in pressure above ambient atmospheric pressure.155Table 1.10. Effect of temperature, level of pressure variation and DCAP on netcarbon dioxide efflux rate (tL kg1 s1) of tomato during constant andvariable pressure treatments.Variable Constant°C kPa1 DCAP2 Meana SD Mean SD10 3.5 5 1.43±0.54 1.66±0.1610 3.5 15 1.70±0.54 1.79±0.6610 6.9 5 1.58±0.02 1.49±0.1410 6.9 15 2.09±0.61 1.53±0.3520 3.5 5 3.82±0.39 2.59±0.1720 3.5 15 3.80±0.21 4.27±0.5420 6.9 5 3.40 ± 0.57 3.85 ± 0.6120 6.9 15 4.48±0.56 4.64±0.58a n=3I Increase in pressure above ambient atmospheric pressure.2 Duration of the interval of constant atmospheric pressure between 5 mmvariable pressure treatments.156Table 1.11. Effect of temperature, level of pressure variation and oxygenconcentration on net carbon dioxide efflux rate (jiL kg1 s1) of tomatoduring constant and variable pressure treatments.Variable Constant°C kPa1 02 (%) Meana SD Mean SD10 3.5 3 2.34±0.34 2.28±0.4310 3.5 21 1.70±0.54 1.79±0.6610 6.9 3 2.16±0.34 1.90±0.5210 6.9 21 2.09±0.61 1.53±0.3520 3.5 3 3.46 ± 1.23 3.35 ± 1.2320 3.5 21 3.80 ± 0.21 4.27 ± 0.5420 6.9 3 3.77± 1.19 2.89 ±0.4520 6.9 21 4.48 ± 0.56 4.64 ± 0.58a1 Increase in pressure above ambient atmospheric pressure.i7Appendix3.Totalionchromatogramsofvolatilesextractedfromonion,tomatoandsweetpepperstoredundervariableandconstantatmosphericpressure.TICofIJATA:ONIOA13A..D8ØE67.OEB6.ØEB5.OES-C r1 -o4.E6C :33.0E62.0E6-Time(mm.)Fig.2.20.Totalionchromatogramofvolatilesextractedfromonionstoredunderconstantpressureat15°Cfor14days.TICofIJATA:QNIOA17A.]J8.0E67.OESB.OEB5.0E6I( -z4.0E6C3.0E62.0E81.OEB_hILLJi_______0•1020304DLTime(mm.)_____________________Fig.2.21.Totalionchromatogramofvolatilesextractedfromonionstoredunderconstantpressureat15°Cfor21days.TICofDRTA:ONIOA11A.D8.0E6-7.0E8B.0E6-a)5.0E81)—iii-4.0E6-Q3.OEB-2.OEB O__..kI.1.1U1.0E6-I—10203040—Time(mm.)Fig.2.22.Totalionchromatogramofvolatilesextractedfromonionstoredundervariablepressureat15°Cfor7days.TICoftJATA:ONIOA12R.IJ8.0E67.0E8S.OES5.OES-C-(ti4.0E8C :33.OEB-2.OES1.0ESJ___Time(mm.)Fig.2.23.Totalionchromatogramofvolatilesextractedfromonionstoredundervariablepressureat15°Cfor14days.TICofDATA:ONIOR1SA.]J8.0E6-7.0E66.0E65.0E64.0E6C :33.0E62.0E61.oE:.203040Time(mine)Fig.2.24.Totalionchromatogramofvolatilesextractedfromonionstoredundervariablepressureat15°Cfor21days.8.OES-TICofDRTA:TOMARO3A.IJ7.0E66.0E6-:::::: 3.OES-2.0E6-1.0E6-IJ JiJjJJ10203040Time(mm.)Fig.2.25.Totalionchromatogramofvolatilesextractedfromtomatostoredunderconstantpressureat15°Cfor7days.Fig.2.26.Totalionchromatogramofvolatilesextractedfromtomatostoredundervariablepressureat15°Cfor3TICofDATA:TOMAAØ2A.D-5OE64.ØEB3.0E62.0E61.0E6ci C rti C -Q ci:10203040Time(mm.)days.TICofDATA:TQMAAO4A.fl8.ØES7.0E66.0E65.ØESCa,4.OEB-(7’c3.ØEB-2.ØES-Time(mm.)Fig.2.27.TotalionchromatogramofvolatilesextractedfromtomatostoredundervariablepressureatI 5°Cfor7days.8.7.S.5.4.3.2.1.ØEBØESØEBE6ESØEBØEBØES 0TICofIJATA:PEPPAO4A.]J0) 0)II) C.) C (11 -o D -Q a:10Ii[20Time(mm.)3040Fig.2.28.Totalionchromatogramofvolatilesextractedfromsweetpepperstoredunderconstantpressureat150Cfor7days.TICoftIRTA:PEPPR1SA.flB.ØEB?.ØESB.ØEG-5.0E6-4.ØEBc 3 -Q30E62:OEBH1.ØEBII•-1—-’---’-r-102030.40_____________________Time(mm.)Fig.2.29.Totalionchromatogramofvolatilesextractedfromsweetpepperstoredunderconstantpressureat150Cfor14days.TICof]JATA:PEPPAØ5A.IJB.0E67OEB6.OESj 3.ØE62.0E62_________Time(min)Fig.2.30.Totalionchromatogramofvolatilesextractedfromsweetpepperstoredundervariablepressureat15°Cfor3days.TICofIJATA:PEPPAØ3A.D8OES6S7.0E6-B.OEB5°5.0E6U27- 0) Co-4.0E6C7:3 -Q3.OES2115 116 I1172.OEB181022232425__10203040LTime(mm.)Fig.2.31.Totalionchromatogramofvolatilesextractedfromsweetpepperstoredundervariablepressureat15°Cfor7days.TICoft3ATA:PEPPA2ØA.IJ8.0E67.0E86.0E65.0E6C Cd0-4.0E6C :33.0E62.ØEB1.OE-Time(mm.)Fig.2.32.Totalionchromatogramofvolatilesextractedfromsweetpepperstoredundervariablepressureat15°Cfor14days.

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