MSU» 5 RETURNING MATERIALS: P1ace in book drop to remove this checkout fr; your record. flfl§§_wil' be charged if book is returned after the daf‘ stamped below. 53E '2 3 {CF -9- “T“ <¢.—-§-.-h.~‘-b co—.n-o —-—‘. . w--—.-—m--— -- MA- A- EFFECT OF HARVEST DATE AND ETHYLENE CONCENTRATION IN CONTROLLED ATMOSPHERE STORAGE ON THE QUALITY OF EMPIRE APPLES By Touran Cheraghi Seifabad A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1988 .- " s ' ) (‘/)(_./l I ’ r A *‘ ABSTRACT Effect of harvest date and ethylene concentration in controlled atmosphere storage on the quality of Empire apples. By Touran Cheraghi Seifabad The main purpose of this study was to investigate the flavor profile of Empire apples as influenced by harvest maturity, ripening, and controlled atmosphere (CA) storage. Empire apples were harvested at two maturity levels as determined by flesh firmness, starch content and internal ethylene. Apples were stored for six months at 3.3‘C in air, low ethylene ((1 PPM) and high ethylene (>100 PPM) with 3% C02, 22 02 and a relative humidity of 98%. The changes in concentrations of volatile compounds due to storage conditions and ripening were determined using dynamic headspace, vacuum distillation and distillation- extraction techniques. Identification and confirmation were determined using capillary gas chromatography and mass spectrometry. Forty-nine compounds were identified in intact Empire apples using the dynamic headspace technique. Compounds identified included 24 esters, 13 alcohols, 7 aldehydes and 2 ketones. The most dominant compounds identified using dynamic headspace were, ethyl propionate, methyl butanoate, ethyl butanoate, methyl 2-methyl butanoate, ethyl 2-methy1 butanoate, butanal, hexanal, butanol, hexanol and 2-pentanol. Fifty-three compounds were identified using vacuum distillation and seventy-seven compounds were identified using the Likens-Nickerson technique. The most dominant compounds obtained using these two techniques were ethyl acetate, ethyl propionate, methyl, ethyl, butyl butanoate, ethyl-Z-methyl butanoate, methyl-2- methyl butanoate, pentyl and isopentyl acetate, ethyl and hexyl acetate, butanal, hexanal, (E)-2-hexenal, 2-hexenal, 1- butanol, 2-pentanol and 3-hexanol. Apples stored in air had the highest concentration of volatiles during storage. Low ethylene CA greatly reduced the volatile content of Empire apples after four months of storage. Those apples considered preclimacteric had a longer shelf life, as determined by total volatile concentration. For all three storage conditions, the concentration of volatiles was at its highest value up to four months, which related to the climacteric rise in respiration. The concentration of volatiles decreased thereafter for the remaining storage period. Ripening at 22 'C had a significant effect on the generation of volatile compounds. However, for apples stored in low ethylene CA the ripening process after four months did not influence the concentration of volatiles. DEDICATION Dedicated to my husband, Peter Jan Kroon, and to my sister, Pooran ACKNOWLEDGMENTS The author wishes to express her sincere thanks to Dr. J.N. Cash whose patience, continued encouragement and guidance was instrumental in the sucessful completion of this dissertation. Genuine appreciation is extented to the members of the research guidance committee, Drs. J. 1. Gray, M. Zabik, P. Markakis and H. Lockhart for their critical review of this dissertation. The author especially wishes to extend her appreciation to Drs. J. 1. Gray, M. Zabik and D. Dilley for allowing the author to use their equipment and their guidance and interest in this research. The author is also indebted to many good friends, Stella Cash, Giso, Dokhy, Kim, Catalina, Mireille, Hossein, Nikrous, Minoo, Dwannie and Petur. For their understanding during my moments of frustration, I offer my heartfelt thanks. The author is also indebted to the long suffering fellow graduate students, Mohamed, Arun, Nayini, Maria-Gloria, Man-lai, Carlos, Charlie, Zaman, Jo-Ann, Joe and work-study students, Bete and Terri, and Margaret for their friendship, technical assistance and above all for their wounderful sense of humor. Finally, to my family for their love, sacrifice, and support. Last but not least, I am forever indebted to my husband, Peter Jan for his love, support, and encouragement through this long and difficult process. -11- TABLE OF CONTENTS INTRODUCTION REVIEW OF LITERATURE Commercial importance of apples Importance of apple flavor Factors affecting the production of apple flavor Internal factors External factors Biogenesis of the flavor components in apples Collection, isolation, and identification of volatile compounds in apples. Headspace analyses Distillation extraction methods Apple flavor analyses prior to gas liquid chromatography Flavor analyses with gas liquid chromatography References VO‘O‘UU’I-L‘ 27 28 37 41 43 50 CHAPTER 1 EFFECT OF MATURITY AT HARVEST, STORAGE CONDITIONS AND POST HARVEST PHYSIOLOGY ON THE QUALITY OF EMPIRE APPLES. Introduction Materails and methods Flesh firmness -111- 63 64 65 70 Sample preparation 1-Amino-cyclopropane-1-carboxylic acid (ACC) assay Protein determination Malic enzyme assay Water soluble polyuronide (WSP) assay Results and discussion References CHAPTER 2 EFFECT OF STORAGE CONDITIONS AND RIPENING ON THE FLAVOR PROFILE OF EMPIRE APPLES USING A DYNAMIC HEADSPACE TECHNIQUE Introduction Materials and methods Materials Dynamic headspace sampling of intact apples Removal and concentration of volatiles Gas chromatographic analyses Gas chromatographic and mass spectrometric analyses of volatiles Mass spectra identification Quantitation of volatile compounds Results and discussion References -iv- 70 70 71 71 71 71 88 92 93 95 95 96 97 100 101 101 101 102 136 CHAPTER 3 EFFECT OF MATURITY AT HARVEST, STORAGE CONDITIONS AND RIPENING ON THE FLAVOR PROFILE OF EMPIRE APPLES USING DISTILLATION EXTRACTION TECHNIQUES Introduction Materials and methods Materials Volatile extraction and collection Method 1: vacuum distillation technique Method II: Likens-Nickerson technique Gas chromatographic analyses of the volatiles Gas chromatographic and mass spectrometric analyses of volatiles Quantitation of volatile compounds Results and discussion Effect of ripening References SUMMARY AND CONCLUSIONS -v- 140 141 143 143 144 144 148 149 149 150 150 197 200 204 LIST OF TABLES REVIEW OF LITERATURE Table Table Table CHAPTER Table Table CHAPTER Table Table CHAPTER Table Table 1. 2. 2 1. 2. 3 1. Retention times on precolumns under simulated loading and water removal conditions (min). Percent recovery (2) of compounds from aqueous solution with Likens-Nickerson apparatus according to Farmer et al. (1973). Volatile components in fresh, stored, and cooked apples and apple juice. Maturity parameters of Empire apples harvested in October 1984. Effect of storage duration and conditions followed by ripening at 22'C on flesh firmness of Empire apples. Percent recovery (2) of selected compounds using simulated dynamic headspace technique. Volatile compounds identified in headspace of Empire apples at harvest, during storage and ripening. Percent recovery (2) for selected compounds using vacuum distillation and Likens- Nickerson techniques. Volatile compounds collected during storage and ripening period at 22'C by vacuum distillation. .v1- 37 39 48 69 73 103 114 151 153 Table 3. Table 4. APPENDIX A Table 1. APPENDIX B Table 1. Volatile compounds collected during storage and ripening period at 22 ’C by Likens- Nickerson extraction distillation technique. Ester/aldehyde ratios at harvest and after four months of storage in air, low and hi h ethylene CA. The ratio is reported after , 7 and 14 days of ripening at 22° C. Chemical analysis of Empire apples after three months of storage in air, low and high ethylene CA and after subsequent ripening at 22 C for 0,7 and 14 days. Numbers are the average of three replicates. Volatiles in the headspace of Empire apples at harvest and ripening at 22' C for 2, 7 and 14 days. Tables 2 to 7. Volatiles in the headspace of Empire apples stored for six months in air at at 3. 3’0 and ripening at 22 'C for 2, 7 and 14 days. Tables 8 to 13. Volatiles in the headspace of Empire apples stored for six months in low ethylene CA ((1 PPM) at 3. 3°C and ripening at 22' C for 2, 7 and 14 days. Tables 14 to 19. Volatiles in the headspace of Empire Table 20. Table 21. Table 22. apples stored for six months in high ethylene CA (>100 PPM) at 3.3 and ripening at 22‘C for 2, 7 and 14 days. Computer program to calculate the concentration of the volatiles. Linear regression for the standards used for the quantitation analyses. Retention times for the standards using bonded, non-bonded and splitless injection with 50 and 60 m Carbowax 20 M capillary columns. -vii- 155 199 209 210 211 217 223 229 230 231 LIST OF FIGURES REVIEW OF LITERATURE Figure Figure Figure Figure Figure Figure Figure CHAPTER Figure Figure 1. 2. 3. 5. Proposed biosynthesis of fruit volatiles (Tressl et a1., 1975). 13 Reaction scheme for conversion of octanoic acid into esters (Tressl and Drawert, 1973). 17 Proposed biosynthesis of volatile compounds in banana from amino acid leucine (Tressl et a1., 1970 ). 19 Proposed biosynthesis of unsaturated esters via beta oxidation of linoleic acid (Jennings and Tressl, 1974). 22 Proposed biosynthesis of unsaturated esters via beta oxidation of linolenic acid (Jennings and Tressl, 1974). 24 Relative integrator reponse for several methods of sampling preparation (Jennings and Filsoof, 1977). 32 Gas chromatogram of blank sample (distilled water) analyzed by various sorbents ( Adams, 1984). 35 Harvest prediction based on the hours to ethylene climacteric and internal ethylene. 67 Changes in flesh firmness of Empire apples harvested on Oct. 2 (A) and Oct. 9 (B), followed by CA storage for 5 months. Storage conditions were: 3.3‘C, 3 2 C02, 2 Z 02 in low (1< PPM) and high (>100 PPM) ethylene, and air. Followed by ripening for 0-day, 7-days and 14-days at 22'C. - air, 0 - high ethylene, o - low ethylene. 74 -viii- Figure 3. Protein content of Empire apples. For storage conditions, refer to Figure 2. 77 Figure 4. Changes in production of 1-amino-cyclopropane-1- carboxylic acid (ACC) content in Empire apples. For storage conditions, refer to Figure 2. 80 Figure 5. Malic enzyme activity of Empire apples. For storage conditions, refer to Figure 2. 83 Figure 6. Water-soluble content polyuronide (WSP) of Empire apples. For storage conditions, refer to Figure 2. 85 CHAPTER 2 Figure 1. Apparatus for collecting volatile compounds from Empire apples by applying the headspace technique. 98 Figure 2. Standard curve for ethyl-Z-methyl butanoate. 105 Figure 3. Gas chromatogram of Empire apples stored for 4 months in air at 3.3‘C with relative humidity of 98 %. After 2 days of ripening at 22‘C. 1.5 ul sample injected. Initial temperature 20'C for 1 min. 20'C ---- 30'C rate of 7 C/min 30'C ---- 180'C rate of 2.5'C/min with holding time at 180‘C of 30 min. 107 Figure 4. Gas chromatogram of Empire apples stored for 4 months in air at 3.3'C with relative humidity of 98 %. After 7 days of ripening at 22°C. For gas chromatographic conditions, refer to Figure 3. 109 Figure 5. Gas chromatogram of Empire apples stored for 4 months in air at 3.3'C with relative humidity of 98 %. After 14 days of ripening at 22'C. For gas chromatographic conditions, refer to Figure 3. 111 Figure 6. Effect of storage conditions (low and high ethylene, and air) on the concentration of esters, 'aldehydes, alcohols and ketones during six months of storage. Apples were ripened at 22'C for 2 days. LCA- Low ethylene CA (3% C02, 2% 02 and (1 PPM ethylene). HCA- High ethylene CA (3% C02, 2% 02 and >100 PPM ethylene). 115 -1};- Figure 7. Figure 8. Figure 9. Figure 10. Figure 11. Figure 12. CHAPTER 3 Figure 1. Figure 2. Effect of storage conditions (low and high ethylene, and air ) on the concentrations of esters, aldehydes, alcohols and ketones during six months of storage. Apples were ripened at 22‘C for 7 days. For storage conditions, refer to Figure 6. 117 Effect of storage conditions (low and high ethylene, and air ) on the concentrations of esters, aldehydes, alcohols and ketones during six months of storage. Apples were ripened at 22‘C for 14 days. For storage conditions, refer to Figure 6. 119 Total changes of esters, aldehydes, alcohols and ketones during low ethylene CA storage and after subsequent ripening for 2, 7 and 14 days at 22'C. 124 Total changes of esters, aldehydes, alcohols and ketones during high ethylene CA storage and after subsequent ripening for 2, 7 and 14 days at 22’C. 126 Total changes of esters, aldehydes, alcohols and ketones during air storage and after subsequent ripening for , 7 and 14 days at 22°C. 128 Effect of ripening time (2, 7 and 14 days) at 22'C on the volatile compounds of Empire apples stored for 4 months in air at 3.3‘C. 1- methyl butanoate 2- ethyl 2-methyl butanoate 3- hexanal 4- 2-hexenal 5- 3-hexanol 6- 2-methyl 1-propanol. 131 Apparatus for collecting volatile compounds from Empire apples by applying vacuum distillation technique. A-Heater, B-Apples+water+salt, C-Thermometer, D-Dewar flask with liquid nitrogen, E-Cooled trap,F-Vacuum Pump and G-Vacuum gauge. 146 Gas chromatograph and mass spectra graph of Empire apples stored for four months in air at 3.3°C and after 7 days of ripening at 22‘C. For GC-MS conditions, refer to text. 158 -x- Figure Figure Figure Figure Figure Figure Figure Figure 10. Total esters, aldehydes, alcohols and ketones obtained by using vacuum distillation technique. Apples were stored for six months in air at 3.3°C and subsequently for 2 days of ripening at 22°C. The numbers are the average of three replicates. 160 Total esters, aldehydes, alcohols and ketones obtained by using vacuum distillation technique. Apples were stored for six months in air at 3.3°C and subsequently for 7 days of ripening. The numbers are the average of three replicates. 162 Total esters, aldehydes, alcohols and ketones obtained by using vacuum distillation technique. Apples were stored for six months in air at 3.3°C and subsequently for 14 days of ripening. Numbers are the average of three replicates. 164 Total esters, aldehydes, alcohols and ketones obtained by using Likens-Nickerson technique. Apples were stored for six months in air at 3.3°C and subsequently for 2 days of ripening. The numbers are the average of three replicates. 166 Total esters, aldehydes, alcohols and ketones obtained by using Likens-Nickerson technique. Apples were stored for six months in air at 3.3‘C and subsequently for 7 days of ripening. The numbers are the average of three replicates. 168 Total esters, aldehydes, alcohols and ketones obtained by using Likens-Nickerson technique. Apples were stored for six months in air at 3.3‘C and subsequently for 14 days of ripening. The Numbers are the average of three replicated. 170 Total esters, aldehydes, alcohols and ketones obtained by using vacuum distillation technique. Apples were stored for six months in high ethylene CA (>100 PPM) at 3.3°C and sybsequently for 2 days of ripening. Numbers are the average of three replicates. 172 Total esters, aldehydes, alcohols and ketones obtained by using vacuum distillation technique. Apples were stored for six months in high ethylene CA (>100 PPM) at 3.3°C and subsequently for 7 days of ripening. Numbers are the average of three replicates. 175 -xi- Figure Figure Figure Figure Figure Figure Figure 11. 12. 13. 14. 15. 16. 17. Total esters, aldehydes, alcohols and ketones obtained by using vacuum distillation technique. Apples were stored for six months in high ethylene CA (>100 PPM) at 3.3°C and subsequently for 14 days of ripening. Numbers are the average of three replicates. 177 Total esters, aldehydes, alcohols and ketones obtained by using Likens-Nickerson technique. Apples were stored for six months in high ethylene CA (>100 PPM) at 3.3‘C and subsequently for 2 days of ripening. Numbers are the average of three replicates. 179 Total esters, aldehydes, alcohols and ketones obtained by using Likens-Nikerson technique. Apples were stored for six months in high ethylene CA (>100 PPM) at 3.3°C and subsequently for 7 days of ripening. Numbers are the average of three replicates. 181 Total esters, aldehydes, alcohols and ketones obtained by using Likens-Nickerson technique. Apples were stored for six months in high ethylene CA (>100 PPM) at 3.3‘C and subsequently for 14 days of ripening. Numbers are the average of three replicates. 183 Total esters, aldehydes, alcohols and ketones obtained by using vacuum distillation technique. Apples were stored for six months in low ethylene CA ((1 PPM) at 3.3°C and subsequently for 2 days of ripening. Numbers are the average of three replicates. 185 Total esters, aldehydes, alcohols and ketones obtained by using vacuum distillation technique. Apples were stored for six months in low ethylene CA ((1 PPM) at 3.3'C and subsequently for 7 days of ripening. Numbers are the average of three replicates. 187 Total esters, aldehydes, alcohols and ketones obtaines by using vacuum distillation technique. Apples were stored for six months in low ethylene (<1 PPM) CA at 3.3'C and subsequently for 14 days of ripening. Numbers are the average of three replicates. 189 -xii- Figure 18. Total esters, aldehydes, alcohols and ketones Figure 19. Figure 20. APPENDIX C Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. Figure 10. obtained by using Likens-Nikerson technique. Apples were stored for six months in low ethylene CA ((1 PPM) at 3.3'C and subsequently for 2 days of ripening. Numbers are the average of three replicates. Total esters, aldehydes, alcohols and ketones obtained by using Likens-Nikerson technique. Apples were stored for six months in low ethylene CA (<1 PPM) at 3.3°C and subsequently for 7 days of ripening. Numbers are the average of three replicates. Total esters, aldehydes, alcohols and ketones obtained by using Likens-Nikerson technique. Apples were stored for six months in low ethylene CA (<1 PPM) at 3.3'C and subsequently for 14 days of ripening. Numbers are the average of three replicates. Mass spectra of methyl propyl 2-methyl butanoate. Mass spectra of ethyl 1-methyl hexanoate. Mass spectra of 2-methyl butyl hexanoate. Mass spectra of 2-methylbutyl 2-methyl propionate. Mass spectra of 2-methylbutyl 3-methyl butanoate. Mass spectra of ethyl 2-methyl butanoate. Mass spectra of 2-methy1 pentanoic acid. Mass spectra of heptyl heptanoate. Mass spectra of 2-butyl 2-octanal. Mass spectra of ethyl 2-methyl benzene. -xiii- 191 193 195 232 234 236 238 240 242 244 246 248 250 Figure 11. Mass Figure 12. Mass Figure 13. Mass Figure 14. Mass Figure 15. Mass spectra spectra spectra spectra spectra of 1,2 dimethyl benzene. of 2-methylpropyl benzene. of 2-methylbuty1 pentanoate. of 2-methyl propyl hexanoate. of 3-methylbutyl butanoate. -xiv- 252 254 256 258 260 INTRODUCTION Until recently, much of the apple research has been aimed at selection of varieties with good appearance, strong disease/injury resistance, good transport and sorting properties but without much attention to flavor quality. The consumer's choice is generally governed by its external appearance, color, shape, size, and freedom from defects. Although these external factors are important, they can not guarantee consumer satisfaction. Internal characteristics of the apple, such as flavor, will ultimately determine its quality, consumer acceptability and price. Fruit flavor is the combined effect of the volatile and non-volatile constituents, sugars, and acids on the sensory organs for taste and aroma. Factors such as variety, climatic variations, husbandry treatment, harvesting and storage conditions influence the chemical composition and thus the flavor of the primary product. However, most fruits are cooked or processed into secondary products, providing the opportunity for additional biological, chemical and physical changes which may also influence flavor. 2 Apples categorized by consumers as low quality, with less than desirable flavor, have often been found to be stored too long in cold rooms or in controlled atmosphere. The food industry has long recognized the need to improve the quality of stored apples to increase consumer acceptance (Liu, 1985). Aroma is one of the key factors in determining fruit flavor quality, which is used as a criterion for the evaluation of flavor influencing parameters. The nature and proportion of aroma constituents formed during maturation and ripening are presumably associated with biochemical reactions involving precursor-product relationship. Flavor extracts from naturally ripened apples are more complex than those of early harvested apples or those stored in controlled atmosphere. Maturation before harvest and subsequent ripening are recognized as important complex physiological processes that directly affect the ultimate quality of fruit (Brown et a1., 1966). Numerous investigations have been conducted related to the composition of the volatile compounds of different apple varieties. The invention and application of gas chromatography in the 1950's and subsequent refinements during the next two decades have contributed to the present knowledge of flavor chemistry. The development of GC-MS coupling has facilitated the identification of more than 4000 flavor ( Van Stern et a1., 1977) compounds in food. There are numerous techniques 3 for the separation and concentration of volatiles. However, according to Jenning et a1. (1977) no single method of isolation provides a satisfactory means of identification of volatiles. Since the content and composition of the volatile compounds differ markedly for each apple cultivar, the method used will depend on the type of sample and the characteristics studied. A study of the changes in flavor constituents during the storage and ripening thereafter is necessary to understand the ultimate quality of stored apples because controlled atmosphere storage often reduces biogenesis of volatile compounds which reduces consumer acceptability. The main objectives of this research were: 1- To study the influence of harvest date and storage conditions on the flavor profile of Empire apples. 2- To investigate the effect of the ripening process on the production of volatile compounds in Empire apples. 3- To study the flavor profile of the intact Empire apples using dynamic headspace techniques. 4- To study the effects of low and high temperature distillation on the flavor profile of Empire apples. REVIEW OF LITERATURE 5 COMMERCIAL IMPORTANCE OF APPLES "The original home of the apple (Malus sylvestris) is not known, but it is thought to be indigenous to the region south of the Caucuses, from the Persian province of Ghilan on the Caspian Sea to Trebizon on the Black Sea" (Smock and Neuber, 1950). In America, there are records as early as 1647 of apples having been grafted on seedling rootstock in Virginia (Hulme and Rhodes, 1963). Apples are one of the major fruits grown in the State of Michigan. More apples are being stored in the United States on a tonnage basis than any other fruit (Michigan Department of Agriculture, 1987). Further, because of a better understanding of physiological processes, improvements in horticultural practices, and ‘advancements in controlled atmosphere storage technologies, apples are now available throughout the year. The world wide distribution of numerous varieties of apples and the commercial economic importance of apples has resulted in the need for extensive research related to factors affecting apple flavor and the isolation and identification of volatile compounds in apples (Broderick, 1965,1974). IMPORTANCE OF APPLE FLAVOR The flavor characteristic that often attracts consumers to taste a particular fruit is its aroma. The aroma which is 6 specific to each variety is due to a complex mixture of volatile compounds: esters, alcohols and aldehydes which are present in the fruit in very small quantities. A better understanding of apple aroma and its subsequent effect on apple flavor can help growers and processors to maximize market potential of this fruit crop and can provide consumers with an acceptable product on a year-round basis. Expansion of the food industry over the years has resulted in a need for new products with improved flavor. One of the most obvious uses of a natural apple flavor is in the manufacturing of concentrated juice from which a full flavored, natural apple juice can be reconsituted by the addition of water (Milleville and Eskew, 1944). Another application of apple flavor is in the preparation of sirups. Apple essence is added to products which do not have any flavor of their own or to enhance the flavor of a particular product (Sugisawa et al., 1962). FACTORS AFFECTING THE PRODUCTION OF APPLE FLAVOR Numerous factors such as variety, climacteric variations, husbandry treatments, harvesting stage and conditions and storage can influence the chemical composition and thus the flavor profile of apples (Gerhardt and Ezell, 1939). Internal Factors Varietal differences among apples such as Golden 7 Delicious, McIntosh and Empire are primarily due to variations in flavor components. In fruits such as apples, respiration rise occurs in the fruit both on the tree and when detached from the tree (Paillard, 1981). As apples develop, biochemical changes gradually increase until the climacteric when there is a rapid evolution of ethylene and carbon dioxide with a maximun of ester production (Hulme et al., 1967). Maturation before harvest and ripening thereafter are recognized as important complex physiological processes that directly affect the ultimate quality of the fruit (Romani et al., 1966). Apples which are picked too early or too late do not develop the full flavor of fully-ripe apples. External Factors Factors such as the temperature of fruit after harvest and before storage affect the shelf life of apples during storage. It is essential therefore that apples are cooled as quickly as possible after harvest. Once apples are, in storage, temperature continues to play a very important role. Low storage temperature slows down the respiration rate which results in low ethylene production. The recommended storage temperature for a variety is one that is most effective in retarding respiration and ripening. For most apples varieties, the optimum storage temperature is 0 to 3.3 °C, with 90% relative humidity (Dewey and Dilley, 1969). In some apples, 8 the mean rate of respiration varies directly with temperature. For apples stored in air, a rise in temperature from 3.3 to 7.2 °C causes a mean percentage increase of C02 output of 40% with 60% 02 uptake. Kidd and West (1939) observed that as the temperature was lowered from 15 to 13‘C, the rate of respiration, ethylene production and volatile production in apples dropped markedly. According to Grevers and Doesburg (1965), the amount of volatiles emanating from apples is greatly dependent on the storage temperature. Apples stored at 3°C had lower levels of total volatiles than those stored at either 10 or 15"C. (Grevers and Doesburg, 1965). Harvesting date is another factor that affects flavor. Early harvested McIntosh apples had lower rates of non- ethylenic volatile production at a storage temperature of 0°C for 150 days than late harvested apples (Brown et al., 1966). Sapers (1977) studied the effect of harvesting date on the volatile components of McIntosh apple juice. He concluded that the sum of volatile components (as measured by peak ratio) increased significantly for apples harvested between mid August and mid September. The greatest increase in volatile components was in the concentration of ethyl butanoate and 1- propyl propionate. Since a significant percentage of the apples produced today are stored in controlled atmosphere (CA) or modified 9 atmosphere (MA) storage, it is important to evaluate the influence of CA and MA storage on apple flavor. Much of the benefit of CA storage is thought to result from the delay in the onset of ripening which results in the loss of the fruit's ablilty to produce volatiles in significant concentrations (Guadagni et al., 1971a; Patterson et al., 1974). Atmospheres containing higher C02 and lower 02 concentrations than air can be used to delay fruit ripening and ethylene production (Paillard, 1981; Little et al., 1982). Increased concentration of C02 and/or decreased concentration of 02 in the storage atmosphere reduces the rate of volatile production due to reduced rates of respiration (Fidler and North, 1969). Apples produce volatile alcohols, aldehydes, ketones, and esters at a much greater rate in air than in gas storage (Meigh, 1957). Apples stored in 2% 02 had a much lower concentration of volatiles than those stored in 19% 02 or in air. This could be primarily due to a lack of substrate or inhibition of enzymes in storage conditions (Patterson et al., 1974). The storage of apples in either 1% or 1.5% 02 atmosphere suppressed the production of ethyl butanoate, hexanal, ethanol and acetaldehyde in comparison with similar fruits stored in either 3% 02 or in air (Lidster et al., 1983). Low ester (ethyl propionate, methyl acetate, ethyl acetate ) production could be due to the reduced rate of alcohol production which 10 is considered to be the precursor to ester and aldehyde production in apples (Knee and Hatfield, 1981). Apples stored for prolonged periods do not develop the same intensity of flavor as those ripened naturally or stored for short periods (Meigh, 1957). Those stored for long periods develop a "banana like" aroma due to high concentrations of 2- and 3-methylbutyl acetate ( Williams and Knee, 1977 ). According to Dirinck et al. (1984a), there was a marked decrease of aroma after long CA storage for both early and late harvested apples. Only the mid-harvest date showed a maximum flavor after prolonged CA storage. Apples stored at 3.5 °C for 12 weeks in 1% 02 and 2% 02, showed variable concentrations of aroma components. Generally, for apples stored in air, the total concentrations for all flavor components was higher compared to those stored in 1% 02 and 2% 02 even when they were subsequently transferred to air (Knee and Hatfield, 1976). As an apple ripens naturally, the quantity of low boiling esters tends to build to a maximum level after a period of several weeks. The actual amount of time depends on the temperature at which apples are held (Drawert et al., 1969; Hatfield and Patterson, 1975). Apple volatiles can be partially regenerated after either CA or MA storage by exposing the fruit to air at 20°C.(Lidster et al., 1983). McIntosh apples stored in CA and subsequently 11 in air were shown to be capable of regenerating ethyl butnoate and hexanal (Lidster et al., 1983). Fruits ripened at 15'C for two weeks had higher levels of volatiles than those stored unripened (Grevers and Doesburg, 1965). Hexyl acetate strongly increased after ripening of the fruits at 15°C for two weeks. Early harvested McIntosh apples stored for 150 days had a lower rate of non-ethylenic compounds than late harvested apples (Grevers and Doesburg, 1965). Apples were ripened at 21 °C for 7 and 14 days to improve their flavor values. Those ripened for 7 days were judged to be mealy and’ had a natural flavor, whereas, those ripened for 14 days were still mealy but had a very poor flavor (Brown et al., 1966). The total sum of esters could be used to compare the quality of apples after they have been removed from storage. Dirinck et al. (1984a,b) using headspace techniques with intact Golden Delicious apples, showed that as ripening at room temperature progressed the sum of esters increased up to 14 days but thereafter decreased up to 22 days. However, by the end of the ripening period, the sum of esters was still higher than at the time of harvest. Nutrients available in the soil influence the production of flavor in many fruits (Childers, 1954; Somogyi et al., 1964). According to Smock and Neubert (1950), heavy nitrogen fertilization resulted in increased production of total volatile material from McIntosh apples. Brown et al. (1968) 12 showed that with phosphor fertilization the rate of production of three volatile esters from freshly harvested Golden Delicious apples increased. BIOGENESIS OF THE FLAVOR COMPONENTS IN APPLES As the Malus fruit develops, biochemical changes gradually increase until the climacteric when there is a rapid evolution of ethylene and carbon dioxide. "Biogenesis is the natural process by which flavor develops with progressive ripening, reaches an optimum value corresponding to the perfect ripening stage and subsequently exhibits a generalized rapid decay" (Feranoli, 1973). The pathway of ester synthesis in apples is not completely understood (Goodenough and Entwistle, 1982), 6;; the primary substances present in the aroma of Golden Delicious apples are esters. Their formation in the fruit seems to be largely dependent on the availability of precursor acids and alcohols ( Yamashita et al., 1975, 1976, 1977; Knee and Hatfield, 1981). A low level of esters in ripe apples could be due to a lack of precursors, low esterifying activity, high esterase activity or high diffusion rates of esters (Knee and Hatfield, 1981). Specific activity of esterase increased considerably from small immature fruits to large fruit at the climacteric stage ( Goodenough and Entwistle, 1982). Figure 1 summarizes possible pathways in the production of volatiles (Tressl et al., 1975). According to 13 Figure 1. Proposed biosynthesis of fruit volatiles ( Tressl et al., 1975 ). 14 LIPI DS POLY SACCHARI DE S PROTEINS ENZYMES LIGNINS l ATP CARBOHYDRATE METABOLISM HALONYL- . ‘\—'v—‘ ACETYL'CO A FATTY ACID ‘A AMINO ACID HETAB OLISM METABOLISM I summ- o-oxnomom A 1 LE? “'11-:9‘72—J cvs-H _ ' rap ACETYL to A TEEI‘TiE‘E VAL TYR HE? Hevxtouu— to A cmmmc ACID },_J M 5; A s. ALIPHATI c m METHYL-BRANCHED AROMATIC ACIDS mavens: ALCOHOLS ALcouou ALCOHO Ls monocansou s ACIDS ACIDS Esra" Atcouou esrens “tens cansouvts cmomrts caesouns cansoms Lacroues momma: 15 Heinze et al. (1954), Romani et al. (1966), and Drawert et al. (1969, 1972), the production of volatiles in ripening pears and apples is initiated by a climacteric rise in respiration, reaching a maximum value in the postclimacteric ripening phase. Early work by Huelin (1952) on whole Granny Smith apples suggested aldehydes as an intermediate in the conversion of acids and alcohols, and acetaldehyde probably being developed from decarboxylation of pyruvic acid and important respiration intermediates. Propionaldehyde was reported for the first time as a flavor component (Huelin, 1952). Full grown unripe apples when treated with acetic, propionic, and butyric acid showed a slight, but sustained increase in respiration rate (De Pooter et al., 1982). Dirinck et al. (1984) showed that intact apples were able to convert added acetic, propionic, butanoic, and carboxylic acids to esters or alcohols or smaller carboxylic acids by beta-oxidation. The greatest changes were observed with butyl acetate, hexyl acetate, butyl propionate, hexyl propionate, butyl butanoate, hexyl butanoate and butyl hexanoate. This indicates that acids (except acetic acid) are reduced to corresponding alcohols, probably by way of aldehydes (De Pooter et al., 1981). When acetic acid was added, only acetates were formed (De Pooter et al., 1983). Acetic acid had the greatest effect and propionic acid the least effect on the 16 production of esters , which indicated that not only the supply of acids is important but also their identity. Carbon 6 to carbon 10 acids were reduced to corresponding alcohols, which then underwent transacylation to esters. Figure 2 illustrates the formation of esters (octanoates ) from octanoic acid by climacteric and postclimacteric banana tissue (Tressl and Drawert, 1973). Knee and Hatfield (1981) concluded that the supply of alcohols is very important to the esterification capacity of apple tissues. Alcohols such as butanol and hexanol are produced from fatty acids (e.g. linoleic and linolenic) supplied to existing tissues (Paillard, 1978, 1979). These 'alcohols are presumably produced by beta-oxidation, followed by a reduction in two stages from acetyl-coenzyme A to aldehyde and then to alcohol. Branched alcohols such as 2- and 3-methyl butanol and 2-methyl-1-propanol, are likely to be produced from amino acids, such as leucine, isoleucine and valine via the corresponding aldehydes (Nursten, 1970 ; Myers et al., 1970; Tressl et al., 1970 ). Figure 3 shows the proposed biosynthesis of volatiles by Tressl et al. (1970). Linoleic and linolenic acid are presumed to be the precursor of carbonyl compounds (Drawert et al., 1962, 1966). Investigation with banana and apple tissues showed a correlation between the production of aldehydes and a decrease in the amounts of linoleic and linolenic content in these 17 Figure 2. Reaction scheme for conversion of octanoic acid into esters ( Tressl et al., 1973 ). l8 0 Gus“ \AM ’0 W 4 411—; C/ en,“ 5: ‘s-cu a-“c-octanolc acid A"o 4! £3 £2 We” ‘o-n MCI, Octanoatos \ 3 293$! \Wcuimc... 6‘ Octyl t .0 Of. 06, £2 0.. as c ‘O \M/‘cuzofl 1-Octnnol Reaction scheme for conversion of octanoic acid into esters, E1, acyI-thiokinase: E3, an-CoA-aloohoI-tnnsacylase; £3, acyI-CoA reducuse; E, alcohol-MAD ouydo- reducuse. 19 Figure 3. Proposed biosynthesis of volatile compounds in banana from amino acid leucine ( Tressl et al., 1970 ). 20 CN- "“- Hfi-é- ca; (3 -C3° 11 , 11 0“ Lemme GLutauaTE w" o aa-KETOGLUTARAIE 11.: -¢':-cu.-E-c:° . on N I-KE TOISOCAHONATE 1 men E1 . cu, «- I I M'é.-cn.°9 . C‘so H CW l—CO. “9:: a FAD MD 9“ “9 '3" I 9 ,0 Y H‘ HrQ'CH-‘G‘C.y c-c-C-C.5_Up u H 115’ an". NAO‘ ‘m-JW-WP ... / a... 5.. mm, m no.5" c". 11 cu. Cu. 9". “PROS-EON E‘ "pi-curd: ac-é-cR-cfin .__. whom-£22-“, n :1 H u u i-Haumutan—I- oi J ”MIMI!!! J-Motnytbutonoata 3 41mm: Dutytyl-CQA “on. W ' ALKVLAIINO W8 ACYLAIING COMPMNTS I ESTER ESTER l-METMVLIU‘I’YL' -J-M£TH¥LDUI’YRATE Conversion of amino acids Into aroma components of banana as illustrated by leucine. E . Maurine aminomnaferaac EC 2 6 l ' mama mum (EC 4.1.1.1); 2,. aIdahdee dehydtouonaaa (EC 1.2.1.3); 5.. alcohol dehy'axomm (EC 1.1.1.1): 1mg. moonin‘eszo'yféf mu: ”Ki. oxidized “pole add: ”<1". reduced upon: acid: FAD, “avian-adenine dInucIeotidc; mo. oxidized 1--” u . 2 diaudaotide; OoA-SH. mm A. 21 fruits (Paillard, 1979). Feys et a1.( 1980) showed that there were increases in the concentrations of hexanal and (E)-2- hexenal when linoleic and linolenic acid were added to apple juice. Koch and Schiller (1964) correlated the sum of 2- hexenal and 2-hexenol to the intensity of apple juice essence. During ripening, fruits such as bananas, apples and strawberries develop the ability to convert unsaturated fatty acids to aldehyde, alcohols, ketones and esters (Tressl and Drawert, 1973; Wade and Bishop, 1978). Alcohols were formed from aliphatic acids through beta-oxidation. Fatty acids with even numbered carbon gave rise to butanol and hexanol, while odd numbered carbon fatty acids generated propanol and pentanol. (Paillard, 1979). The formation of 2-hexena1 and hexanal from linoleic and linolenic acids in apples and bananas is a typical example of the enzymatic activity of these fruits during ripening. (Tressl and Drawert, 1973). Proposed biosynthesis of unsaturated esters via beta-oxidation from linoleic and linolenic acids is given by Jennings and Tressl, (1974) (Figures 4 and 5). The addition of butanol led to enhanced levels of butyl butanoate. Acetates were formed when 2-methyl propanol and 2- or 3-methyl butanol were added to the cortical tissue (Myers et al., 1970). Hexyl acetate was detected in the gas during storage when hexanol was applied. The persistance of higher levels of hexyl acetate in air than in 2% oxygen is presumably 22 Figure 4. Proposed biosynthesis of unsaturated esters via beta oxidation of linoleic acid ( Jennings and Tressl, 1974 ). 23 >0 WUUVW‘C’ ' ‘S-CoA LINOLEYL—CoA METHYL AND ETHYL CIS—S, CIS—8 TETRADECADIENOATE {,0 WC \O-R METHYL AND ETHYL CIS—4 DECENOATE AAFAP“ \O_ R-OH R “-0“ - \- /w=v~c’ 95 c W ‘04, METHYL AND ETHYL TRANS—2, CIS-6 DODECADIENOATE E1 .3 A3 cas _ A2 TRANS ENOYL—CoA ISOMERASE £2 . ACYL—CoA DEHYDROGENASE , \5- 00A FADH2£ [WK \ S- CoA R-OH NV'=’\/IC”0 ‘O-R METHYL, ETHYL, PROPYL, BUTYL AND HEXYL TRANS-2, CIS-4 DECADIENOATE 24 Figure 5. Proposed biosynthesis of unsaturated esters via beta oxidation of linolenic acids ( Jennings and Tressl, 1974 ). 25 40 MC \S-CoA LINOLENYL—COA 40 /\=/\=/\=:/‘C \ S-CoA R- OH C/o O/W C\O-R V V ,VW‘C \ s- CoA ETHYL TRANS—2 l CIS—6,CIS-9 40 DODECATRI ENOATE V V V‘C . \S-CoA FAD E 2 FADH2 R-OH 40 L 40 V V V‘C 3" V V VAC ‘8- 00A \O-R METHYL AND ETHYL TRANS—2, C [8—4, CIS—7 DECATREINOATE A3 (3'3 — A2 TRANS ENOYL—CoA ISOMERASE ACYL CoA DEHYDROGENASE 26 due to the higher endogenous production of this ester (Knee and Hatfied, 1981). Apple tissue has the ability to metabolize primary alcohols to acetate esters and aldehydes. Aldehydes will be produced if alcohol dehydrogenase is active in the tissue (Patterson et al., 1974). Alcohol dehydrogenase activity continues to increase from the initial stage to the fully ripened stage. Alcohol oxidoreductase (ADH) will reduce aldehydes to their corresponding alcohols (Yamashita et al., 1976). Peel tissues were able to produce butyl acetate at a rate of 0.4-2.0 nmol gIh- without any trend in time when either butanol or 2-metyl propanol was used as a substrate (Knee and Hatfield, 1981). Williams' and Knee's (1977) experiments with apple disks showed that if substrates in the form of vapor are applied, the peel is more able to produce flavor compounds such as butanol from butyl acetate and butyl butyrate than peeled apple disks . Similar findings were reported by Guadagni et al. (1971a,b). Ethanol is esterified with formic, acetic, butanoic and hexanoic acids to form ethyl formate, ethyl acetate, ethyl butanoate, ethyl hexanoate. The esterification of methanol with formic and hexanoic acids resulted in the formation of methyl formate and methyl hexanoate (Sugisawa et al., 1962). 27 Guadagni et al. (1971) suggested that the esterifying enzymes are present in CA storage and the lack of ester production is due to a lack of precursors. Acetate, propionate, n-butanoate, 3-methylbutanoate (iso- valerate) and hexanoate (n-caproate ) esters were formed when strawberry fruits were incubated with alcohols (Yamashita et al., 1975). This conversion is enzymatic since heated strawberries were not able to produce alcohols or esters. (Yamashita et al. 1976) Hexanal, (E)-2-hexenal and (Z)-3-hexenal quickly appeared in the destroyed apple cells through enzymatic oxidation degradation of their precursor linoleic and linolenic acids (Drawert et al., 1966 ). (E)-2-hexenal and n-hexanal, which showed important fluctuations in concentrations, depended on the degree of maturity and were formed enzymatically during disintegration (Drawert et al., 1966). COLLECTION, ’ISOLATION, AND IDENTIFICATION OF VOLATILE COMPOUNDS IN APPLES There are many factors that influence flavor analysis in food and food products. Of these many factors, one of the more important, yet also most difficult to analyze, is the profile of volatile organic compounds present. The difficulty arises from the fact that there may be many volatiles present at very low concentrations in a complicated matrix. 28 A number of different preparation techniques have been used for the analysis of flavor volatiles, including solvent extraction, steam distillation, equilibrium headspace and dynamic headspace sampling (Bemelmans, 1978; Sugisawa, 1981). Headspace Analyses In flavor research analysis of the composition of volatile components, the vapor phase above the food is referred to as "headspace analysis". Headspace analysis offers the possibility for isolation of the volatiles without sample destruction or artifact formation (Land, 1975). Fruit samples can be evaluated for varietal differences, seasonal variations, ripening studies, or the detection of storage abuses. Gas chromatographic headspace analysis based methods, simplifies and shortens the determination of low concentrations of volatile compounds in this complex matrix (Ettre et al., 1983; Dirinck et al., 1984a,b). In studying the aroma properties and quality of food materials, direct headspace analysis (static headspace) is the simplest and fastest method of analysis ( Weurman, 1961; Martin, 1969; Gasco' et al., 1969; Guadagni et al., 1971; Schreyen et al., 1976; Yamashita et al., 1977; McNally and Grob, 1985). Unfortunately, this method has not provided the sensitivity needed for trace analysis and is not well suited to modern capillary column gas chromatography (Nawar, 1966 ; Reineccius, 1983). In order to obtain a complete picture of 29 volatiles present in a particular system, more than one sampling technique must be employed (Wyllie et al., 1978). One of the first techniques for trapping apple volatiles during storage was demonstrated by Turk et al. (1951), using direct trapping from the atmosphere of an apple storage into dry ice or liquid air. Meigh (1956, 1957) and Heinze et al. (1953) collected volatiles using activated charcoal. They argued that by using this procedure, the natural flavor of apple would not be altered. From the results of Meigh (1957) on the emanation collected in cold traps, Grevers and Doesburg (1962) calcluated the mean ratio of ester/ free alcohols to be 4/1 (calculated as butyl acetate). Volatile components of fresh and frozen strawberries were studied by Teranishi and Buttery (1962) using an aromagram (direct injection of headspace). Direct headspace analysis, even though it has the advantages of simplicity, is usually restricted to compounds with boiling points below 15030 and requires high instrument sensitivity (Levins and Ikeda, 1968). The typical aroma compounds are often present at trace levels. Therefore, intense enrichment of the "aroma-fraction" is necessary (Adams, 1984). Dynamic headspace (purge and trap) analysis is based on the principles of concentrating, stripping and trapping of volatile compounds. The concentration of compounds present only in trace amounts in the headspace of apples necessitates the use of low capacity 30 and high resolution capillary columns (Simpson, 1979). This technique is one of the most suitable methods in the analysis of flavor components in food (Novotny et al., 1974; Simon et al., 1980; Dirinck et al., 1981). Volatile compounds will partition out of a sample into the vapor phase above a sample at a rate dependent on a variety of factors (Westendrof, 1984). These factors include the volatility of the compound, solubility in the sample matrix, homogeneity of the matrix, temperature and sample container configuration. By sweeping gas over the sample and trapping the resultant vapor on the porous polymer traps a considerable concentration can be achieved. Compounds that have a poor solubility will be purged more efficiently than compounds of high solubility (Westendrof, 1984). Adsorption on charcoal from a stream of gases and subsequent desorption is used in the enrichment, isolation and determination of volatile compounds from food (Turk et al., 1951; Heinze et al., 1953). Charcoal was used because of its high adsorption capacity and apparent absence of involvement in artifact formation (Paillard, 1965; Jennings and Nursten, 1967; Tang and Jennings, 1967). Activated carbon has been used as a selective adsorbent because of its low affinity for water and great chemical stability (Heins et al., 1966; Paillard, 1978). The charcoal was either solvent extracted or thermally desorbed with back flushing (inert gas) to recover the 31 adsorbed volatiles (Cronin, 1982). Synthetic porous polymers have been found suitable for collection of volatiles from headspace of samples (Simon et al., 1980; Schaefer, 1981; Heydanek and McGorrin, 1981 a,b; Galt and MacLeod, 1984; Olafsdottir et al., 1985). Adsorbent traps offer the advantages of providing a water-free flavor isolate (Sydor and Pietrzyk, 1978). It is also possible to enrich volumes of 10 L or more so that volatiles with headspace concentration of less than 1 ng/l can be identified (Drawert and Christoph , 1984). Bulter and Burke (1976) and Jennings and Filsoof (1977) evaluated the chromatographic capacity and efficiency of a number of porous polymers and concluded that no single one was universally suitable . Volatile components are either heat desorbed (Dirinck et al., 1981) or removed by various extraction techniques ( Guadagni et al., 1971; Clark and Cronin, 1975; Williams et al., 1977; De Pooter et al. 1981). Jennings and Filsoof (1977) have provided detailed information on various porous polymers (Figure 6). Among these different polymers, Tenax-GC (Zlakis et al., 1973; Schaefer, 1981) and recently Tenax-TA (MacLeod and Ames, 1986) are most commonly used. Tenax-GC (a polymer based on 2,6 diphenyl-p-phenylene oxide) has a very low affinity for low molecular weight alcohols and water (Jennings et al., 1972; Kuo et al., 1977). 32 Figure 6. Relative integrator reponse for several methods of sampling preparation ( Jennings and Filsoof, 1977 ). 33 LALA ._L_I.|.l. a Lilly ‘LY-lm ncot sobficn boodapoca, haodspoce. heodspoca. headspace, mot solution 100;»!!! moo: IOOppm aqueous, IOOppm comma, 407. N00 mutated 80% N00 saturated .11z .llodo ['1 W 9 zones 6C. Poropoh O Tenor 6C, solvent distillation- "aaaanca aaaonca solvers! aattocflon extraction «Indian 34 Retention of the low molecular weight alcohols is negligible, since they do not contribute to the aroma of the product (Adams, 1984). Tenax-GC does not produce major artifacts as other porous polymers sometimes do under certain operating conditions ( Figure 7). Lewis and Williams (1980) observed 59 compounds when Tenax-GC traps were regenerated above their normal operating temperature (250°C). Tenax-GC traps are useful when high boiling compounds are a major concern. Simon et al. (1980) used Tenax-GC to trap volatiles from carrot roots. They found a significant correlation between Tenax-GC and distillation for all the volatile compounds. Retention times exhibited on Tenax-GC were considerably shorter than Porapak and Chromosorb 102 used for trapping volatile compounds from frozen and canned corn (Bokyo et al., 1978). Table 1 summarizes retention times for Porapak Q, Chromosorb 102 and Tenax-GC for selected compounds. Direct injections are sometimes satisfactory with the use of a "cryogenic procedure." (Rushneck, 1965; Heins et al., 1966). The cold trap will quite efficiently collect headspace vapor irrespective of compound polarity and boiling point. The sample is condensed in the initial portion of the column. This is accomplished by lowering the initial column temperature 50 to 150 °C below the temperature required to elute the sample under isothermal conditions ( McEwen, 1964.; Heins et al., 1966, Grob and Grob, 1969). Rushneck (1965), using the 35 Figure 7. Gas chromatogram of blank sample (distilled water) analyzed by various sorbents ( Adams, 1984). 36 [ PorapakN ._.JA A A #14114 “:1 J] PorapakQ ! _._1-44 1-..- . Tonax GC -41 1 A; J I; [ l -Chromosorb 106 I! 0 1'0 i0 ‘ 3'0 RotontIon Mme I mln. 37 cryogenic procedure to identify the volatiles in cigarette smoke, showed that the eluting peaks were sharper and more compounds could be identified. Also, because of the higher gas flow the retention times of those eluting peaks were shortened by 40:. Table 1. Retention times on precolumns under simulated loading and water removal conditions (min). Compound Porapak Q Chromosorb 102 Tenax-GC Methanol 2.3 1.3 0.4 Ethanol 8.5 5.3 1.1 Formic acid 14.6 12.0 2.0 Acetaldehyde 3.6 2.1 0.5 Propanal 18.0 12.0 2.0 Methyl formate 6.5 3.5 0.8 Ethyl formate 33.0 16.5 2.0 Precolumn Conditions: . Column temperature 50 C Injection port temperature 120’C Detector temperature 160°C He flow rate 12 ml/min Distillation Extraction Methods The headspace methods are used very commonly in flavor studies to good advantage. However, one must be aware of their limitations since the higher boiling compounds which may be important in the overall flavor of apples are missing. In 38 order to study the complete flavor profile of food, one of the distillation-extraction techniques should be used. Distillation is the most widely used technique to isolate volatiles from apples and other fruits (Stevens et al., 1966; Flath et al., 1967, 1969). The distillation-extraction technique was first reported by Likens and Nickerson (1964). Modification of the original Likens-Nickerson apparatus allows distillation and extraction simultaneously. With this technique, large quantities of samples can be extracted with only a small volume of organic solvent (Nursten and Woolfe, 1972). This small quantity of solvent and the general convenience of the device are reasons for its frequent use (Guadagni et al., 1971). Shortcomings of this method include a moderately long isolation time , potential artifact formation due to heat during distillation- extraction (Alberola et al., 1978) and low percent recovery (Maarse, 1971). Another disadvantage is that water will be present in the distillate, requiring further solvent extraction (Heath and Reineccius, 1986). Table 2 summarizes the percent recovery of compounds studied by Farmer et al. (1973). 39 Table 2. Percent recovery (%) of compounds from aqueous solution with Likens-Nickerson apparatus according to Farmer et al. (1973). Carbon number 1-Alkanol 2-Alkanone Alkanal Alkanen 3 trace 4 trace trace trace trace 5 73 79 101 64 6 97 104 91 94 7 101 101 101 103 8 102 94 94 94 9 99 97 83 90 10 94 11 104 The artifacts formed due to heat during distillation- extraction are elimitated when this apparatus is operated under vacuum (Land, 1984 ). Reduced pressure operation minimizes thermally-induced artifact formation. This technique has been used by numerous investigators to isolate volatile components from food. Vacuum distillation procedures used to collect volatile compounds in the a cold liquid trap from orange juice produce the isolate that had the full flavor of fresh orange juice ( Merritt et al., 1959; Angelini et al. 1967; Alberola et al, 1978). Extraction methods are used in flavor research either to isolate volatiles directly from food or to recover them from diluted aqueous distillates. These extraction methods are based on the favorable distribution coefficient of the 40 volatiles between solvent and food or the distillate. There are numerous organic solvents that are used in extraction and concentration of volatile compounds. Solvents used for aroma research have been reviewed by Weurman (1969). Pentane, isopentane, diethyl ether, dichloromethane, benzene, liquid COZ and freon 11 are examples of solvents which could be used to extract these volatiles from food and distillates (Levins and Ikeda, 1968; Schultz et al., 1967). Extraction of aroma compounds from apple juice with 2—methylbutane (isopentane) was described by Mehlitz and Gierschner (1962). Isopentane is shown to give good recovery using the Likens-Nicherson apparatus (MacLeod and Cave, 1975). Isopentane is known to reject ethanol which may mask important minor components, or limit the extent to which volatiles may be concentrated (Schultz et al., 1967). It is somtimes necessary to concentrate the distillate obtained from distillation-extraction techniques because the concentration of volatiles may be too low for gas chromato- graphic analysis. Nursten and Woolfe (1971) used temperatures of 45 to 50°C to concentrate the solvent (pentane) containing flavor components from apples. Concentration is usually achieved by purging with a gentle stream of nitrogen. A comparison of several techniques (direct, dynamic head- space; distillation-extraction) carried out with model system solutions showed that distillation-extraction gave results 41 which more closely agreed with those obtained by direct injection (Alberola et al., 1978). Direct injection (on-column injection) is commonly used to determine the flavor quality of vegetable oils (Legendre et al., 1979). This technique is most suitable for samples which are thermally stable and do not contain a large amount of water. However, with the modification of the injection port a thermally unstable sample could be analyzed (Legendre et al., 1979). There are numerous methods available for isolation, collection, and extraction of volatile compounds from food. Each method has its benefits and limitations. To achieve reliable data, however, it may be necessary to use more than one method of analysis. APPLE FLAVOR ANALYSES PRIOR TO GAS LIQUID CHROMATOGRAPHY Power and Chesnut (1920, 1922) were among the first investigators to study flavor in apples and apple products. They obtained volatile carbonyl compounds by passing air over the whole fruit and subsequently over a bisulfite solution to collect different volatiles. They identified acetaldehyde, and pentyl esters of formic, acetic and hexanoic acids, and traces of octanoic acid in various apple varieties (1920). They also identified geraniol, a terpene alcohol in McIntosh apples (1922). White (1950) examined the volatile fractions of apple juice and found acetaldehyde, acetone, hexanal, and 2-hexenal. 42 The dinitrophenylhydrazone of these compounds was separated and identified by melting points. White (1950), using apple-essence which was fractionated at atmospheric pressure produced an apple oil. This oil was fractionated under vacuum and fractions were treated to form derivatives which were identified using paper chromatography. Alcohols were identified as dinitrobenzoates and carbonyl compounds as dinitrophenylhydrazones. Esters split into alcohols and acids. The following compounds were identified: methanol, ethanol, n-propanol, 2-propanol, butanol, isobutanol, 2-methy1-1-butanol, hexanol (92% of total), acetaldehyde, acetone, hexanal, 2-hexena1 (6% of total), ethyl butanoate and ethyl hexanoate (2%). Thampson (1951) identified hexanol and hexanoic acid as products of whole apples. Aliphatic acids containing one, two, three, four, five, and six carbon atoms have been identified by White (1950), Thampson (1951), and Thampson and Huelin (1952). Huelin (1952) used paper chromatography to identify acetaldehyde and propanal from whole. Granny Smith apples stored at O‘C for four to six months and ripened at 30 °C. He reported that aldehydes might be intermediates in the interconversion of acids and alcohols. Acetaldehyde was probably formed from the decarboxylation of pyrvic acid which is an important intermediate of respiration. Acetone was 43 possibly the product of fatty acid metabolism. These early investigations were limited by the available techniques for analysis of volatiles. (Power and Chestnut, 1920, 1922; Walls, 1942; White, 1950; Thampson, 1951; Thampson and Huelin, 1951; Huelin, 1952; Heinze et al., 1953; Heinze et al., 1954; Meigh, 1956; Meigh, 1957; Fidler, 1958; Wenzel, 1962; Gasco' et al. 1969; Meigh, 1964; Gasco' and Barrera, 1972). FLAVOR ANALYSES WITH GAS LIQUID CHROMATOGRAPHY The use of modern analytical techniques, particularly coupled gas chromatography-mass spectometery, has revealed the complexity of aroma in food and food products ( Drawert et al., 1962 ; Grevers and Doesburg, 1962; Kieser and Pollard, 1962 ; Matthews et al., 1962; Mehlitz and Grieschner, 1962; Koch and Schiller, 1964 ; McGregor et al., 1964; Merritt et al., 1964; Dirinck et al., 1977 a, b; Neubeller et al., 1979; Peredi et al., 1981; Dimick et al., 1983). Since James and Martin succeeded in making a prototype gas chromatograph in 1952, this instrument has become the most powerful tool in the analysis of flavors. In the 1960's, the development of the high resolution column and gas chromatography-mass spectrometery techniques permitted the identification of a tremendous number of flavor components of various substances such as esstential oils, fruit essences and many other food products. Flame ionization detection is particularly suitable for 44 flavor analysis because of its high sensitivity to organic compounds, and its insensitivity to inorganic gases and water (Nawar, 1966). With this type of detector, flavor chemists were able to separate and identifiy volatile components at the parts per billion levels (Teranishi and Mon, 1977). Flavor samples are often exceedingly complex. Their separartion depends on many factors, the most important of which is the type of column being used. Chromatographic columns are usually divided into two classifications: packed and open tubular (capillary). Packed columns are larger in diameter, require higher flow rates and can handle large sample sizes. Open tubular or capillary columns, while of smaller bore size, are usually of greater length which results in greater resolution (higher number of theoterical plates), (Cronin, 1974). Class or fused silica columns are most commonly used in flavor analysis because fused silica columns have the advantage of being inert (Shibamoto, 1984). In addition to column construction (i.e. packed or capillary), selection of the stationary phase, flow rate, and control of column temperature becomes very important. Bonded phase columns can withstand higher operating temperatures. However, a coated phase column is superior to a bonded phase column in the reproducibility of retention time. Resolution is equally good for both types of column (Shibamoto , 1984). Takeoka et al. (1985) used a bonded phase column (Durable 45 Wax) for flavor analysis of German wine. This work resulted in a clean separation of methyl acetate, ethyl acetate, methanol and ethanol using an initial temperature of 35°C. The presence of ethyl alcohol did not interfere with the separation pattern of wine. There are basically two types of injections: split and splitless injection. Spliting may easily cause quantitative errors. Sample losses due to spliting are troublesome when using expensive or toxic compounds. In splitless injection there is complete elimination of sample losses, and analysis of very diluted samples is possible. In head space analysis, the splitless mode is commonly used (Grob and Burrus, 1965; Grob and Grob, 1969). Willis et al. (1968) reported the use of a trapping loop. A small capillary length column which is cooled during injection and after introduction of the sample was quickly heated to start separation. This technique is very suitable for diluted samples. A narrow starting band can be achieved either by the injection of a narrow band (split injection), or focusing before the chromatographic process (splitless injection and in-column injection) (Jennings and Takeoka, 1984). The gas chromatograph is a differentiator. At best, it reveals how many components are in a mixture, and how much of each component is present. Even the most impressive chromatograms obtained with high efficiency capillary columns 46 are of little use without the possible identification of separated peaks. Due to sample size requirements for capillary columns (Merritt and Brobertson, 1982), mass spectrometry is one of the methods used for identification purposes. The first mass spectrograph, built by Aston and Dempster before 1920, is an instrument which separates atomic and molecular particles according to their masses. During the past 20 years, mass sepctrometry has been used widely to determine organic structures. Mass spectra provide information based on the mode of fragmentation of organic compounds which have been ionized usually by electron bombardment. Interpretation of the mass spectrum of an unknown compound is always easier when additional information , such as infrared, nuclear magnetic resonance and other chromatographic data are also available. Coupling a gas chromatograph with a mass spectrometer provides additional information about flavor compounds in food. (Flath et al., 1969; Nursten and Woolfe, 1972; Williams et al., 1977; Williams and Tuchnott, 1978; Schreier et al., 1978; Williams et al., 1980). Flavor compounds in apples have been identified using one or more of the techniques described . Since the work of Power and Chesnut in 1920, the number of flavor compounds identified in apples has grown rapidly. These compounds consist of esters, alcohols, aldehydes, ketones, acids and hydrocarbons. Depending on the variety of apples the concentration of these 47 volatile compounds vary. A list of esters, aldehydes and ketones that have been identified by various researchers is summarized in Table 3. 48 Table 3. Volatile components in fresh, stored and cooked apples and apple juice. Esters Carbonyl compounds Alcohols ethyl formate acetaldehyde ethanol propyl formate propanal 1-propanol utyl formate -propanal 2-propanol isobutyl formate 2-methylpropanal 2-methyl propanol 2-butyl formate 2-oxopropanal 2-methyl 2-propanol isopentyl formate butanal 2-butanol hexyl formate (E)-2-butanal 2-methyl butanol methyl acetate 2-methylbutanal 3-methy1 butanol ethyl acetate 3-methylbutana1 2-methyl 2-butanol propyl acetate pentanol 1-pentanol isopropyl acetate hexanal 2-pentanol butyl acetate 2-hexenal 3-pentanol isobutyl acetate (E)-2-hexenal 3-methyl pentanol 2-butyl acetate (E)-3-hexena1 1-hexanol 2-methylbutyl acetate heptanal 2-hexanol pentyl acetate 4-heptanal 2-hexenol isopentyl acetate (E)-2-heptenal (E)-2-hexenol hexyl acetate octanal (Z)-2-hexenol (E)-2-hexenyl nonanal 3-hexenol acetate decanal (Z)-3-hexenol (Z)-2-hexenyl acetate (E)-2, (Z)-4 decadienal (E)-3-hexenol heptyl acetate (E)-2, (E)-4- I-heptanol octyl acetate decadienal 2-heptanol nonyl acetate undecanal 1-octanol decyl acetate dodecanal 2-octanol bezyl acetate benzaldehyde 1,3-octandeiol phenethyl acetate 2-phenylacetaldehyde 1-nonanol perillyl acetate 2-propanone 2-nonanol ethyl propionate 2-butanone 1-decanol propyl propionate 3-hydroxybutan-2-one 2-decanol butyl propionate 2,3-butanedione 1,10-decanediol isobutyl propionate 2-pentanone 1-undecanol pentyl propionate 3-pentanone 2-undecanol isopentyl propionate 4-methypentan-2-one 1-dodecanol hexyl propionate 2-hexanone 2-dodecanol ethyl 2-methyl- 2-heptanone I-tetradecanol propionate 3-heptanon 1-octadecanol ethyl 2-hydroxy- 4-heptanone eraniol propionate 2-octanone g-phenylethanol methyl butanoate 7-methyloctane-4-one ethyl butanoate acetophenone propyl butanoate 49 Table 3 (cont.). Volatile components in fresh, stored, and cooked apples and apple juice. Esters Miscellaneous isopropyl butanoate furan butyl butanoate furfural isobutyl butanoate 5-hydroxymethyl furfural pentyl butanoate S-methylfurfural isopentyl butanoate hexyl butanoate ethyl 2-butanoate methyl 2-ethylbutanoate ethyl 2-methy1butanoate propyl 2-methylbutanoate pentyl 2-methyl butanoate 2-methylbutyl 2-methylbutanoate methyl 3-methylbutanoate ethyl 3-methylbutanoate isopentyl 2-methylbutanoate isopentyl 3-methylbutanoate hexyl 2-methylbutanoate methyl pentanoate ethyl pentanoate propyl pentanoate butyl pentanoate pentyl pentanoate isopentyl pentanoate hexyl pentanoate methyl hexanoate ethyl hexanoate butyl hexanoate pentyl hexanoate hexyl hexanoate 2-methylbutyl hexanoate ethyl heptanoate ethyl octanoate butyl octanoate pentyl octanoate isopentyl octanoate hexyl octanoate ethyl nonanoate ethyl decanoate butyl decanoate pentyl decanoate isopentyl decanoate ethyl dodecanoate ethyl 2-phenylacetate 2-acetyl furan furfuryl alcohol 50 REFERENCES Adams, S. 1984. Analysis of volatile aroma components in fruit and vegetable juices by gas/liquid extraction, sorptive collection, cryogenic focusing and high-resolution gas chromatography. Proc. Int. Symp., p. 67. Alberola, J., Izquierdo, L. J., and Espana, V. 1978. The volatile fraction of orange juice. Methods for extraction and study of composition. In "Flavor of Food and Beverages," G. Charalambous and G. Inglet (Ed.), p. 283. Academic Press, London. Angelini, P., Forss, D. A., Bazinet, M. L., and Merritt, C. 1967. Methods of isolation and identification of volatile compounds in lipids. J. Amer. Oil Chem. Soc. 44: 26. Bemelmans, J. M. 1978. Review of isolation and concentration techniques. In "Progress in Flavour Research," D. G. Land and H. E. Nursten (Ed.), p. 79. Applied Science Publishers Ltd., London. Boyko, A. L., Morgan, M. E., and Libbey, L. M. 1978. Porous polymer trapping for GC/MS analysis of vegetable flavors. In 'Analysis of Fruits and Beverages," G. Charalambous (Ed.), p. 57. Academic Press, London. Broderick, J. J. 1965. It started with the apple. Amer. Perfume Cosmetics 80: 33. Broderick, J. J. 1974. Has apple research helped?. The Flavor Industry, (July-Aug.), p. 184. Brown, D. S., Buchanan, J. R., and J. R. Hicks. 1966. Volatiles from apple fruits as related to variety, maturity, and ripening. J. Amer. Soc. Hort. Sci. 88: 98. variety, season, maturiy and storage. Proc. Amer. Soc. Hort. Sci. 93: 705. Bulter, L. D., and Burke, M. F. 1976. Chromatographic characterization of porous polymers for use as adsorbents in sampling columns. J. Chromatogr. Sci. 14: 117. Childers, N. F. 1954. Fruit nutrition. Horticultural Publications, Rutgers University, p. 910. Clark, R. G., and Cronin, D. A. 1975. A new techniqe for trapping and sensory evaluation of flavor volatiles. J. Sci. Fd. Agric. 26: 1009. 51 Cronin, D. A. 1974. Some factors affecting the properties of thin films of carbowax 20 M intended for deactivation of glass capillary columns. J. Chromatogr. 148: 379. Cronin, D. 1982. Techniques of analysis of flavours. Chemical methods including sample preparation. In "Food Flavours Part A. Introduction,‘ I. D. Morton and A. J. MacLeod (Ed.), p. 15. Elsevier Scientific Publishing 00., Amsterdam, The Netherlands. De Pooter, H. L., Dirinck, P. J., Willaert, G. A., and Schamp, N. M. 1981. Metabolism of propionic acid by Golden Delicious apples. J. Phytochem. 20: 2135. De Pooter, H. L., Montens, J. P., Dirinck, P. J., Willaert, G. A., and Schamp, N. M. 1982. Ripening induced in pre- climacteric immature Golden Delicious apples by propionic and butyric acids. J. Phytochem. 21: 1015. De Pooter, H. L., Montens, J. P., Willaert, G. A., Dirinck, P. J., and Schamp, N. M. 1983. Treatment of Golden Delicious apples with aldehydes and carboxylic acids: Effect on the headspace composition. J. Agric. Fd. Chem. 31: 813. Dewey, D. H., and Dilley, D. R. 1969. Managing and operating a controlled atmosphere storage for apples. Horticultural Report 10: 1. . Dimick, P. S., Hoskin, J. C., and Acree, T. E. 1983. Review of apple flavor-state of the art. CRC Crit. Rev. Food Sci. Nutr. 18: 387. Dirinck, P. J., Schreyen, L., and Schamp, N. M. 1977 a. Flavor quality of apples and tomatoes. 15th Symp. Int. Comm. Int. Ind. Agric. Aliment., p. 427. Dirinck, P. J., Schreyen, L., and Schamp, N. M. 1977 b. Aroma quality evaluation of tomatoes, apples, and strawberries. J. Agric. Fd. Chem. 25: 759. Dirinck, P. J., De Pooter, H. L., Willaert, G. A., and Schamp, N. M. 1981. Flavor quality of cultivated strawberries: the role of the sulfur compounds. J. Agric. Fd. Chem. 29: 319. Dirinck, P. J., De Pooter, H. L., Willaert, G. A., and Schamp, N. M. 1984a. Application of a dynamic headspace procedure in fruit flavor analysis. In "Analysis of Volatiles. Methods. Applications," P. Schreier (Ed.), p. 381. W. de Gruyter, New York. 52 Dirinck, P. J., Kutom, A. H., and Schamp, N. M. 1984b. Quantitative dynamic headspace analysis for objective mearsurement of rancidity in oil. In "Progress in Flavour Research," J. Adda (Ed.), p. 505. Elsevier Scienctific Publishing Co., Amsterdam, The Netherlands. Drawert, F., RaPP. A., and Bachmann, O. 1962. Gascheromato- graphische untersuchung der aromastoffe und alkohole von fruchten. In "Volatile Fruit Flavours," H. Luthi (Ed.), p. 235. Juris, Zurich, Switzerland. Drawert, F., Heimann, W., Emberger, R., and Tressl, R. 1966. Uber die biogenese von aromastoffen bei pflanzen und fruchten, 2 enzymatische bildung von hexen-Z-al, hexanal und anderen vorstufen. Liebigs Ann. Chemie. 614: 200. Drawert, F., Heimann, W., Emberger, R., and Tressl, R. 1969. Gas chromatographic investigation of vegetable aroma. II. Concentration, separation and identification of apple flavor components. J. Chromatogr. 2: 57. Drawert, F., Heimann, W., Emberger, R., and Tressl, R. 1972 Biogenesis of aromatics in plants and fruits. XIV. Significance of the climacteric conditions for the development of aromatics in apples and bananas. Chem. Mikrobiol. Technol. Lebensm. 1: 201. Drawert, F., and Christoph, N. 1984. Significance of the sniffing technique for the determination of odor thershold and detection of aroma impacts of trace volatile. In "Analysis of Volatiles. Methods. Application," P. Schreier (Ed.), p. 269. W. de Gruyter, New York. Ettre, L. S., Kolb, B., and Hurt, S. G. 1983. Techniques of headspace gas chromatography. J. Amer. Lab. Oct.: 76. Farmer, J. W., Hulme, A., and Burt, J. R. 1973. Efficiency of Likens Nickerson distillation method for carbon compounds up to C11. Chem. Ind., March, p. 279. Feranoli, G. 1973. "Feranoli's Handbook of Flavor Ingredients," T. E. Furia and Bellanca (Ed.). Chemical Rubber Co., Cleveland, Ohio. Feys, M., Tobback, P., and Maes, E. 1980. Volatiles of apples (var. 'Schone VanBoskoop'): isolation and identification. J. Fd. Technol. 15: 485. Filder, J. C. 1958. The Metabolism of acetaldhyde by plant tissue. J. Exp. Bot. 19: 41. 53 Fidler, J. C., and North, C. J. 1969. Production of volatile organic compounds by apples. J. Sci. Fd. Agric. 20: 521. Flath, R. A., Black, D. R., Guadagni, D. G., McFadden, W. H., and Schultz, T. H. 1967. Identification and organoleptic evaluation of compounds in apple essence. J. Agric. Fd. Chem. 15: 29. Flath, R. A., Black, D. R., Forrey, R. R., McDonald G. M., Mon, T. R., and Teranishi, R. 1969. Volatiles in Gravenstein apple essence identified by GC-MS. J. Chromatogr. Sci. 7: 508. Galt, A. A., and MacLeod, G. 1984. Headspace sampling of cooked beef aroma using Tenax-GC. J. Agric. Fd. Chem. 32: 59. Gasco', L., Barrera, R., and de la Cruz, F. 1969. Gas chromato- graphic investigation of the volatile constitutents of fruit aromas. J. Chromatogr. Sci. 7: 226. Gasco', L., and Barrera, R. 1972. The use of derivatives for the gas chromatographic identification of alcohols, primary and secondary amines, and thiols in food aromas. J. Anal. Chem. Acta. 61: 253. Gerhardt, F., and Ezell, B. D. 1939. A method of estimating the volatile products liberated from stored fruit. J. Agric. Res. 58: 493. Goodenough, P. W., and Entwistle, T. 1982. The hydrodynamic properties and kinetic constants with natural substrates of the esterase from Malus pumila fruit. Eur. J. Biochem. 127: 145. Grevers, G., and Doesburg, J. L. 1962. Gas chromatographic determination of some volatiles, emanated by stored apples. In "Volatile Fruit Flavours," H. Luthi (Ed.), p. 319. Juris, Zurich, Switzerland. Grevers, G., and Doesburg, J. J. 1965. Volatiles of apples during storage and ripening. J. Food Sci. 30: 412. Grob, K. J., and Burrus, F. J. 1965. Gas chromatography of cigarette smoke, part III. Separation of the overlap region of gas and particulate phase by capillary columns. J. Gas Chromatogr. 3: 52. 54 Grob, K., and Grob. G. 1969. Splitless injection on capillary columns, Part I-the basic technique; steroid analysis as an example. J. Chromatogr. 7: 584. Guadagni, D. G., Bomben, J. L., and Hudson, J. S. 1971a. Factors influencing the developmemt of aroma in apple peels. J. Sci. Fd. Agric. 22: 110. Guadagni, D. G., Bomben, J. L., and Harris, J. G. 1971b. Recovery and formation of aroma development in apple peel. J. Sci. Fd. Agric. 22: 115. Hatfield, S. G., and Patterson, B. D. 1975. Effect of CA storage on apple aroma. Rep. East Malling Res. Sta., p. 80. Heath, H. B., and Reineccius, G. 1986. Flavor and its study. In "Flavor Chemistry and Technology," p. 3.AVI Publishing Co., Westport, CT. Heins, J. T., Maarse, H., Ten Noever de Brauw, M. C., and Weurman, C. 1966. Direct food vapor analysis and compound identification by a coupled capillary GLC-MS arrangement. J. Gas Chromatogr. 4: 395. Heinze, R. E., Baker, C. E., and Quackenbush, F. W. 1953. The chemical composition of apple storage volatiles. %;acégs, alcohol and esters. Proc. Am. Soc. Hort. Sci. : 7. Heinze, R. E., Baker, C. E., and Quackenbush, F. W. 1954. Carbonyl compounds in apple storage volatiles. J. Agric. Fd. Chem. 2: 1118. Heydanek, M. G., and McGorrin, R. J. 1981 a. Gas chromatography- mass spectroscopy investigation on the flavor chemistry of oat groats. J. Agric. Fd. Chem. 29: 950. Heydanek, M. G., and McGorrin, R. J. 1981 b. Gas chromatographic mass spectroscopy identification of volatiles from rancid oat groats. J. Agric. Fd. Chem. 29: 1093. Huelin, F. E. 1952. Volatile products of apples. III- identification of aldehydes and ketones. Aust. J. Sci. ReSo B. 5: 328. Hulme, A. C., and Rhodes, M. J. 1963. Pome fruits. In "Biochemistry of Fruits and Vegetables," A.C. Hulme (Ed.), p. 85. Academic Press, London. 55 Hulme, A. C., Rhodes, M. J., and Wooltorton, L. S. 1967. The respiration climacteric in apple fruits: some possible regulatory mechanisms. J. Phytochem. 6: 1343. Jennings, W. G., and Nursten, H. E. 1967. Gas chromatographic analysis of diluted aqueous systems. Anal. Chem. 39: 521. Jennings, W. G., Wohleb, R., and Lewis, M. J. 1972. Gas chromatographic analysis of headspace volatiles of alcoholic beverages. J. Food Sci. 37: 69. Jennings, W. G. and Tressl, R. 1974. Production of volatile compounds in the ripening Bartlett pear. Chem. Microbiol. Technol. Lebensm. 3: 52. Jennings, W. G. and Filsoof, M. 1977. Comparison of sample preparation techniques for gas chromatographic analysis. J. Agric. Fd. Chem. 25: 440. Jennings, W. G. and Takeoka, G. 1984. State of the art fused silica capillary gas chromatography: flavour problem applications. In " Analysis 0 Volatiles. Methods. Applications," P. Schreier (Ed.), p. 64. W. de Gruyter, New York. Kidd, F., and West, C. 1939. The Production of volatiles by apples: Effects of temperature, maturity, technique of estimation, etc. Rept. Food Invest. Bd.(Gt. Brit.), p. 136. Kieser, M. E., and Pollard, A. 1962. The examination of some fruit juice volatiles by use of the flame ionization detector. In "Volatile Fruit Flavours," H. Luthi (Ed.), p. 249. Juris, Zurich, Switzerland. Knee, M., and Hatfield, S. G. 1976. A comparison of methods for measuring the volatile components in apple fruits. J. Fd. Technol. 11: 485. Knee, M., and Hatfield, S. G. 1981. The metabolism of alcohols by apple fruit tissue. J. Sci. Fd. Agric. 32: 593. Koch, J., and Schiller, H. 1964. Beitrag zur kenntnis des apfelaromas. Z. Lebensm. Unters. Forsch. 125: 364. Kuo, P. P., Chain, E. S., De Walle, F. B., and Kim, J. H. 1977. Gas stripping, sorption, and thermal desorption procedures for preconcentrating volatile polar water- soluble organics from water samples for analysis by gas chromatography. J. Anal. Chem. 49: 1023. 56 Land, D. G. 1975. Techniqes for assesing odour: uses and limitations. Proc. Int. Symp. Aroma Research. Pudoc., Wageningen, The Netherlands. Land, D. G. 1984. Flavour research in 1984- where now and where next? In "Progress in Flavour Research ," J. Adda (Ed.), p. 615. Elsevier Science Publishers B.V., Amsterdam. Legendre, M. G., Fisher, G. S., Fuller, W. H., Dupuy, H. P., and Rayner, E. T. 1979. Novel technique for the analysis of volatiles in aqueous and non aqueous systems. J. Amer. Oil Chem. Soc. 56: 552. Levin, R. J., and Ikeda, R. M. 1968. The separation of volatiles from solids in the injection port of a gas chromatograph. J. Gas Chromatogr. 6: 331. Lewis, M. J., and Williams, A. A. 1980. Potential artifacts from using porous polymers for collecting aroma compounds. Jo SCio Fd. AgtiCo 31: 10170 Lidster, P. D., Ligthfoot, H. J., and McRae, K. B. 1983. Production and generation of principal volatiles in apples stored in modified atmosphere and air. J. Food Sci. 48: 400. Likens, S. T., and Nickerson, G. B. 1964. Detection of certain hop oil constituents in brewing products. Proc. Amer. Soc. Brew. Chem. 5: 13. Little, C. R., Faragher, J. D., and Taylor, H. J. 1982. Effects of initial oxygen stress treatments in low oxygen modified atmosphere storage of 'Granny Smith' apples. J. Amer. Soc. Hort. Sci. 107(2): 320. Maarse, H. 1971. Samenstelling van de vluchtige olie van onganum vulgane 1. ssp. vulgane gedurende de ontwikkeling van de plant. Ph.D. thesis, University of Groningen, The Netherlands. MacLeod, A. J., and Cave, S. J. 1975. Volatile flavor components of egg. J. Sci. Fd. Agric. 26: 351. MacLeod, G., and Ames, J. 1986. Comparative assesment of the artefact background on thermal desorption of Tenax-GC and Tenax-TA. J. Chromatogr. 355: 393. Martin, J. M. 1969. Gas chromatographic qualitative and semiqualitative analysis of apple aroma by means of retention indexes. J. Anal. Chem. Acta. 48: 169. 57 Matthews, J. S., Sugisawa, H., and McGregor, D. R. 1962. The flavor spectrum of apple-wine volatiles. J. Food Sci. 27: 355. McEwen, D. 1964. Backflushing and two-stage operation of capillary columns in gas chromatography. J. Anal. Chem. 36(2): 279. McGregor, D. R., Sugisawa, H., and Matthews, J. S. 1964. Apple juice volatiles. J. Food Sci. 29: 448. McNally, M. E., and Grob, R. L. 1985. Current application of static and dynamic headspace analysis. A review. Part II: nonenviromental applications. J. Amer. Lab. Feb: 106. Mehlitz, A., and Gierschner, K. 1962. Weitere untersuchungen uber aromakonzentrate aus fruchtssaften. In "Volatile Fruit Flavours," H. Luthi (Ed.), p. 301. Juris, Zurich, Switzerland. Meigh, D. F. 1956. Volatile compounds produced by apples. I- Aldehydes and ketones. J. Sci. Fd. Agric. 7: 396. Meigh, D. F. 1957. Volatile compounds produced by apples. II- Alcohols and esters. J. Sci. Fd. Agric. 8: 313. Meigh, D. F. 1964. The natural coating of the apple and its influence on scald in storage. I-Fatty acid and hydrocarbons. J. Sci. Fd. Agric. 15: 436. Merritt, C., Bresnick, S. R., Basinet, M. L., Walsh, J. T., and Angelini, P. 1959. Determination of volatile components of foodstuffs. Techniques and their application to studies of irradiated beef. J. Agric. Fd. Chem. 7: 784. Merritt, C., Walsh, J. T., Fross, D. A., Angelini, P., and Swift, S. M. 1964. Wide range programmed temperature gas chromatography in the separation of very complex mixtures. J. Anal. Chem. 36: 1502. Merritt, C., and Brobertson, D. H. 1982. Techniques of analysis of flavours. gas chromatography and mass spectrometery. In "Food Flavours. Part A:Introduction," I. D. Morton and A. J. MacLeod (Ed.), p. 49. Elsevier Scientific Publising Co., Amsterdam, The Netherlands. Michigan Department of Agriculture, 1987. Michigan agricultural statistics 1987. 58 Milleville, H. P., and Eskew, R. K. 1944. Apple flavor. Wasted essence is recoverable; has commercial possibilities. J. Fd. Packer. Nov., p.33. Myers, M. J., Issenberg, P., and Wick, E. L. 1970. L-Leucine as precursor of isoamyl alcohol and isoamyl acetate. Volatile aroma consitutents of banana fruit discs. J. Phytochem. 9: 1693. Nawar, W. W. 1966. Some consideration in interpretation of direct headspace gas chromatographic analyses of food volatiles. J. Fd. Technol. Feb : 115. Neubeller, J., Buchloh, G., and Dhuria, H. S. 1979. Behavior of volatile substances and lipids of apples during cold storage. Mitt. Klosterneuberg. 8: 227. Novotny, M., McConnell, M. L., and Lee, M. L. 1974. Some aspects of high resolution gas chromatographic analysis of complex volatile samples. J. Agric. Fd. Chem. 22(5): 765. Nursten, H. E. 1970. Volatile compounds: the aroma of fruits. In "The Biochemsitry of Fruits and their Products," A.C. Hulme (Ed.), p. 239. Academic Press, London. Nursten, H. E. and Woolfe, M. L. 1972. An examination of the volatile compounds present in cooked Bramley's seedling apples and the changes they undergo on processing. J. Sci. Fd. Agric. 23: 803. Paillard, N. 1965. Analyse des produits emis par les pommes. Fruits 20: 189. Paillard, N. 1978. Biosynthesis of apple volatiles: comparison of behaviour of different apple varieties. Int. Fed. of Fruit Juice Producers. 15th Symposium: Flavours of Fruits and Fruit Juices, p. 25. Paillard, N. 1979. Biosynthese des produits volatiles de la pomme: formation des alcools et des esters a partir des acid gras. J. Phytochem. 18: 1138. Paillard, N. 1981. Factors influencing flavor formation in fruits. In "Flavor' 81," P. Scheier (Ed.), p. 479. W. de Gruyter Publishing Co., Berlin. Patterson, B. D., Hatfield, S. G., and Knee, M. 1974. Residual effects of controlled atmosphere storage on the production of volatile compounds by two varieties of apples. J. Sci. Fd. Agric. 25: 843. 59 Peredi, K., Vamos-Vigyazo, L., Kiss-Krutz, N. 1981. Flavour losses in apple juice manufacture. Die Nahrung 25: 573. Power, F. B., and Chesnut, V. K. 1920. The odor constituents of apples. Emanation of acetaldehyde from ripe fruit. J. Amer. Chem. Soc. 42: 1509. Power, F. B., and Chesnut, V. K. 1922. The odorous constituents of apples. Evidence of the presence of geraniol. J. Amer. Chem. Soc. 44: 2938. Olafsdottir, G., Steinke, J. A., and Lindsay, R. C. 1985. Quantitative performance of a simple Tenax-GC adsorption method for use in the analysis of aroma volatiles. J. Food. Sci. 50: 1431. Reineccius, G. 1983. Determination of flavor components. Presented at 43th Annual Meeting Inst. of Food Technologists, New Orleans, LA. Romani, R. J., Lilly, L., and Ku, . 1966. Direct gas chromato- graphic analysis of volatiles produced by ripening pears. J. Food. Sci. 31: 558. Rushneck, D. R. 1965. Cryogenic injection and chromatographic separation of cigarette smoke. J. Gas Chromatogr. 3: 318. Sapers, G. M. 1977. Volatile composition of McIntosh apple juice as a function of maturity and ripeness indices. J. Food. Sci. 42: 44. Schaefer, G. 1981. Comparison of adsorbents in head space sampling. In " Flavour'81," P. Schreier (Ed.), p. 301. W. de Gruyter Publising Co., Berlin. Schlutz, W. G. 1966. Liquid C02 for selective aroma exteraction of flavor. Presented at the 26th Annual Meeting of Inst. of Food Technologists, Portland, Oregon, May 1966. Schreier, P. Drawert, F. and, Schmid, M. 1978. Changes in the composition of neutral volatile components during the production of apple brandy. J. Sci. Fd. Agric. 29: 728. Schultz, T. H., Flath, R. A., Black, D. R., Guadagni, D. G., Schlutz, W. G., and Teranishi, R. 1967. Volatiles from Delicious apples essence-extraction methods. J. Food. Sci. 32: 279. 60 Schreyen, L., Dirinck, P., Van Wassenhove, F. and, Schamp, N. 1976. Analysis of leek volatiles by head space condensation. J. Agric. Fd. Chem. 24: 1147. Shibamoto, T. 1984. Application of high resolution capillary columns on flavor and fragrance analysis. In 'Analysis of Volatiles. Methods. Applications," P. Schreier (Ed.), p. 233. W. de Gruyter Publishing Co., Berlin. Simpson, R. F. 1979. Influence of gas volume sampled on wine headspace analysis using preconcentration on Chromosorb 105. J. Chromatogr. 12: 733. Simon, P. W., Lindsay, R. C., and Peterson, C. E. 1980. Analysis of carrot volatiles on porous poylmer traps. J. Agric. Fd. Chem. 28:549. 'V/Smock, R. M., and Neubert, A. M. 1950. "Apples and Apple _ Products". Interscience Publisher, New York. / V'Somogyi, L. P., Childers, N. F., and Chang, S. S. 1964. Volatile constituents of apple fruits as influenced by fertilizer treatments. Proc. Amer. Soc. Hort. Sci. 84: 51. Stevens, K. L., Bomben, J., Lee, A., and McFedden W. H. 1966. Volatiles from grapes, Muscat of Alexandria. J. Agric. Fd. Chem. 14: 249. Sugisawa, H., McGregor, D. R., and Mathews, J. S. 1962. Apple juice volatiles. Int. Fruchtsaft Union Ber. Wiss. Tech. Komm. 4: 351. Sugisawa, H. 1981. Sample preparation: isolation and concentration. In "Flavor Research. Recent Advances," R. Teranishi, R. A. Flath and H. Sugisawa (Ed. ), p. 15 Marcel Dekker, New York. Sydor, R., and Pietrzyk, D. J. 1978. Comparison of porous copolymers and related adsorbents for the stripping of low molecular weight compounds from a flowing air stream. Anal. Chem. 50: 1842. Takeoka, G., Ebeller, S., and Jennings, W. G. 1985. Capillary gas chromatographic analysis of volatile compounds. J. Amer. Chem. Soc. 104: 95. Tang, C. S., and Jennings, W. G. 1967. Volatile components of apricot. J. Agric. Fd. Chem. 15: 24. 61 Teranishi, R., and Buttery, R. 1962. Aromagrams-direct vapor analysis with gas chromatography. Int. Fruchtsaft Union Ber. Wiss. Tech. Komm. 4: 257. Teranishi, R., and Mon, T. R. 1977. Large bore capillary column and low pressure drop packed column. J. Anal. Chem. 36: 1490. Thampson, A. R. 1951. Volatile products of apples. 1. Indentification of acids and alcohols. Aust. J. Sci. Res. B. 4: 283. Thampson, A. R. and Huelin, F. E. 1951. Volatile products of apples. II. Production of volatile esters of Granny Smith apples. Aust. J. Sci. Res. 4: 544. Tressl, R., Drawert, F., Heimann, W., and Embergerr, R. 1970. Uber die biogenesis von aromastoffen bei pflanzen und fruchten. VIII. Zur biogenese der im bananen gefundenen aromastoffe. Z. Lebensm. Unters. Forsch. 144: 4. Tressl, R. and Drawert, F. 1973. Biogenesis of banana volatiles. J. Agric. Fd. Chem. 21: 560. Tressl, R., Holzer, M., and Apetz, M. 1975. Biogenesis of volatiles in fruit and vegetables. In "Aroma Research," H. Maarse and P. J. Groenen (Ed.). Proc. Int. Symp. Aroma Res. Pudoc., Wageningen, Netherlands. Turk, A., Smock, R. M., and Taylor, T. I. 1951. Mass and infrared spectra of apple vapor. J. Fd. Technol. 5: 58. Wade, N. L., and Bishop, D. G. 1978. Changes in the lipid composition of ripening banana fruits and evidence for an associated increase in cell membrane permeability. B. B. A. 529(3): 454. Walls, L. P. 1942. The Nature of the volatile products from apples. J. Pomol. Hort. Sci. 20: 59. . Wenzel, F. W. 1962. The aroma and the flavor of florida orange juice and concentrate. In "Volatile Fruit Flavours," H. Luthi (Ed.), p. 205. Juris, Zurich Switzerland. Westendrof, R. G. 1984. Trace analysis of volatile organic compounds in food by dynamic headspace gas chromatography. Presented at 35th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Atlantic City, N. J., March 1984. 62 Weurman, C. 1961. Gas-liquid chromatographic studies on the enzymatic formation of volatile compounds in raspberries. J. Fd. Technol. 15: 531. Weurman, C. 1969. Isolation and concentration of volatiles in food odor research. J. Agric. Fd. Chem. 17: 370. White, J. W. 1950. Composition of a volatiles fraction of apples. Food Res. 15: 68. Williams, A. A., Tuchnott, O. G., and Lewis, M. J. 1977. 4- methoxyally benzene: an important aroma component of apples. J. Sci. Fd. Agric. 28: 185. Williams, A. A., and Knee, M. 1977. The flavor of Cox's Orange Pippin apples and its variation with storage. Ann. Appl. Biol. 87: 127. Williams, A. A., and Tuchnott, O. G. 1978. The volatile aroma components of fermented ciders: minor natural components from the fermentation of sweet coppin apple juice. J. Sci. Fd. Agric. 29: 381. Williams, A. A., Lewis, M. J., and Tucknott, O. G. 1980. The neutral volatile components of cider apple juices. J. Food. Chem. 6: 139. Willis, D. E. 1968. Trapping techniques for open tubular chromotographic columns.J. Anal. Chem. 40(10): 1597. Wyllie, S. G., Alves. M., Filsoof, M., and Jennings, W. G. 1978. Headspace sampling: uses and absues. In "Analysis of Food and Beverages. Headspace Techniques," G. Charalmbouse (Ed.), p. 1. Academic Press, New York. Yamashita, I., Nemoto, Y., and Yoshikawa, S. 1975. Formation of volatile esters in strawberries. J. Agric. Biol. Chem. 29(12): 2303. \/ Yamashita, I., Nemoto, Y., and Yoshikawa, S. 1976. Formation of volatile alcohols and esters from aldehydes in straw- berries. J. Phytochem. 15: 1633. \\/ Yamashita, I., Nemoto, Y., and Yoshikawa, S. 1977. Studies on flavor development in strawberries. 4. Biosynthsis of volatile alcohol and esters from aldehyde during ripening. J. Agric. Fd. Chem. 25: 1165. Zlakis, A., Lichtenstein, H. A., and Tishbee, A. 1973. Solid adsorbent. J. Chromatogr. 6: 67. CHAPTER 1 EFFECT OF MATURITY AT HARVEST, STORAGE CONDITIONS AND POST HARVEST PHYSIOLOGY ON THE QUALITY OF EMPIRE APPLES 63 64 INTRODUCTION Increased awareness of the role of ethylene in the ripening of apples during controlled atmosphere (CA) storage has led to the development and implementation of means to attenuate the synthesis or action of ethylene (Bangerth et al., 1984). Ethylene is the fruit ripening hormone (Burg and Burg, 1965 , 1969) and is responsible for initiation of the ripening capacity of apples during development on the tree and during storage. Much apple storage research has been conducted in recent years employing low 02 levels (Dilley, 1962; Knee, 1980; Lidster et al., 1980; Lidster, 1982), rapid controlled atmosphere (Lau, 1982), and ethylene scrubbing (Blanpied, 1985). Oxygen is required for the synthesis and action of ethylene, so it is important to quickly lower the oxygen level in storage to the safest minimum level (2%) (Burg and Burg, 1965; Hulme et al., 1971; Lidster et al., 1980). Carbon dioxide is a natural competitive inhibitor of ethylene action and should be kept at the highest safe level (3-5%). Since ethylene synthesis and action are temperature dependent, and ethylene production is autocatalytic, it is generally recognized that for long-term storage apples should be harvested and placed under CA while they are still at the preclimacteric stage, and cooled as soon as possible to lower 65 the respiration rate and ethylene synthesis (Bangerth et al., 1984). Therefore, during CA storage the ethylene level within the fruit should be kept below 1 ul/l (Liu, 1978; Blanpied et al., 1982; Lange and Pics, 1984) to effectively delay ripening. Change in flesh firmness is a major post-harvest criterion for "ripening" of apples and many other fruits. The softening process is associated with other physiological and chemical changes, such as an increase in the activity of cell wall-degrading enzymes (Dilley, 1966, 1972; Knee, 1975) which in turn might result from ethylene-induced enzyme synthesis (Hulme, 1954; Frankel et al., 1968). The decrease in flesh firmness of apples is correlated with an increase in the content of polyuronide water soluble polyuronide (Dilley and Klein, 1969; Knee, 1973; Bartley, 1974). The main objective of this study was to determine the effect of maturity at harvest, storage conditions and ripening on the quality of Empire apples. MATERIALS and METHODS Empire apples were grown in the Horticulture Research Center (HRC) at Michigan State University in East Lansing, Michigan. Maturity of fruit at harvest was assessed by determining the development of the autogenous ethylene climacteric and the onset of the induced ethylene climacteric 66 for three, 10-fruit samples at weekly intervals during the 1984 harvest season. The level of ethylene in the internal atmosphere of the fruit during development on the tree was also measured. Ethylene was determined with a Varian series 1700 gas chromatograph equipped with a 1 m long by 2 mm wide column of activated alumina, and a flame ionization detector. Nitrogen gas was used as the carrier gas. Figure 1 illustrates how harvest was predicted based on internal ethylene concentration and also the time required for apples to reach 0.1 ul/l ethylene. Apples were harvested on Oct.2 and Oct. 9, 1984 as indicated in Figure 1. Table 1 illustrates the maturity parameters for apples harvested on Oct. 2 and 9. Harvested fruits were randomly seperated into 20 liter plastic containers and stored at 3.3 °C. After cooling for one day, the containers were sealed with covers fitted with inlet and outlet tubes and ventilated with a gas mixture prepared from liquified N2 , C02 , and air to provide 3% C02 and 2% 02. The gas mixture was humidified and brought to dew point at 3.3 °C. The gas mixture was then distributed to the containers via a calibrated capillary flow rate control system. High (>100 PPM) and low (<1 PPM) ethylene levels were established by ventilating half the containers at 30 ml/min (high ethylene level) and half at 300 ml/min (low ethylene level). Additional fruits were maintained in air at 3.3 °C. 67 Figure 1- Harvest prediction based on the hours to ethylene climacteric and internal ethylene. 68 internal ethylene (ppm) hours to ethylene climacteric 026- 3 l—) l---) {-170 021- ~14S 0.16- 420 0.11- ~95 0.06- -70 0.01- —45 cl“? . . r , . 9.4-0 69 Table 1. Maturity parameters of Empire apples harvested in October, 1984. Harvest Date October 2 October 9 Flesh firmness(lbs.-force) 18.0:1.4 17.8:1.7 Starch index * 3.7 4.5 Internal ethylene (ul/l) Median 0.097 0.226 Range 0.038-0.158 0.016-2.56 Mean 0.095 0.96 Malic enzyme activity 3.8 3.6 (units/s.d.wt) *Starch Index of Ontario Ministry of Agriculture: 1-immature, 9-overmature. 70 Fruits were examined monthly over a five-month period of storage. Flesh Firmness: Flesh firmness was determined with an Effegi penetrometer with an 11 mm diameter tip mounted in a drill press. Two determinations were made per fruit after removing the epidermis. The same fruits were used for determination of starch index (Priest and Lougheed, 1981). Sample Preparation: Freeze dried samples were prepared every month for five months after 0, 7 and 14 days of subsequent ripening at 22°C for measurement of 1-amino-cyclopropane-1- carboxylic acid (ACC) content (Lizada and Yang, 1979), protein content, malic enzyme activity (ME), and water-soluble polyuronide content (WSP) (Irwin, 1981). One gram of freeze dried tissue was extracted by mortar and pestal, mixed with 20 ml of a liquid medium consisting of: 100 mM Tricine-KOH, 4 mM mercaptoethanol, 5 uM pyridoxal phosphate, 50 mM magnesium chloride, and 0.1% v/v Triton X-100. The pH of the extraction medium was 8.35. The suspension was centrifuged at 10,000 x G for 15 minutes, and the supernatant solution was used to measure ACC content, protein and malic enzyme (ME) activity. 1-Amino-cyclopropane-1-carboxylic Acid (ACC) Assay: ACC content in freeze-dried apple tissues was determined according to the method of Lizada and Yang (1979). This method is based on the principle that ACC is converted to ethylene by 71 NaOCl in the presence of Hg (Bangerth et al., 1984). Protein Determination: Protein in apple tissues was determined by the Lowery method (Lowery et al., 1951) and the Bio-Rad protein assay dye agent (1985) according to Bradford (1976). Malic Enzyme Assay: The assay was performed spectrophoto- metrically at 340 nm according to Klein and Dilley (1973). One ME unit is defined as the amount of enzyme causing 0.01 absorbance change per minute (Ochoa et al., 1948). Water-Soluble Polyuronide (WSP) Assay: The pellet from the enzyme extract was suspended with 95% ethanol and centrifuged at 1000 x G for 15 min. This was repeated after the second alcohol extraction. The pellet was extracted with 25 ml boiling water, centrifuged and the supernatant solution was used for WSP determination. Water-soluble polyuronides in freeze dried apple tissue were determined according to Kosh and Hess (1965). Absorbance of samples was measured at 525 nm employing a calibration curve using galacturonic acid in the range of 10-90 ug/l. RESULTS and DISCUSSION The most significant benefit of low ethylene CA storage of Empire apples was the effective control of fruit softening. Flesh firmness was evaluated at three stages: 0, 7, and 14- 72 days after apples were transferred from the storage atmosphere to 22 °C air every month for a duration of five months (Table 2). Ethylene production capacity is a good parameter to assess maturity, ripening and storability of apples (Frenkel et al., 1968). Table 1 illustrates that the internal ethylene at harvest on Oct. 2 ranged from 0.04 to 0.16 ul/l and fruits were judged to be preclimacteric and suitable for long term storage. Fruits sampled on Oct. 9 had begun to ripen and were judged to be suitable for short term CA storage. After five months of storage at low and high ethylene CA, the internal ethylene level was nearly at the initial value for zero day holding period at 22°C. Early harvested fruits were firmer than late harvested ones (Figure 2; Table 1). Liu (1978) reported that after five months of storage and a 1-day holding period at 21°C , early harvested McIntosh apples were firmer and had higher soluble solids and acidity than late-harvested fruits. In this study, apples held at 22°C for 7 days or 14 days lost flesh firmness during the ripening period. The firmer the apples immediately after storage, the greater the firmness loss during the subsequent 7-days holding period at 22°C for the apples harvested on Oct. 2. However, 0- day and 7 days holding periods after three months of storage did not affect the eating quality of apples harvested on Oct. 9 (Figure 2). 73 TABLE 2. Effect of storage duration and conditions followed by ripening at 22°C on flesh firmness ** of Empire apples. Post-Storage Harvest Storage Holding Duration 22 C (Months) (days) October 2 October 9 Storage condition * Air LCA HCA 731! LCA HCA 0 16.2 19.8 18.7 15.2 18.0 18.4 7 13.4 16.4 17.4 12.8 16.0 14.6 14 12.7 13.7 13.8 12.0 13.1 13.4 0 1204 1804 1803 1.2.5 16o7 17o6 7 11.7 12.9 13.9 12.4 16.5 14.1 14 11.5 12.3 12.5 11.5 12.1 12.7 0 11.4 17.9 17.9 15.0 18.9 18.9 7 11.3 13.5 13.2 12.3 16.6 15.9 14 9.8 11.8 11.1 10.5 12.6 11.5 0 11.3 16.6 17.7 11.5 16.3 14.9 7 10.5 13.9 12.2 10.1 12.8 12.6 14 9.5 12.3 11.7 8.8 11.2 10.9 0 10.4 16.6 15.6 10.8 14.2 14.4 7 11.0 13.1 12.1 9.9 13.2 12.3 14 10.3 11.7 10.7 9.5 11.4 11.0 * CA conditions were 2% 02, 3% C02 at 3.3°C. 20 L chambers with 60 fruits were ventilated at 30 or 300 ml/min. for high (HCA) and low (LCA) ethylene levels, respectively. ** Flesh firmness at harvest was 18.0 (lbs.-force) and 17.8 on Oct. 2 and Oct. 9, respectively. 74 Figure 2- Changes in flesh firmness of Empire apples following CA storage for 5 months. Storage conditions were: 3.330, 3 z coz, 2 z 02 in low (1(PPM) and high (>1PPM) ethylene, and air. Followed by ripening for O-day, 7-days and 14-days at 22°C. 2:3; mmm 2cm 2 3 AW... 3. 2.- s- ,3- /oao< -------------- 2... :1 3.. 8.. m a. w a. M . D m ----- - q - u N +aq4.: 76 Fruits stored in air for five months lost their flesh firmness very quickly. Late-harvested apples were softer after five months storage for all treatments with a holding period of 7 and 14 days. Apples harvested on both Oct. 2 and Oct. 9 and stored either in low or high ethylene CA were less ripe than fruits stored in air. Liu (1977) reported that ethylene removal from CA fre- quently, but not always, resulted in greater flesh firmness retention in different apple cultivars like McIntosh. However, these data indicate that removal of ethylene from storage, particularly for the second harvest, did not retain flesh firmness during five months of storage. This is because ethylene removal has its maximum effect when fruits are picked in a preclimacteric state. CA storage (3.3‘b, 2% 02, 3% C02) suppressed the rate of protein accumulation. Apples ripened upon removal from storage showed a high net protein synthesis. Throughout the post- storage holding period, the protein content of both first and second harvested apples increased sharply but this increase did not correlate with delayed holding times. Apples stored in air, however, had a higher rate of protein synthesis than those stored either in low or high ethylene CA (Figure 3). The ACC content of apples was very low when measured immediately after harvesting for both harvests. The ACC content remained at a low and constant level in both low and 77 Figure 3- Protein content of Empire apples. For storage conditions, refer to Figure, 2. ”m2 ah. I. o- W“. v. .3 E F a v. ”H oAclInll \ \ allla\ / 11* \\w .-\.I 1.1/IA \.\ \ \.\\ .- A .l l. .. \ \ w ...... a . . . o- w n o \ M- .\/ . a .\ /% . ”H. m\my L m... a\\ \ .\.o\.m/. w _ . . . . . . . o —-b N u.) p U'I —-§ N Lu a U1 ‘- N- w- p- m §+aa25=§:33 79 high ethylene CA for zero day holding time at 22°C. Apples stored in air at 3.3°C accummulated ACC in a linear manner from the beginning of the storage period. During ripening at 22°C for 7 and 14 days, there was a climacteric rise in the ACC content for all treatments. A higher ACC value was observed for the second harvest. Apples harvested on Oct. 2 and stored in air at 3.3°C showed a steady increase in ACC content during the first three months of storage, then reached the maximum level, and subsequently stayed constant for the remaining period. However, for the second harvest the ACC content after two months of storage continued to increase for the remaining period of storage (Figure 4). Mansour et al. (1986) examined the internal ethylene content and the ACC content from different tissues of Golden Delicious apples. They concluded that the internal ethylene content increased for 20 days at 24°C to reach a maximum level, but the ACC content of all the tissues continued to rise until 32 days and fell thereafter. Flesh softening is a major criterion for 'ripening' of many fruits. The softening process is generally attributed to an increase in the activity of cell wall-degrading enzymes presumably resulting from ethylene-stimulated protein synthesis. The dominant protein in pome fruits which increases in activity during the ripening process (Dilley, 1962; Hulme 80 Figure 4- Changes in production of 1-amino-cyclopropane-1- carboxylic acid (ACC) content in Empire apples. For storage conditions, refer to Figure, 2. :3 >93 2. N8 a a3. a SE .3 3% > Am? 8o. . Al/NIKXL q J11 - q q - ll- 1 a a a? \ 3o- mo. + a\ all 0 H/QVGIHII \IRO\+ *Awww ”DNA £28m 23:8 .5832 L. . c a N A r. 82 and Woolterton, 1962) is malic enzyme (Frenkel et al., 1968). The synthesis of malic enzyme takes place during the early- climacteric stage prior to any marked physical changes in the fruit tissue. Hulme and Woolterton (1962) suggested that the climacteric rise in whole fruit might be caused by an increase in malic enzyme activity. Specific activity of malic enzyme was observed to increase in post climacteric fruits (Dilley, 1966). In this study, malic enzyme activity in freeze-dried powders of Empire apples showed the same trend as the protein content of apples under all storage conditions (Figure 5). WSP residues increased in concentration as apples softened with ripening. This is due to cell wall degrading enzymes which results in loss of flesh firmness (Bartley, 1974). The WSP content for first harvested apples and no post storage holding period increased for both low and high ethylene CA. This rise was higher for those apples stored in high ethylene CA compared to low ethylene CA which correlated with flesh firmness. Apples stored in air had a sharp increase in WSP level after one month, then declined and stabilized after the second month. The same pattern was observed with flesh firmness which had a sharp decline until the second month and remained unchanged thereafter (Figure 6). Chen and Borgie (1985) reported that pear fruits stored in air for four or five months showed little or no change in WSP level during ripening. They suggested that there 83 Figure 5- Malic enzyme activity of Empire apples. For storage conditions, refer to Figure, 2. M 88 3m 5: \ a mg. 38 , o a8. be Q83 F an? > / use- . \. allo/ \L 88. a\a \ \w/ . mulli/ m\. 38..\ .[\ Tall. o 4 q # q uuuuuuuuu .58. as. .\ _. al.. .. MMUVRWKIWMVWN o l. N. W M. o. l. M W M. m l. w w M. m «825m 9.328 583...... 85 Figure 6- Water-soluble content polyuronide (WSP) of Empire apples. For storage conditions, refer to Figure, 2. ooooo ssssss 87 was either no further release of polyuronides from the cell wall or that the release of polyuronides from the cell wall had reached an equilibrium with the degradation of WSP into the uronic acid monomer. The data for both harvests suggests such an explanation. However, more research should be done in this area with regard to the measurement of WSP degradation process and/or uronic acid determination. The WSP level in fruits harvested on Oct. 9 and stored for five months increased up to the second month and then declined. Post storage holding for 7 and 14 days did not affect the WSP level substantially. In conculsion, there are a few points one should bear in mind with regard to storage of Empire apples. The harvest date is a crucial factor influencing the holding quality of apples in any kind of storage enviroment. Those apples harvested before the onset of the climactric rise in ethylene production (Oct. 2) had very good storage quality. The commercial application of low ethylene CA storage for Empire apples is feasible. Low ethylene CA storage has very significant benefits for Empire apples. Because of low internal ethylene at harvest and the constant removal of ethylene from the storage, low ethylene CA is very feasible. However, the major disadvantage of employing low ethylene CA is the slowness of producing normal non-ethylene volatiles upon removal of apples from storage (Patterson et al., 1974; Dilley at al., 1982). 88 REFERENCES Bangerth, F., Bufler, G. and, Halder-Doll, H. 1984. Experi- ments prevent ethylene biosynthesis and/or action and effects of exogenous ethylene on ripening and storage of apple fruits. In "Ethylene: Biochemical, Physiological, and Applied Aspects," Y. Fuchs and E. Chalutz (Ed.), p. 291. Martinus Nijhoff/Dr. W. Junk, The Hague. Bartley, J. M. 1974. Beta-galactosidase activity in ripening apples. J. Phytochem. 13: 2102. Bio-Rad. 1985. Bio-Rad protein assay instruction manual. Blanpied, G. D., Turk, J. R., and Dougles, J. B. 1982. Low ethylene CA storage for apples. In "Controlled Atmosphere for Storage and Transport of Perishable Agricultural Commodities," D.G. Richardson and M. Meheriuk (Ed.), p. 337. Timber Press, Beaverton, OR. Blanpied, G. D. 1985. Low ethylene CA storage for "Empire" apples. Horticulture report No. 126. Proceedings of the 4th National Controlled Atmosphere Research Conference, July 23-26, p. 95. Bradford, M. M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing tge principle of the protein-dye binding. Anal. Biochem. 7 : 48. Burg, S. P., and Burg, E. A. 1965. Ethylene action and the ripening of fruits. Science 148: 1190. Burg, S. P., and Burg, E. A. 1969. Interaction of ethylene, oxygen and carbon dioxide in the control of fruit ripening. Qual. Plant. Mater. Veg. XlX 1-3; 185. Chen, P. W., and Borgie, D. M. 1985. Changes in water soluble polyuronides in the pulp tissue of ripening 'Bosc' pears following cold storage in air or in 1% oxygen. J. Amer. Hort. Sci. 110(5): 667. Dilley, D. R., 1962. Malic enzyme activity in apple fruits. Nature 196: 387. 89 Dilley, D. R., 1966. Enzymes. In "The Biochemistry of Fruits and Their Products," A.C. Hulme (Ed.), p. 195. Academic Press, London. Dilley, D. R., and Klein, I. 1969. Protein synthesis in relation to fruit ripening. Qual. Plant. Mater. Veg. 29: 55. Dilley, D. R. 1972. Post harvest fruit preservation: protein synthesis, ripening, and senescence. J. Food Sci. 37: 518. Dilley, D. R., Irwin, P. L., and McKee, M. W. 1982. Low oxygen, hypobaric storage and ethylene scrubbing. In Controlled Atmospheres for Storage and Transport of Perishable Agricultural Commodities," D. G. Richardson and M. Meheriuk (Ed.), p. 327. Timber Press, Beaverton, OR. Frenkel, C., Klein, 1., and Dilley, D. R. 1968. Protein syn- thesis in relation to ripening to pome fruits. Plant. Physiol. 43: 1146. Hulme, A. C. 1954. The climacteric rise in respiration in relation to changes in the equilibrium between protein synthesis and breakdown. J. Expt. Bot. 5: 159. Hulme, A. C., and Woolterton, L. S. 1962. Separation of the enzyme present in the mitocondrial fraction from apple peel. Nature 196: 388. Hulme, A. C., Rhodes, M. T., and Wooltorton, L. S. 1971. The effect of ethylene on the respiration rate, ethylene production, RNA and protein synthesis for apples stored in low oxygen and in air. J. Phytochem. 10: 1315. Irwin, P. L. 1981. Factors affecting effusivity and ripening behavior of 'Empire' apples-fruits. Ph.D. thesis, Michigan State University, East Lansing, MI. Klein, 1., and Dilley, D. R. 1973. Malic enzyme isoenzyme in pome fruits. FEBS LETT. 29: 305. Knee, M. 1973. Polysaccharide changes in cell walls of ripening apples. J. Phytochem. 12: 1543. Knee, M. 1975. Changes in structural polysaccharides of apples ripening during storage. Facteurs et regulation de la maturation des fruits. Colloq. Intern., CNRS 238: 241. 90 Knee, M. 1980. Physiological responses of apple fruits to oxygen concentrations. Ann. Appl. Biol. 96: 243. Koch, J, and Hess, D. 1965. Ein beitrag zur peklinbestimmung in "fruchtsaften". Z. Lebensm. Unters. Forsch. 126:_25. Lange, E., and Fica, J. 1984. The storage response of Golden and Jonathan applles to ethylene removal from controlled atmosphere and prestorage stort term high COZ treatment. Fruit Sci. 11: 159. Lau, O. L. 1982. The use of rapid CA to maximize storage life of apples. In "Controlled Atmospheres for Storage and Transport of Perishable Agricultural Commodities," D. G. Richardson and M. Meheriuk (Ed.), p. 201. Timber Press, Beaverton, OR. Lidster, P. D., Forsyth, F. R., and Lightfoot, H. J. 1980. Low oxygen and carbon dioxide atmosphere for storage of McIntosh apples. Can. J. Plant Sci. 60: 299. Lidster, P. D. 1982. Low oxygen atmospheres to maintain apple quality in storage. In "Controlled Atmospheres for Storage and Transport of Perishable Agricultural Commodities," D. G. Richardson and M. Meheriuk (Ed.), p. 109. Timber Press, Beaverton, OR. Liu, F. W. 1977. The ethylene problem in apple storage. Mich. State Univ. Hort. Rep. 28: 86. Liu, F. W. 1978. Effect of harvest date and ethylene concentration in controlled storage on the quality of 'McIntosh' apples. J. Amer. Soc. Hort. Sci. 103(3): 388. Lizada, M. C., and Yang, S. F. 1979. A simple and sensative assay for 1-aminocyclopropane-1-carboxylic acid. Anal. Biochem. 100: 140. Lowery, H. 0., Rosebrough, N. J., Farr, A. L., and Randall, S. J. 1951. Protein measurement with folin phenyl reagent. J. Biol. Chem. 193: 265. Mansour, R., Latch, A., Vaillant, V., Pech, J., and Ried, M. S. 1986. Metabolism of 1-aminocylcopropane-1-carboxylic acid in ripening fruits. Physiol. Plant. 66: 495. Ochoa, S., Mehler, H., and Kornberg, A. 1948. Biosynthesis of dicarboxylic acid by carbon dioxide fixation. J. Biol. Chem. 174: 979. 91 Patterson, B. D., Hatfield, S. G., and Knee, M. 1974. Residual effect of controlled atmosphere storage on the production of volatile compounds by two varities of apples. J. Sci. Fd. Agric. 25: 843. Priest, K. L., and Lougheed, E. C. 1981. Evaluating apple maturity using the starch iodine test. Ontario Ministry of Agriculture and Food, March. CHAPTER 2 EFFECT OF STORAGE CONDITIONS AND RIPENING ON THE FLAVOR PROFILE OF EMPIRE APPLES USING A DYNAMIC HEADSPACE TECHNIQUE 92 93 INTRODUCTION Apples are stored at low temperature and in various gas mixtures to extend their shelf life. Low storage temperature, high C02 and low 02 concentrations reduce the respiration rate which further delays ripening and extends shelf life. A reduction in the concentration of 02 decreases the rate of accumulation of ethylene, and an increase in the concentration of CO2 further reduces it. These conditions delay the production of volatiles (Kidd and West, 1954; Fidler and North, 1969). Low 02 levels in controlled atmosphere (CA) storage have a greater effect on reduced volatile production than high levels of C02 (Patterson et al., 1974). Guadagni et al. (1971) found that fruits from a commercial CA which were stored for extended periods were lacking ester production when transferred to air. The level of ethylene during storage paid an important part in extending shelf life due to the prevention of the ripening process. Liu (1985) reported that low ethylene CA storage preserved the flesh firmness of McIntosh apples more effectively compared to conventional CA stored apples. However, apples stored in low ethylene CA storage did not develop a full flavor compared to the apples stored in the conventional CA. Low ethylene CA storage reduced the production of the short chain esters, but did not 94 suppress the production of lipid oxidation products (Yahia et al., 1985). After prolonged CA storage, apples do not develop aroma in the same way as fruits ripened naturally ( Patterson et al., 1974; Williams and Knee, 1977; Lidster et al., 1983). According to Williams and Knee (1977), ester patterns changed from short chain esters to "banana like compounds", such as 2- and 3-methylbutyl acetate which tended to increase in concentration by the end of storage. Meigh (1956, 1957) studied the effect of cold and CA storage on the production rate of alcohols, aldehydes, and esters. He concluded that apples stored in air produced volatiles at a greater rate than those stored in CA. Grevers and Doesburg (1962) studied the volatile content of four varieties of apples during storage and found that acetic acid and hexyl acetate increased with extended storage. Grevers and Doesburg (1964) also studied the effects of storage temperature and ripening on the volatiles from two varieties of apples. Apples stored at lower temperature produced lower levels of total volatiles compared to higher temperature storage. When apples are ripened at temperatures above 7°C, they undergo climacteric rise which is probably initiated by ethylene production. Patterson et al. (1974) concluded that low temperatures greatly reduced the volatile production in both Golden Delicious and Orange Cox's Pippin varieties. 95 The striking changes in the production of volatiles by apples following long periods in cold and CA storage indicate that the measurement of volatiles may be useful in evaluating the shelf life of fruits. Specific objectives of the study were to determine the effect of storage conditions and ripening on the flavor quality of intact Empire apples. MATERIALS AND METHODS MATERIALS Empire apples were harvested on Oct. 2 , 1984 at the Michigan State University Horticulture Farm, East-Lansing, Michigan. Maturity at harvest was determined by internal ethylene, flesh firmness and starch content (as described previously in Chapter 1). Fruits were randomized into three groups. One group was stored in a air-cooled room and the other two groups were stored in CA at 3.3°C, 3% C02 and 2% 02 with relative humidity of 98%. A low ((1 PMM) and high (>100 PPM) level of ethylene was established. Apples were removed from storage every month for a duration of six months. To study the effect of ripening, after removal of apples from storage every month, apples were kept at 229C for 2, 7 and 14 days. Three replicates of six apples were used for the collection of volatiles. 96 Tenax-GC is frequently used to trap volatile compounds (Kuo et al., 1977; Boyko et al., 1978; Wellnitz-Ruen et al., 1982; Josephson et al., 1983; Bassette, 1984), mainly because of its high thermal stability, and its low affinity for low boiling compounds. The properties of Tenax-GC were studied by Sakodynkii et al. (1974) and Butler and Burke (1976). The composition of the collected headspace vapor varied with the flow rate of the sweeping gas, collection time, sampling time, and trap capacity (Wyllie et al., 1978 ). Standards were purchased from Sigma Chemical Co. (St. Louis, MO). DYNAMIC HEADSPACE SAMPLING OF INTACT APPLES To evaluate the volatile composition of Empire apples as a function of storage and ripening, a dynamic headspace sampling of intact apples was performed. A maturity index generally functions if it is non-destructive and allows for repetitive monitoring of fruits as they ripen or respond to imposed conditions. Headspace analysis of volatile compounds meet these requirements (Romani et al., 1966). For dynamic headspace sampling without fruit disintegration, six apples of approximately the same size were placed in 8 L desiccators which were flushed continously with air purified by passing through activated carbon, at a 97 flow rate of 15 m1/min. Before sampling, the pure air flow was increased to 100 ml/min. After 30 min, the traps were attached to the outlet of the desiccators and after readjusting the air flow to 15 ml/min, sampling was continued for six hours. Two traps in series were used to collect the volatiles. The traps were prepared by filling pyrex glass tubing (0.6 mm x 10 cm) with approximetaly 0.2 g of 80/100 mesh Tenax-CC (Supelco, Ann Arbor, MI). The tubes were plugged at both ends with silanized glass wool. The traps were conditioned at 190°C at a flow rate of 40 ml/min of purified nitrogen for eight hours. These traps were put in dry ice to prevent loss during collection . At the end of the six hour collection period, the traps were connected to a vacuum pump for 15 min to remove the volatiles in the desiccators. Figure 1 shows the design of the headspace apparatus for collecting the volatile compounds. The adsorption tubes were tightly closed and kept in the freezer. The extracts were stored until analysis was conducted. REMOVAL AND CONCENTRATION OF VOLATILES Two traps were removed from the freezer and kept at room temperature for 15 min. Volatiles were eluted from the Tenax traps by backflushing the traps with 2 ml volumes of isopentane. The content of the traps was subsequently added to the solvent containing the volatiles. The content of the 98 Figure 1. Apparatus for collecting of volatile compounds from Empire apple by applying the headspace technique. 99 >3_<>.—mo zmmorm .9282 x <>_.x 190 MM: 65% um u 1 “H <>occz ocio ._. L. 100 traps was removed with help of purified nitrogen. The Tenax slurry was centrifuged at 650 rpm for 1 min. This extraction process was repeated four times. The fifth and sixth extraction did not contain any volatiles. A total of 8 ml of isopentane extract was collected which was further reduced with a gentle flow of nitrogen to 1 ml. The final extract was kept in the freezer until analysis. 1.5 ul of extract were used for gas chromatographic analysis. GAS CHROMATOGRAPHIC ANALYSES Analyses of the volatiles were performed on a Hewlett Packard Model 5850 A equipped with a flame ionization detector. A bonded fused silica capillary column, 60 m by 0.25 mm, coated with Carbowax 20 M (Supelco Inc., Bellefonte, PA) was used. The inlet and the detector were maintained at 200°C. The inlet pressure was adjusted to achieve an average linear velocity of 0.7 ml/min as measured by methane injection. The helium flow rate was 28 ml/min with a column head pressure of 1.8 kg/cm3, which gave a flow rate of approximately 0.75 ml/min through the column. The detector was supplied with 240 ml air/min. The temperature program was 20 C for 1 min, and a rate of 7°C/min up to 30°C followed by a rate of 2°C/min up to 180°C with a holding time of 30 min. The purge was off for 0.5 min and open thereafter, operating under splitless mode. The outlet signal was fed through a digital integrator to the recorder. 101 GAS CHROMATOGRAPHIC AND MASS SPECTROMETRIC ANALYSES OF VOLATILES The system used for GC/MS analysis of the volatiles was a Nermag R-10-10C interfaced with a Delsi splitless capillary GC and a bonded phase Carbowax 20 M (60m x 0.25 mm). The temperature program was 40°C for 3 min, and a rate of 2.5 °h/min up to 180 °C with a holding time of 25 min. The instrument was operated in the electron impact mode at an ionization voltage of 70 eV. The ion source temperature was 200 °C. A scan of 1 sec was used over a mass range of 40-250 amu. MASS SPECTRA IDENTIFICATION Identification of volatile compounds was determined by the mass spectra of the reference compounds from The National Institute of Health (NIH) and The Environmental Protection Agency (EPA) libary. Mass spectra of compounds were made for those compounds which were not included in NIH-EPA libary. Confirmation of compounds was presumed when an 80% match with the reference compound was determined. QUANTITATION OF VOLATILE COMPOUNDS The standard curve of all the standards was obtained by injecting various concentrations of pure standards (90 to 99%) 102 using the same procedure as described in the gas chromatographic analyses section. Experimental area values were plotted against the known concentrations of standards. The standard curves were calculated using the algorithm for least squares estimation of non-linear parameters of the Marquard-compromise method (Marquard, 1963). Calculations were performed on a Zenith data system model 148 microcomputer, using a non-linear regression subroutine of the integrated graph statistical program Plotit (Einsensmith, 1981). The concentrations (ug/g) of volatile compounds were determined using both gas chromatographic and mass spectrophotometric analyses. RESULTS AND DISCUSSION Percent recoveries of hexanal, ethyl hexanoate, 3- methyl-I-butanol, (E)-2-hexenal, methyl acetate, butanoic acid, and phenyl ethyl acetate from Tenax-CC traps were measured. A known quantity of these standards was introduced into three desiccators (three replicates) and simulated sampling for these compounds was extracted with isopentane. These standards were selected based on their retention times, with methyl acetate having the lowest retention time (5.28 min) and phenyl ethyl acetate the highest (69.88 min). Digital integrator response was measured and compared to a standard curve prepared by direct injection. Table 1 shows the percent 103 Table 1. Percent recovery (%) of selected compounds using simulated dynamic headspace technique. Compounds Percent Recovery (%) Methyl acetate 61.6 Hexanal 45.2 3-Methyl 1-butanol 54.4 Ethyl hexanoate 46.5 (E)-3-hexen-1-ol 43.7 Butanoic acid 44.0 Ethyl phenyl acetate 45.7 104 recoveries of these standards. Various flow rates during trapping were used: 50 ml/min, 20 ml/min, and 15 ml/min. The best recovery was achieved using a flow rate of 15 ml/min for six hours. Thus, when establishing conditions for analysis, it appears that greater recoveries were obtained using lower purge rates and longer collection time. The influence of flow rate and sampling time was studied by various investigators (Jennings et al., 1972; Novotny et al., 1974; Buckholz et al., 1980; Olafsdottir et al., 1985; Mills, 1986). They concluded that the total area under the chromatogram increased as the gas flow rate increased. Generally, for a flow rate of 50 ml/min purged samples, collection times up to 300 min resulted in increasingly efficient recovery of all the selected compounds. A typical standard curve for ethyl-Z-methyl butanoate, an important flavor compound in Empire apples, is shown in Figure 2. Coefficient correlation values (r) and the mathematical equations for all of the standards are summarized in the appendix B (Tables 20, 21, and 22). A typical chromatographic pattern of volatile compounds of Empire apples obtained by the headspace method is illustrated in Figures 3, 4 and 5. These samples have been stored for four months in air at 3.3°C and subsequent ripening at 22°C for 2, 7 and 14 days. 105 Figure 2. Standard curve for ethyl 2-methyl butanoate. 4E+06- 3E+OG- ZE+06- Detector Response (oreo) 1E+06- 106 OI) LO 2fo Concentration (ppm) 1 3O 40 107 Figure 3. Gas chromatogram of Empire apples stored for 4 months in air at 3. 3 °C with relative humidity of 98 % after 2 days of ripening at 22° C. 1. 5 ul sample injected. Initial temperature 20' C for 1 min. 20----30 ramp of 7 °C/Min 30----180 ramp of 2. 5° C/Min holding time at 180 30 min. 108 . 9 56.24 66 27 5.56 2 57.67 7.“ 7 46 " 9.67 61.14 I 9'79 62.13 -, 1..., ”13.39 13.62 14.24 ' 15.78 15.42 " 67.77 16.66 ' 16.61 71.46 73.16 22.45 75.16 25.56 26.66 76.39 2811 79.97 36.26 64.33 35.34 37.64 39.17 92.66 43.69 109 Figure 4. Gas chromatogram of Empire apples stored for 4 months in air at 3.3 with relative humidity of 98 % after 7 days of ripening at 22°C. 1.5 sample injected. For gas chromatographic conditions, refer to Figure 3. 11() 1.77 3.17 54.55 56.28 62.16 '67579 66.51 73.21 78.42 84.37 35.65 92.85 43.72 111 Figure 5. Gas chromatogram of Empire apples stored for 4 months in air at 3.3°C. with relative humidity of 98 % after 14 days of ripening at 22°C. 1.5 sample injected. For gas chromatographic conditions, refer to Figure 3. 112 $6.28 62.16 8 .49 73.21 26.33 78.‘3 ‘11 [C 9" --==— :16 64.46 33.35 33 16 f 34.66 35.46 39.11 92.69 46.45 43.74 113 Fourty-nine compounds were identified from the dynamic headspace collection of volatiles during storage and ripening at 22 ‘0 after 2, 7 and 14 days. The compounds identified included: 24 esters, 13 alcohols, 7 aldehydes and 2 ketones (Table 2). The most abundant volatile constituents consisted of esters, alcohols and aldehydes, with minor amounts of ketones. Of the volatile compounds detected and identified in Empire apples, ethyl propionate, methyl butanoate, ethyl butanoate, methyl-Z-methyl butanoate, ethyl-Z-methyl butanoate, propyl acetate, ethyl acetate, butyl acetate, pentyl acetate, butanal, 2-methyl butanal, hexanal , butanol, hexanol and 2- pentanol were found to be the major constituents. Most compounds identified were esters of butanoic acid and acetic acid. Empire apples are a hybrid of Red Delicious and McIntosh apples. In McIntosh apples, esters are mainly of butanoic acid. As the ripening process proceeds, the total concentration of volatiles increased significantly which agrees with the findings of Brown et al. (1966). This is due to the higher respiration rate during the ripening process, which affects the biogenesis of the volatile compounds. The increase in the concentration of total volatiles is valid for all the storage conditions. Figures 6, 7, and 8 illustrate the effect of storage conditions during six months of storage and after 2, 7 114 Table 2. Volatile compounds identified in headspace of Empire apples at harvest, during storage and ripening. Esters Aldehydes Alcohols Methyl acetate 2-Methyl butanal 1-Butanol Ethyl acetate Z-Hexenal 2-Methy1-1-butanol Propyl acetate Pentanal 3-methyl-1-butanol Butyl acetate Isopentanal Z-Methyl-l-propanol Hexyl acetate Phenyl acetaldehyde 1-Pentanol Pentyl acetate Butanal Z-Pentanol Isopentyl acetate Heptanal 3-Pentanol Ethyl phenyl acetate Ethyl propinoate 2-Methyl-1-pentanol Methyl butanoate I-Hexanol Ethyl butanoate Z-Hexanol Methyl-Z-methyl butanoate 3-Hexanol Ethyl 2-methy1 butanoate (Z)—3-hexen-1-ol Propyl butanoate (E)-3-hexen-101 Butyl butanoate 3-Methylbuty1 butanoate (Isopentyl butanoate) Ketones 2-Methylbutyl-2-methyl butanoate Ethyl formate Butyl formate 2-Pentanone Methyl pentanoate 3-Heptanone Ethyl-Z-methyl pentanoate 2-Methylbutyl pentanoate Ethyl hexanoate (Ethyl caproate) Miscellaneous Butyl hexanoate (Butyl caproate) Ethyl Z-methyl benzene 1,2 Dimethyl bezene 2-Methyl propyl benzene Figure 6. 115 Effect of storage conditions ( low, high ethylene and air) on the concentration of the esters, aldehydes, alcohols and ketones during the six months of storage. Empire apples were ripened at 22°C for 2 days. LCA-low ethylene controlled atmosphere ( 3% C02 : 2% 02 and (1 PPM ethylene). RCA-High ethylene controlled atmosphere ( 3% C02 : 2% 02 and )1 PPM ethylene ). 116 14- % h. m . mw//////. 6 m .m .m. m _“.m,//////A a m m a my////////2 K A A E \\\\.//////////. 5 _ . . _ VkaZ/é ________=_.\\\\\\\\\\\\\\\\\\\ k //////////.////////////////////.////. «mg 4 _=====§§m%§§ om7//////// 3 w.../%//////////4 ////// 2 § JV .1 / .2 “motor Eggso — _ 4 — a _ — _ _ H q _ q B .0. H m 9 8 7 6 5 4 3 2 .1 0 AEan cozobcoocoo Storage duration (months) Figure 7. 117 Effect of storage conditions ( low, high ethylene and air ) on the concentrations of the esters, aldehydes, alcohols and ketones during the six months of storage. Apples were ripened at 22°C for 7 days. LCA-low ethylene controlled atmosphere ( 3 2 C02 : 2 X 02 and <1 PPM ethylene ). HCA- high ethylene controlled atmosphere ( 3 2 C02 : 2 z 02 and >1 PPM ethylene ). 118 Aldehydes V/l 7// ’l/. 'IA “71/”. “MW/é ===V\\\\\\\.m//////////////////////////#/.//MM "y/ x. — Ketones - Aldehydes - Esters .////////////.///////////////////////////////. “my/é mmw/é iy////A 3 <0: f”. <3 “7». .2 xx? V & smote: \\\\\m,/////////////////////////////// 70- 65- 60- 55- 501 d _ _ _ — q — a q _ 5 0 5 O 5 0 5 O 5 0 1| 11 4. 4. 3 3 2 2 AEaav cozobcmocoo Storage duration (months) 119 Figure 8. Effect of storage conditions ( low, high ethylene and air ) on the concentrations of esters, aldehydes, alcohols and ketones during six months of storage. Apples were ripened at 22°C for 14 days. LCA-low ethylene controlled atmosphere ( 3 1 C02 : 2 X 02 and < 1 PPM ethylene ). RCA-high ethylene controlled atmosphere ( 3 1 C02 : 2 2 02 and > 1 PPM ethylene ). Concentration (ppm) 140- 120- 100- 80‘ 4o- 20— Harvest 120 Storage duration (months) - Ketones — Alcohols — Aldehydes - Esters .2 5 < 5 I / -’ 16 w / 7‘9 7 / I A f ’ / I M 1 2 3 4 5 6 121 and 14 days of ripening at 22°C. Apples stored in air and ripened naturally during storage and thereafter, had the highest concentration of the esters. Apples stored in high ethylene CA had higher concentrations of the volatiles after ripening, relative to those stored in low ethylene CA. This clearly indicates the importance of the ethylene level in CA storage. Methyl-Z-methyl butanoate and ethyl-Z-methyl butanoate are particularly prominent and appear to be associated with maturation and ripening. The methyl-Z-methyl butanoate concentration increased from 0.82 ug/g after 2 days of ripening to 8.45 ug/g after 14 days. Similarly, the ethyl-2- methyl butanoate concentration increased from 0.49 ug/g to 20.30 ug/g. Brown et al. (1966) reported that many volatile compounds reach a maximum level at a time which coincides with the respiratory climacteric. The total volatiles and subsequently the aroma of fruits such as apples are developed after the climacteric rise with a maximum of ester production. While many compounds may contribute to the apple-like aroma, ethyl-Z-methyl butanoate is responsible for the special aroma of apples as indicated by Flath et a1. (1969). These compounds are produced only in higher amounts during ripening, are related to the climacteric rise during respiration and are only present in small concentrations during growth or at the time of harvest. 122 Methyl , ethyl and butyl acetate , pentyl, and isopentyl acetate are particularly prominent and appear to be associated with the maturation and ripening process. Both butyl acetate and pentyl acetate were depressed following storage, which is in agreement with Brown '8 (1966) report. Ethyl acetate, methyl butanoate and hexyl acetate levels changed substantially after six months of storage. Ethyl formate and pentyl butanoate disappeared totally during storage, and after ripening at 229C for 14 days. In a study of flavor changes during storage and ripening of Golden Delicious apples, Dirinck et al. (1984) suggested that the total esters could be used as a criterion for the fruity character of apple aromas. They showed the sum of esters increased during ripening up to 14 days and decreased therafter. In this study, butyl acetate, hexanal, butanal, ethyl butanoate, methyl-Z-methyl butanoate, and ethyl-Z-methyl butanoate had the highest value up to 14 days. High 002, low 02 and low ethylene levels during storage reduce the level of volatiles, particularly the level of methyl-Z-methyl butanoate and ethyl-Z-methyl butanoate. A low ethylene level had the greatest effect in the reduction of volatile concentrations. The concentration of ethyl-Z-methyl butanoate was especially low when compared to air and high ethylene CA storage after six months. Both butanal and butyl acetate increased during the early 123 stage of storage and then declined to almost zero after six months of storage. This reduction was more severe for low ethylene CA storage. Propyl acetate stayed unchanged during storage in air, decreased in concentration in high ethylene CA storage, and increased in low ethylene CA storage. Ripening did not have any affect on the propyl acetate concentration for apples stored in low ethylene CA. The concentration of propyl acetate for apples stored either in air or in high ethylene CA increased during ripening. Aroma estimation by headspace of Cox's Orange Pippin apples placed in CA storage showed depressed rates of volatile production following ripening (Paillard, 1981). 5% C02 and 22 02 had the greatest effect on volatile production while ethylene had an opposite effect on the level of several volatiles, such as ethyl butanoate and hexanal. (Hatfield et al., 1974) An increase in the concentration of volatiles during ripening could be due to an increase in the activity of some enzymes. The ester content increases in concentration as the fruit ripens (Broderick, 1974), which is responsible for fruity aroma of apples. The effect of ripening for each storage condition on the sum of esters, aldehydes, alcohols and ketones is shown in Figures 9, 10 and 11. 124 Figure 9. Total esters, aldehydes, alcohols and ketones changes during low ethylene controlled atmosphere storage and after subsequent ripening for 2, 7 and 14 days at 22‘C. 125 é 7 AW; ”/ w; ;Mm7/,//M/////,. mV/é “mg/é x72 4 V\\\\\\. h,V///////////fi///////////////////////.///////////////////////A All lil- 11111, 11111111111111.1111: 2 \km¢////////A I Ketones - Alcohols — Aldehydes - Esters \ =_.\. d1 _ 0 2 AEQQV cozobcoocoo A 0 30- 10- Storage duration (months) 126 Figure 10. Total esters, aldehydes, alcohols and ketones changes during high ethylene controlled atmosphere storage and after subsequent ripeningfor 2, 7 and 14 days at 22 °C. 127 60- 50‘ 'I’l ”l 7/A Ketones Alcohols .m/////////.. .7////. 5 //// ///////////////////////////M/M/1////M/i/M/VV/Iiin////////NM/H . r 4 :VW\\ /////A. - Aldehydes - Esters =====V\\\\\\\\\\\\\\. 7 \\\\aM//////////////////////////V///////H 14 10- O _ . . _ 0 0 0 4 2 AEQQV cozobcmocoo Storage time (months) 128 Figure 11. Total esters, aldehydes, alcohols and ketones changes during normal air storage and after subsequent ripening for 2, 7 and 14 days at 22°C. 129 - Ketones — Alcohols - Aldehydes ==V\\\\\\\\\ w/a ./ I - Esters ’IAl// . //////. 6666 666% “ "7% 6. “w 7//¢///////////////////////////////. m7////////////6 mg I. M nm6/////,/////////// 7 §m7///////////M 2 m7. 14o~ 120- . . a O 0 8 6 100- AEQQV cozobcmocoo 40- Storage duration (months) 130 Based on the data presented by Flath et al. (1969), hexanal, 2-hexenal and ethyl-Z-methyl butanoate were considered most applelike by the taste panelists. The effect of ripening on the concentration of methyl butanoate, ethyl-2- methyl butanoate, hexanal, 2-hexana1 and 2-methy1 propanol after four months of storage in air is shown in Figure 12. 3-Methylbutyl and hexyl butanoate are said to contribute to the odorous character of stored apples (Jakob et al., 1969). The concentrations of these two esters increased after four months of storage. According to Sapers et al. (1977), the concentrations of acetaldehyde, ethyl acetate, 1-butana1, ethanol, 2- and 3-methylbutanal tended to increase during storage. However, due to the low affinity of Texax-GC for either ethanol or acetaldehyde, these compounds were not identified in the headspace of Empire apples. But the concentrations of ethyl acetate and 1-butanol increased during ripening after 7 and 14 days. Methyl and ethyl acetate and ethyl propionate tended to increase during the early stages of cooled storage and then decline to very low levels. This pattern was also observed by Forsyth et al. (1969) and Lidster et al. (1983) . However, their levels increased after ripening compared to those at harvest. High C02 and low 02 concentrations had the greatest effect on the level of these three esters. Transfer from CA storage to air stimulated the production of the volatiles at a level comparable to cooled air storage. 131 Figure 12. Effect of ripening time (2,7 and 14 Days) at 22' C on the volatile compounds in Empire apples stored for 4 months in air at 3. 3C 132 .6; 11234.55 :2: '4 100- 10- AEaav cozobcmocoQ mom O.1~ 0.01 14 Days of ripening 133 Hexyl acetate was below the detection limit ((0.001 ug/g) at harvest and during the early stages of storage. Its concentration increased after three months of storage in air and declined to a very low level thereafter. Grevers and Doesburg (1962) reported similar results. Hexyl acetate totally disappeared after two months storage in low ethylene CA. The reverse happened for apples stored in high ethylene CA. Another interesting observation was the presence of hexanal after four months of storage in air. Hexanal increased from 0.76 ug/g after 2 days of ripening to 28.87 ug/g after 7 days and finally to 41.41 ug/g after 14 days of ripening. The concentration of hexanal was very low for apples stored under low ethylene conditions. Similar results for hexanal were reported by Yahia et al. (1985) for McIntosh apples stored either in air or in a low ethylene CA. However, apples stored in low ethylene CA had a much lower concentration of hexanal than either air or high ethylene CA storage and ripening did not enhance its concentration (Appendix B, Tables 1-19). This is contrary to results reported by Yahia et a1. (1985). Apples that have been stored for a long period in CA and are then transferred to air, loose their ability to regenerate volatile compounds (Lidster et al., 1983). In this study, low ethylene CA storage suppressed the production of short chain esters. This suppression was reversed when apples were 134 transferred from low ethylene CA to air. However, after six months storage they had low levels of esters and ripening did not greatly affect their concentration, even though it did increase the concentration of total volatiles after 14 days of ripening. Apples stored in air ripened naturally during storage and the level of the total volatiles increased gradually over the period of storage. At two months of storage, the apples had a high level of volatiles mainly due to the high concentrations of methyl-Z-methyl butanoate and ethyl-Z-methyl butanoate. The high level of total volatiles at four months storage is mainly due to high concentrations of ethyl propionate, ethyl butanoate and hexanal. After four months, the total volatile production decreased rapidly due to prolonged storage. As shown in Figure 9, low ethylene CA storage severely suppressed the total production of volatiles. However, when apples were transferred to air at 2290, they were able to generate volatiles to a level slightly less than for apples before storage at harvest. As shown in Figure 10, high ethylene CA storage did not severely affect the total level of volatiles of the apples stored for four months, relative to the apples at harvest. Apples stored in high ethylene CA had a better capacity to generate volatiles compared to those stored in low ethylene CA, but still less than those stored in air. 135 Liu et al. (1985) showed that McIntosh apples stored in . low ethylene CA for four months had much lower levels of the odor-active volatiles compared to those stored in air. They concluded that after apples have been transferred to air at 20 in they were able to generate volatiles to the same extent as apples before being placed in storage. However, in this study Empire apples that have been stored for four months had comparable levels of volatiles to those at harvest. 136 REFERENCES Bassette, R. 1984. Measuring flavor changes with vapor sampling and GLC analysis. J. Food Protect. 47: 410. Boyko, A. L., Morgan. M. E., and Libbey, L. M. 1978. Porous polymer trapping for GC/MS analysis of vegetable flavours. In "Analysis of Food and Beverages: Headspace Techniques," G. Charalambous (Ed.), p. 57. Academic Press, New York. Broderick, J. J. 1974. Apple-has research helped? Flavor Industry, (July-Aug), p. 184. Brown, D. S., Buchanan, J. R., and Hicks, J. R. 1966. Volatiles from apples as related to variety, maturity and ripeness. Proc. Amer. Soc. Hort. Sci. 88: 98. Buckholz, L. L., Withycombe, D. A., and Daun, H. 1980. Application and characteristics of polymer adsorption method used to analyze flavor volatiles from peanuts. J. Agric. Fd. Chem. 758: 760. Bulter, L. D., and Burke, M. F. 1976. Chromatographic characte- rization of porous polymers for use as adsordents in sampling columns. J. Chromatogr. Sci. 14: 117. Einsensmith, S. 1981. Plotit, an integrated graph-statistical program. Ann Arbor, MI. Dirinck, P., De Pooter, H., Willaert, G., and Schamp, N. 1984. Application of a dynamic headspace procedure in fruit flavor analysis. In "Analysis of Volatiles Methods Applications," P. Schreier (Ed.), p. 381. W. de Gruyter, New York. Fidler, J. C., and North, C. J. 1969. Production of volatile organic compounds by apples. J. Sci. Fd. Agric. 20: 521. Flath, R. A., Black, D. R., Forrey, R. R., McDonald, G. M., Mon, T. R., and Teranishi, R. 1969. Volatiles in Gravenstein apple essence identified by GC-MS. J. Chromatogr. Sci. 7: 508. 137 Forsyth, F. R., Eaves, C. A., and Lightfoot, H. J. 1969. Sensory quality of McIntosh apples as affected by removal of ethylene from controlled atmosphere. Cand. J. Plant Sci. 49: 567. Grevers, G., and Doesburg, J. J. 1962. Gas chromatographic determination of some volatiles, emanated by stored apples. In " Volatile Fruit Flavours," H. Luthi (Ed.), p. 319. Juris, Zurich, Switzerland. Grevers, G., and Doesburg, J. J. 1964. Volatiles of apples during storage and ripening. J. Food. Sci. 30: 412. Guadagni, D. G., Bomben, J. L., and Hudson, J. S. 1971. Factors influencing the development of aroma in apple peel. J. Sci. Fd. Agric. 22: 110. Hatfield, S. G., and Patterson, B. D. 1974. Facteurs et Requlation de la Maturation des Fruits. Colloques Internationeaux CRNS, p. 57. Jakob, M. A., Hippler, R., and Luethi, H. R. 1969. Occurrence of hexyl-Z-methylbutyrate in apple aroma. Mitt. Geb. Lebensmittelunter. Hyg. 60(3): 223. Jennings, W. G., Whohleb, R. H., and Lewis, M. J. 1972. Gas chromatographic analysis of headspace volatiles of alcoholic beverages. J. Food Sci. 37: 69. Josephson, D. B., Lindsay, R. C., and Stuiber, D. A. 1983. Identification of compounds characterizing the aroma of fresh whitefish (coregonus clupeaformis). J. Agric. Fd. Chem. 31: 326. Kidd, F., and West, C. 1954. Quality in Cox's Orange Pippin apples. Agriculture 52: 419. Kuo, P. P., Chian, E. S., De Walle, F. B., and Kim, J. H. 1977. Gas stripping, sorption, and thermal desorption procedures for preconcentrating volatile polar water-soluble organics from water samples for analysis by gas chromatography. J. Anal. Chem. 49: 1023. Lidster, P. D., Lightfoot, H. J., and McRae, K. B. 1983. Production and regeneration of principal volatiles in apples stored in modified atmospheres and air. J. Food Sci. 48: 400. 138 Liu, F. W. 1985. Low ethylene controlled atmosphere storage of McIntosh apples. In "Ethylene and Plant Development," T. A. Roberts and G. A. Tucker (Ed.), p. 385. ' Butterworths, London. Marquard, D. W. 1963. An algorithm for least squares estimation of non-linear parameters. J. Siam. 11: 431. Meigh, D. F. 1956. Volatile compounds produced by apples. I- Aldehydes and ketones. J. Sci. Fd. Agric. 7: 396. Meigh, D. F. 1957. Volatile compounds produced by apples. II- Alcohols and esters. J. Sci. Fd. Agric. 8: 313. Mills, 0. E. 1986. Headspace sampling method for monitoring flavour volatiles of protein products. J. New Zealand Dairy Sci. Tech. 21: 49. Novotny, M., McConnell, M. L., and Lee, M. L. 1974. Some aspects of high resoluation as chromatographic analysis of complex volatile samp es. J. Agric. Fd. Chem. 22: 765. Olafsdottir, G., Stenink, J. A., and Lindsay, R. C. 1985. Quantitative performance of a simple Tenax-CC adsorption method for use in the analysis of aroma volatiles. J. Food Sci. 50: 1431. Patterson, B. D., Hatfield, S. G., and M. Knee. 1974. Residual effects of contolled atmosphere storage on the production of volatile compounds by two varieties of apples. J. Sci. Fd. Agric. 25: 843. Paillard, N. 1981. Factors influencing flavor formation in fruits. In "Flavour'81," P. Schreier (Ed.), p. 479. W. de Gruyter Co, Berlin. Romani, R. J., Lilly, L., and Ku, L. 1966. Direct gas chromatographic analysis of volatiles produced by ripening pears. J. Food Sci. 31: 558. Sakodynskii, K., Palina, L., and Klinskaya, N. 1974. A study of some properties of tenax, a porous polymer sorbent. J. Chromatogr. 7: 339. Sapers, G. M., Abbott, J., Massie, D., Watada, A., and Finney, E. E. 1977. Volatile composition of McIntosh apple juice as a function of maturity and ripeness indices. J. Food Sci. 42: 44. 139 Weelintz-Ruen, W., Reineccius, G. A., and Thomas, E. L. 1982. Analysis of the fruity off-flavor in milk using headspace concentration capillary column gas chromatography. J. Agric. Fd. Chem. 30: 512. Williams, A. A., and Knee, M. 1977. The flavor of Cox's Orange Pippin apples and its variation with storage. Proc. Assoc. Appl. Biol. 87: 127. Wyllie, S. G., Alves, M., Filsoof, M., and Jennings, W. G. 1978. Headspace sampling: uses and abuses. In "Analysis of Food and Beverages. Headspace Techniques," G. Charalmbouse (Ed.), p. 1. Academic Press, New York. Yahia, E. H., Liu, F. W., Acree, T. E., and Butts, R. 1985. Odor-active volatiles in McIntosh apples stored in stimulated low ethylene controlled atmosphere. Horticulture report No. 126. Proceedings of the 4th National Controlled Atmosphere Research Conference, July 23-26. CHAPTER 3 EFFECT OF MATURITY AT HARVEST, STORAGE CONDITIONS AND RIPENING ON THE FLAVOR PROFILE OF EMPIRE APPLES USING DISTILLATION EXTRACTION TECHNIQUES 140 141 INTRODUCTION Michigan ranks third in the production of apples in the United. States. Of the apples harvested in Michigan in 1986, 39.5 percent were used for the fresh market and the rest were used for apple juice and for canned or frozen apples. Apples are stored either in cooled rooms or controlled atmosphere (CA) upon harvest. Low storage temperature , high 002, and low 02 extends the storage life of most fruits by delaying the respiration rate and decreasing ethylene production which induces ripening (Knee, 1982). It is well documented that ethylene is responsible for ripening. Retardation of fruit ripening by an inhibitor of ethylene synthesis (Bangerth, 1978), ethylene action (Janes and Frenkel, 1978) and hypobaric storage (Burg and Burg, 1966) have been confirmed as regulators of ripening in climacteric fruits. Storage temperature and gas composition are the determining factors in the effectiveness of low ethylene CA storage (Kidd and West, 1939; Fidler and North, 1969; Peacock, 1972). If apples are kept in high C02 and/or low 02, the accumulation of ethylene is prevented and the onset of the ripening process is delayed. Low 02 storage of apples is commercially successful, even though ethylene levels of several hundred PPM accumulate during storage (Knee, 1985). 142 Many varieties of apples and pears held at low but non- injurious temperature show greater ethylene production compared to fruits held at higher temperature ( Fidler and North, 1971; Looney, 1972; Knee et al., 1983 ). Fidler and North (1969) concluded that a significant loss of flavor from exposure of unripe apples to ethylene in refrigerated air storage is unlikely. However, most of these studies lack an extensive look at the flavor of apples stored in low ethylene CA. Ethylene removal may not be feasible when low temperature or CA has to be maintained (Knee, 1985). Volatile compounds in fruits are produced during ripening as a result of a higher respiration rate. Apples that have been stored for prolonged periods of time do not have the same flavor as those that have been ripened naturally or stored for a shorter period. However, in most cases the flavor of stored apples can be regenerated if the apples are subsequently kept at a higher temperature for ripening. Not every variety of apple has the same ablity to retain an acceptable flavor or to generate volatiles after long periods of storage, even when ripened at a higher temperature. CA storage of apples in low ethylene is a new concept on the commercial scale. Numerous investigations have reported conflicting results regarding the removal of ethylene from storage (Forsyth et al., 1969: Knee, 1975; Liu, 1979 ). Low ethylene CA storage has been proven to be effective if the 143 apples are stored during their preclimacteric stage. Once ethylene production has started, removal of ethylene becomes a physical impossibility (Knee and Hatfield, 1981). Since the advent of gas chromatography (Van Straten et al., 1977), the volatile components of apple and apple juice have been investigated many times but few of these investigations have been concerned with the effect of storage and ripening on the flavor. The initial objective of the study was to determine the flavor profile of Empire apples during air, low and high ethylene CA storage and their ability to generate flavor compounds. A second objective was to determine the affect of maturiy at harvest on the flavor of Empire apples during storage and ripening. MATERIALS AND METHODS MATERIALS Empire apples were harvested at two maturity levels determined by flesh firmness, starch content, and internal ethylene concentration. Apples were harvested on Oct. 2 and 9 from ten trees at Michigan State University Horticultural Farm, East-Lansing, Michigan. Apples harvested on Oct. 2 were considered preclimacteric since their ethylene level was less than 1 PPM (see chapter 1), and were considered suitable for long term storage. Apples harvested on Oct. 9 had an average 144 internal ethylene level of .226 PPM and were considered suitable for short term storage. Fruits were collected and randomized on the same day. Sixty apples were randomly selected and placed in plastic containers, with an inlet and an outlet to establish the CA storage. Thirty containers were used for each storage condition. These containers were then divided into three groups. One group was placed in a air- cooled room at 3.3°C, and the other two groups were stored in CA with 3% C02, 2% 02, a temperature of 3.3°C and a relative humidity of 98%. Two ethylene levels were established: low ethylene (<1 PPM) and high ethylene (>100 PPM). These conditions were established twenty—four hours after harvest. To determine the effect of storage time , apples were removed monthly from storage for a duration of six months. After the apples were removed from air, high and low ethylene CA storage, they were placed for a duration of 2, 7 and 14 days at 22°C for evaluation of volatile compounds during the ripening process. VOLATILE EXTRACTION AND COLLECTION METHOD 1: VACUUM DISTILLATION TECHNIQUE Vacuum distillation techniques have been employed to isolate volatile components from food (Merritt et al., 1959; Angelini et al., 1967). For this study apples were cleaned, cored with a 0.6- 145 inch diameter cork borer, and quartered. One hundred and fifty grams of cored and sliced apples, together with 150 m1 of distilled water containing 10 grams of salt were blended by using a Waring blender at high speed for 1 min. Salt was added to prevent browning during blending. Following blending, 100 grams of this mixture were transferred to a 500 ml round bottom flask. The collection of volatile compounds was carried out for four hours using a mineral oil vacuum distillation technique. This apparatus is shown in Figure 1. During the collection period, the vacuum was set at 20 mm Hg and the flasks containing the samples were held in a water bath maintained at 40:2°C. Two ml of double glass distilled water were added to the traps to create a coating area for the volatiles. The traps were kept in liquid nitrogen. After four hours, the vacuum was released gradually. Twenty m1 of isopentane were added to the traps. The content of the traps was then transferred to a separatory funnel which was shaken for five min. The pooled isopentane fractions were dried over anhydrous sodium sulfate. This process was repeated four times. A total of 80 ml of isopentane extract was collected. The volume of the extract was then reduced to 10 ml with a Kuderna-Danish concentrator. The water bath temperature was maintained at 70'C. During distillation, the level of the water in the heating bath was kept just below that of the isopentane extract. The volume of the extract was further 146 Figure 1. Apparatus for collecting volatile compounds from Empire apple by applying the high vacuum distillation technique. A-Heater, B-Apples+water+salt, C-ThermometerD-Dewar flask with liquid nitrogen, E-Cooled trap,F-Vacuum Pump and G-Vacuum gauge. 148 reduced to 1.5 ml with a gentle flow of nitrogen. These extracts were stored in a freezer (-20°C) until the time of analysis. 1.5 ul of the extract was used for gas chromatographic analysis. METHOD II:LIKENS- NICKERSON TECHNIQUE Three hundred grams of cleaned, cored and sliced apples were used for the collection and extraction of the volatiles. Three hundred ml of double glass distilled water containing 15 grams of salt were added to the apples and blended for two min at high speed in a Waring blender. The puree was placed in a 2 L boiling flask. The Likens-Nickerson extractor was assembled as described by Likens and Nickerson (1964) and as modified by MacLeod and Cave (1975). Distilled water served as the high density layer, and isopentane (Fisher) as the low density layer. Twenty-five ml of isopentane were used, and the distillation was carried out for four hours. A hot jacket was used to adjust the isopentane distillation rate at 1 ml/min before starting distillation of the aqueous apple slurry. The apple puree had to be heated slowly, since foaming was a major problem. When all the apple had broken down, after approximately one hour, the foaming subsided. A dry ice- acetone condenser was attached to prevent the loss of volatiles. Following distillation/extraction for four hours, the 149 isopentane was dried over anhydrous sodium sulfate. It was noted at this time that the residues did not possess any appreciable aroma resembling apples. The extract was then concentrated using the procedure described in method I. GAS CHROMATOGRAPHIC ANALYSES OF THE VOLATILES Analyses of the volatiles were performed on a Hewlett Packard Model 5850 A equipped with a flame ionization detector. A non-bonded fused silica capillary column, 0.25 mm x 60 m, coated with Carbowax 20 M (Supelco Inc., Bellfonte, PA) was used. The inlet temperature was 200'C and the detector temperature was maintained at 275°C. The inlet pressure was adjusted to achieve an average linear velocity of 0.7 ml/min as measured by methane injection. The helium flow rate was 28 ml/min with column head pressure of 1.8 kg/cmi A column flow rate of 0.75 ml/min was established. The detector was supplied with 240 ml/min air. The temperature program was 40°C for 15 min, and a rate of 2.5'C/min up to 180°C with a holding time of 45 min. A split ratio of 1:20 was used. The outlet signal was fed through a digital integrator to the recorder. GAS CHROMATOGRAPHIC AND MASS SPECTROMETRIC ANALYSES OF VOLATILES The system used for GC/MS analysis of the volatiles was a Nermag R-10-10C interfaced with a Delsi splitless capillary GC and a bonded phase Carbowax 20 M (60m x 0.25 mm). The temperature program was 40°C for 3 min, and a rate of 2.5 150 'b/min up to 180 °C with a holding time of 25 min. The instrument was operated in an electron impact mode at an ionization voltage of 70 eV. The ion source temperature was 200°C. A scan of 1 second was used over a mass range of 40-250 amu o QUANTITATION OF VOLATILE COMPOUNDS Quantitation of volatiles identified in Empire apples using vacuum distillation and Likens-Nickerson extraction distillation was determined according to the procedure described in Chapter 2. RESULTS AND DISCUSSION The percent recoveries obtained for both vacuum distillation and Likens-Nickerson extraction/distillation techniques are presented in Table 1. Superior extracts were obtained more readily and more efficiently by using the Likens-Nickerson apparatus. This technique only required a small volume of solvent to extract large quantities of sample. The small quantity of solvent and general convenience of this device are reasons for its frequent use (Bemelmans, 1978). Extracts obtained with vacuum distillation resembled fresh apple aroma more closely than extracts obtained by the Likens-Nickerson technique. This is primarily due to the low temperature extraction of volatiles using vacuum distillation. 151 Table 1. Percent recovery (2) for selected compounds using vacuum distillation and Likens-Nickerson techniques. Compounds Vacuum Distillation Likens-Nickerson Percent Recovery (2) Ethyl acetate 65.3 62.9 2-Methy1 butanal 43.5 49.7 Propyl acetate 65.5 69.0 Ethyl-Z-methyl butanoate 78.7 89.8 Ethyl caproate 65.8 69.9 1-Pentanol 58.7 63.8 152 Extracts obtained using the Likens-Nickerson technique had a slightly cooked flavor, due to the higher temperature applied during extraction. The major difference between these two techniques and the headspace technique consists of the lower concentrations of low boiling point compounds such as methyl actate, ethyl acetate, methyl- 2-methyl butanoate and ethyl formate. However, extracts obtained by method I had an aroma closer to the headspace technique compared to extracts obtained by method II. The higher concentrations of low boiling compounds in the extracts obtained by the headspace technique could explain the fresh aroma of the headspace extract. Fifty-eight compounds were identified in the extract obtained from vacuumn distillation by GC and coupled GC-MS techniques. The compounds identified included 26 esters, 8 aldehydes, 14 alcohols and 5 ketones (Table 2). Most of the compounds identified in this work have been identified in various other apple varieties. However, 3 esters not identified in apples before were reported in this work. This could be due to varietial differences. These compounds are: Methyl propyl 2-methyl butanoate, 2-methyl propyl hexanoate, and methyl 2-ethyl heptanoate. The mass spectrograph of these compounds are given in Figures 1-15 in Appendix C. Butyl 3-methy1 butanoate was only reported previously by Jacob et al. 1969 and this report confirms their findings. 153 Table 2. Volatile compounds collected during storage and ripening period at 22‘C. by vacuum distillation. Esters Aldehydes Alcohols Methyl acetate Butanal Butanol Ethyl acetate Pentanal 1-Pentanol Propyl acetate Isopentanal 2-Pentanol Butyl acetate Hexanal 3-Pentanol Pentyl acetate 2-Hexenal 1-Hexanol Isopentyl acetate (E)-2-hexenal 2-Hexanol Hexyl acetate Heptanal 3-Hexanol Ethyl phenyl acetate Methyl butanoate Ethyl butanoate Propyl butanoate Butyl butanoate Pentyl butanoate Phenyl acetaldehyde Nonanol Methyl 2-methyl butanoate Ethyl 2-methy1 butanoate Methyl propyl 2-methyl butanoate * Butyl 3-methyl butanoate Propyl formate Butyl formate Pentyl formate Ethyl propionate Butyl pentanoate Ethyl hexanoate Hexyl hexanoate 2-Methyl propyl hexanoate * Methyl 2-etyhl heptanoate * 2-Methyl propanol 2-Methyl butanol 3-Methyl butanol 2-Methyl pentanol (Z)-3-hexen-l-ol (E)-3-hexen-1-ol Ketones Miscellaneous 2-pentanone Ethyl 2-methyl benzene 3-Pentanone 1,2 Dimethyl benzene 2-Hexanone Tridecane 2-Heptanone Pentadecane 3-Heptanone Octadecane * These compounds have been identified in apples for first time. 154 Seventy-seven compounds were identifield by GC and coupled GC-MS from the extract obtained by the Likens- Nickerson extraction/distillation technique. The compounds identified include: 39 esters, 9 aldehydes, 19 alcohols and 5 ketones (Table 3). Out of these compounds, 7 esters, 1 aldehyde, 4 alcohols and 4 others have been reported for the first time in apple extract. These compounds are: 2-methyl 2-methyl butyl propionate, 3-methyl butyl propionate, methyl propyl 2-methyl butanoate, 2-methyl hexanoate, ethyl 1-methyl hexanoate, 2-methyl propyl hexanoate, 2-methyl butyl hexanoate, 2-butyl 2-octanal, 2-cyclo hexanal, isooctanol, 4- methyl 4-octanol, 7-methyl 4-octanol, 6-ethyl undecane, 2- methyl pentanoic acid, 1,4 dimethyl benzene, and ethyl propyl benzene. The most dominant volatiles in both extracts are esters, alcohols and aldehydes. The esters which contribute to the flavor of Empire apples are: ethyl acetate, ethyl propionate, methyl butanoate, ethyl butanoate, butyl butanoate, methyl-2- methyl butanoate, ethyl-Z-methyl butanoate, pentyl acetate, isopentyl acetate, and hexyl acetate. Of the aldehydes and alcohols, butanal, hexanal, (E)-2 hexenal, 2-hexenal, 1- butanol, 2-pentanol and 3-hexanol were observed in the highest concentrations. The concentrations of these volatiles in apples stored in air were higher compared to those stored in high and low ethylene CA. The concentrations of these 155 Table 3. Volatile compounds collected during storage and ripening at 22‘C by Likens-Nickerson extraction technique. Esters Aldehydes Alcohols Methyl acetate Butanal Butanal Ethyl actate Pentanal l-Pentanol Propyl acetate IsOpentanal 2-Pentanol Butyl acetate Hexanal 3-Pentanol Pentyl acetate 2-Hexanal 1-Hexanol Isopentyl acetate (E)-2-hexenal 2-Hexanol Hexyl acetate Heptanal 3-Hexanol Ethyl phenyl acet Phenyl acetaldehyde 1-Heptanol Ethyl propionate 2-Methyl 2-methyl- butyl propionate * 3-Methyl butyl- propionate * Methyl butanoate Ethyl butanoate Propyl butanoate Butyl butanoate Pentyl butanoate Isopentyl butanoate Hexyl butanoate Methyl 2-methyl butanoate Methyl propyl 2- 2-Butyl 2-octanal * 2-Methyl propanol 2-Methyl butanol 3-Methyl butanol 2-Methyl pentanol (Z)-3-hexenol (E)-3-hexenol 2-Cyclo hexenal * Nonanol Isooctanol * 4-Methy1 4-octanol * 7-Methyl 4-octanol * MisEElIaneous methyl butanoate * Ketones 3-Methyl propyl 3- methyl butanoate Z-Pentanone Methyl butyl 3-Pentanone 2-methyl butanoate 2-hexanone Ethyl 2-methyl butanoate 2-Hexanone Ethyl 3-methyl butanoate 2-Heptanone Butyl 3-methyl butanoate 3-Heptanone Propyl formate Butyl formate Pentyl formate Methyl pentanoate Propyl pentanoate Butyl pentanoate Ethyl hexanoate Butyl hexanoate Hexyl hexanoate 2-Methyl hexanoate * 2-Methyl propyl hexanoate * 2-Methyl butyl hexanoate * Ethyl 1-Methyl hexanoate * Heptyl heptanoate 1,4 Dimethyl benzene* 6-Ethyl undecane * 9-Hexadecenoic acid Ethyl propyl benzene * 2-Methyl pentanoic acid * * Compounds reported for the first time in apples. 156 volatiles increased over 14 days, which is due to ripening. (E)-2-hexenal is not present in the whole apples (see Chapter 2) but is formed very rapidly upon crushing (Dimick et al., 1983). As in many other apple varietes, low concentrations of (E)-2-hexenal, hexanol and ethyl 2-methyl butanoate greatly contribute to the flavor of Empire apples (Flath et al., 1967). According to Kim and Grosch (1979), the formation of (E)-2-hexena1 and hexanal in apples is due to lipoxygenase activity which has the ability to form hydroperoxides, thus producing 13-hydroperoxyoctadeca-9-11 dienoic acid from linoleic. The typical flavor compounds of apples are not produced during growth, and they are not present at a great level at harvest. The biogenesis of volatiles in apples is a dynamic system. It is produced during a short period, and is related to climacteric rise (Tressl et al., 1975). The respiration rate increases during storage and especially during ripening at higher temperatures. Apples stored in air at 3.3 ‘0 had a much higher concentration of volatile esters, aldehydes and alcohols compared to those stored in low and high ethylene CA. This is due to the natural ripening process of apples. It is well documented that 02 is required for the conversion of ACC to ethylene and ethylene has long been known to induce ripening ( Adams and Yang, 1979). Apples stored in all three storage 157 conditions underwent a slow ripening process due to the low temperature, and consequently, the total amount of volatiles produced was low during the first month of storage. A typical GC-MS of apples harvested on Oct. 2 after four months of storage in air at 3.3°C and after ripening at 22 °C for 7 days is shown in Figure 2. Figures 3, 4, and 5 illustrate the total amount of volatile esters, aldehydes, alcohols and ketones obtained from extract by vacuum distillation technique. Figures 6, 7, and 8 illustrate the results for extracts obtained by the Likens-Nickerson technique. These figures are for apples stored in air at 3.3°C after a ripening process of resp. 2, 7, and 14 days. In all cases, the total amount of volatile compounds increased until the fourth month of storage for apples harvested on Oct. 2. Apples harvested on Oct. 9 had higher levels of volatiles at harvest and had the highest total level of volatiles after three months of storage. Apples harvested on Oct. 2 reached their climacteric rise after four months of storage due to the lower internal ethylene level at harvest. The initial internal ethylene for apples harvested on Oct. 2 averaged 0.097 PPM, and for apples harvested Oct. 9 averaged 0.226 PPM, with a flesh firmness of 18.00 and 17.75, respectively. Generally, apples stored in high ethylene CA had lower total volatiles compared to those stored in air. This is due to the relatively high C02 (3 Z) and low 02 (2 %) levels, 158 Figure 2. Gas chromatograph and mass spectra graph of Empire apples stored for four months in air at 3.3 'C and after 7 days of ripening at 22°C. For GC-MS conditions, refer to text. 159a aHouO mme DNLfiummmG AmwNOUMdV swam Hmmm wwam mm»m mam» mm.mm pawm.sm aim» . m0 H0 Amubb mmamm wm.ms smsms a a Amqu n 0» Ad . l.-I .. l a . r. .55. .p. .6781: r . 1F... U0u00 $0.00 00~00 Ha00u00 an0u00 159b wlmm DmIZuLLmL Amwdmmmmu d m cm mmmm immi Ammo snow mama mwu» U pamwsum u.xc.pm L H.Hm.um H.ua.mm . Hupaupm f H.um.wm . ».mm.mm r . C a amuse unmonoo Humm.oo - - - unwowos ».wm.oo ~.xo.oa pnxm.oo 160 Figure 3. Total esters, aldehydes, alcohols and ketones obtained by high vacuum distillation technique. Apples were stored for six months storage in air at 3.3‘C and subsequently for 2 days of ripening at 22‘C. The numbers are average of three replicates. 161 _sM6e __.\\\.M6//6 40- m w m. m KSM665 m m m m =§m666 _ _ _ _=§smw66666.. Emmmmwi 666666666666 Emmmmm 66666666666663 =§§§MM666666 3 W/ 1 __ 668: é a o _ “motor .. AEQQV cozozcmocoo Storage Months 162 Figure 4. Total esters, aldehydes, alcohols and ketones obtained from high vacuum distillation technique. Apples were stored for six months in air at 3.3‘C and subsequently for 7 days of ripening at 22°C. The numbers are the average of three replicates. 163 60! 50- - Ketones - Alcohols - Aldehydes =.§\\\\\\\6 S r e t S E _ §IV//////%//////////////////////////////6 $6 //////////////////////////////////////////63 u___.\\\\\\\\\\\\\\s . .___.\\\\\\\\\\\. _____§ ____.\\\\\\\s hf 66666666666666. 2 6///////////////////////////////////6.. s66 __\..6///6 =wa ___.\\.nu..6//////////6 ___s.m6///6 4 ////////////////////6 Storage duration (months) __ 6.28: ==§\\\\\\\\\\\\s 6///// o .V/////////////////6 _ smote: n=V\\\\\\\\\\\\\\. - 4 q m m A863 cozobcmocoo 204 _ . _ 0 0 Figure 5. 164 Total esters, aldehydes, alcohols and ketones obtained by high vacuum distillation technique. Apples were stored for six months in air at 3.3‘C and subsequently for 14 days of ripening at 22‘C. Numbers are the average of three replicates. 165 — Ketones - Alcohols ==§ _\m//6.6 __\\\n11//// m _.\.m /////////65 ...... _=\\\\\\\\\\1“I///////5 _ _\\\R /////////////////64 6/////////////////////////////,/////////////////////////64 __\\\\\\ /////////////////63 =\\\\\\\\\:////// //////////////63 - Aldehydes g . .7 //////////////,////////////////////////////62 //////////////////////////2 /////////////////6.1 .///////////////.1 //////////////6o 7//////////60 \\\\\\\\\\\\§ ==§ _=w\\\\\\ __ 62cc: =5§ 2826: =..§ 1001 90- 80- 7o- - a _ q _ . _ u q _ 0 0 O 0 0 O 6 5 4 3 1 20- AEQQV cozobcmocoo Storage duration (months) 166 Figure 6. Total esters, aldehydes, alcohols and ketones obtained by Likens-Nickerson extraction technique Apples were stored for six months in air at 3.3°C and subsequently for 2 days of ripening at 22‘C. The numbers are the average of three replicates. 167 - Aldehydes Esters - Ketones - Alcohols i _\\.m//66 __\\\\\\M7////66 _\\m6/////66 =\\\m6/////6.s __\\\\\m /////////////////6 6//////////////////////////////////////////////6.4 .6/ i /////////////////////////////////////,3 ___\\\\\\\\\\m6///////////////////63 __ 6.... $1 2...... g 40- q 0 2 AEQQV cozobcmocoo 30- 10- O Storag: Months Figure 7. 168 Total esters, aldehydes, alcohols and ketones obtained by Likens-Nickerson extraction technique. Apples were stored for six months in air at 3.3’C and subsequently for 7 days of ripening at 22°C. The numbers are the average of three replicates. 169 ===§ 6.76 . 5 L_7///////////6 ____sw7//6 4 §1 17//////////////////////////////////6 ___§W666666666666666 ... ___§“7666666 . 76666666667666 2 ._._\\\\\\\\\\\\\\6 //////////////////////////////6 _.\\\N %//////////////////////6 l 1 ___§\\\\..II4%/////////////////,//////////6 _. .63.. _____6\\\\\\\\\\\\\. 666/6 0 :62... ==6\\\\\\\\. 166/6 =.§ Esters ___V\\\\\\\\\. - Ketones - Alcohols - Aldehydes 601 50- d q q q u a d d u 0 0 0 0 0 4 3 2 1 AEaav cozobcmocoo Storage duration (months) Figure 8. 170 Total esters, aldehydes, alcohols and ketones obtained by Likens-Nickerson extraction technique. Apples were stored for six months in air at 3.3'C and subsequently for 14 days of ripening at 22‘C. Numbers are the average of three replicated. Concentration (ppm) 100 90 80 7O 60 50 4O 30 2O 1O Harvest l Harvest II 171 — Ketones Alcohols - Aldehydes - Esters 1 2 3 4 5 Storage duration (months) 172 which reduce the respiration rate and delay ethylene production. In addition, high C02 retards the effect of endogenous ethylene. Knee (1980) suggested that since the affinity of cytochrome oxidase for 02 is so high, the inhibition of ethylene under commerical low 02 storage is unlikely. However, in this study it was shown that high C02 and low 02 levels delayed the ripening process and reduced the level of volatiles. After six months of storage, apples in a high ethylene CA had a much lower volatile content than those stored in air (Figures 9, 10, and 11 for extracts using method I and Figures 12, 13, and 14 for extracts using method II). Low ethylene CA storage had a greater effect on the total level of volatiles in Empire apples. These apples had the least amount of volatiles compared to apples in either air or high ethylene CA storage (Figures 15, 16, and 17 for extracts using method I and Figures 18, 19, and 20 for extracts using method II). This clearly indicates that high C02 , low 02 in combination with low ethylene CA storage has a determining effect on the final flavor of Empire apples. Particularly, low ethylene CA greatly reduced the level of volatiles after six months of storage (Figures 15-20). The ACC content of apples stored in low ethylene CA was lower compared to both air and high ethylene CA stored apples. During storage, the ACC content of apples stored in either low or high ethylene CA stayed almost unchanged for the first three months of storage 173 Figure 9. Total esters, aldehydes, alcohols and ketones obtained by high vacuum distillation technique. Apples were stored for six months in high ethylene CA (>100 PPM) at 3.3’C and sybsequently for 2 days of ripening at 22°C. Numbers are the average of three replicates. 174 __ .826: . .62.: =§ ==§\\\\\\\\\\\\\\. ==§ Ketones Alcohols dehydes \ \x. ___.\\\\\1 _____V\\\ \. ==V\\\\\\\\\\\\\\\. SW76 §WV6 _\\m66 .7/////////////////6 m ////////////////6. 1/////////////6 ___§W6666 ... 166666666 ,//////////////////////////////////6 . 3 2 66 2%” // 11’ I F/ F” 30? _ 0 2 AEQQV cozobcmocoo — O 1 Storage duration (months) 175 Figure 10. Total esters, aldehydes, alcohols and ketones obtained by high vacuum distillation technique. Apples were stored for six months in high ethylene CA (>100 PPM) at 3.3°C and subsequently for 7 days of ripening at 22°C. Numbers are the average of three replicates. 176 __ c820: . .moboz i=1 - Ketones - Alcohols - Aldehydes - Esters =§\\\\\\\\\\\\\\\1 ___\\\\\\\\\\\\\\\\\\\\ 50- d O 4. AEQQV cozobcmocoo Storage duration (months) 177 Figure 11. Total esters, aldehydes, alcohols and ketones obtained by high vacuum distillation technique. Apples were stored for six months in high ethylene CA (>100 PPM) at 3.3°C and subsequently for 14 days of ripening at 22°C. Numbers are the average of three replicates. 178 50 Harvest ll '2’; — Ketones E O :1: 4O — Alcohols 30 - Aldehydes Esters Concentration (ppm) N O 10 O ' 1 2 3 4 5 6 Storage duration (months) 179 Figure 12. Total esters, aldehydes, alcohols and ketones obtained by Likens-Nickerson extraction technique. Apples were stored for six months in high ethylene CA (>100 PPM) at 3.3'C and subsequently for 2 days of ripening at 22‘C. Numbers are the average of three replicates. 180 666 SW66 _ _ _ __4\§M6//////6 =V\\\\\.W6////6/////////////A Aldehydes Ketones Alcohols Esters . g 6/////////////////////////////6 a§ ./////////////////////////////////6 =w\\\\\\\.W6//////////////////6 ____§ .///////////////////6 _=.\\\\\\1\1\\\& ==§ 6///////////////6 .7//////////6 ___.\\\\\\\. __ 36:2. §H 6666 2...... §1 6///////////,////////,/6 301 _ 0 2 AEQQV cozobcoocoo _ 0 1 0 6 5 4 3 2 1 0 Storage duration (months) 181 Figure 13. Total esters, aldehydes, alcohols and ketones obtained by Likens-Nikerson extraction technique. Apples were stored for six months in high ethylene CA (>100 PPM) at 3.3°C and subsequently for 7 days of ripening at 22°C. Numbers are the average of three replicates. 182 _ =\.I..6 _\\\m7///5 _ . . . =\\\\.W6//,6/65 _=\\\\\\\\.m6///66///64 S§ 66666666664 ===.\\\\\\\\\\\\\\\\ . 76666666666663 ___.\\\\\\\\\\\\\x 6666666663 _____6\ . 666666662 ___\\\\\\\\\\l6////////////////2 __=_\\\\. \x n //////,6/////6 |\l\\\\‘ [666 Aldehydes Ketones Alcohols Esters __ ...32. é . 66666666 263:. § M66666666 SO- _ O 4. 41 a a _ q _ O O O 3 2 1 AEQQV cozobcmocou I. 0 -7////////////61 0 Storage duration (month:) 183 Figure 14. Total esters, aldehydes, alcohols and ketones obtained by Likens-Nickerson extraction technique. Apples were stored for six months in high ethylene CA (>100 PPM) at 3.3'C and subsequently for 14 days of ripening at 22°C. Numbers are the average of three replicates. Concentration (ppm) 184 50 Harvest ll _ - Ketones '53 $3 40 3‘3 - Alcohols 30 - Aldehydes Esters 20 10 0 1 2 3 4 5 6 Storage duration (months) Figure 15. 185 Total esters, aldehydes, alcohols and ketones obtained by high vacuum distillation technique. Apples were stored for six months in low ethylene CA (<1 PPM) at 3.3°C and subsequently for 2 days of ripening at 22°C. Numbers are the average of three replicates. 186 __ 528: ____§ - Ketones __N\\\\\\\. __=.\\\\\\\. ____N\\\\\\\\\\\\\ ____N\\\\\\\\. - Alcohols Lil“ /, !,',','. .Nfl. Nm/fl 6666666” 766666 - Aldehydes Esters 6666/66 66666, 6666/66 666/6 66666662 ___.\\\\. /66 =\\\\ ”__N\\\\. 76666666666660 é _ 2822i . §l l6666666/o 30- _ 0 2 AEQQV cozobcmocoo _ 0 1 O 1 Storage duration (months) Figure 16. 187 Total esters, aldehydes, alcohols and ketones obtained by high vacuum distillation technique. Apples were stored for six months in low ethylene CA ((1 PPM) at 3.3°C and subsequently for 7 days of ripening at 22°C. Numbers are the average of three replicates. Concentration (ppm) 50- 4o- 30- 20- 10- Harvest l MM] Harvest ll \\I \‘ V// 1 2 188 3 \fi ‘1 4 — Ketones - Alcohols - Aldehydes — Esters 5 6 Storage duration (months) Figure 17. 189 Total esters, aldehydes, alcohols and ketones obtaines by high vacuum distillation technique. Apples were stored for six months in low ethylene. (<1 PPM) CA at 3.3°C and subsequently for 14 days of ripening at 22'C. Numbers are the average of three replicates. 190 NW6 __NW6 6 4 _____N\\\\\\\\ 66666 _==_N\\\\\\\\\\\\\\t 66666666666666 3 ==N\\\\\\\\\\\\. 666666666 _____N\\\\\\\\\t .6666 2 _____N\\\\\\\\M 666 ==N\\N 666 __ ES: g 6666666666666 0 2322.. ====N\\\\\\\\\\\\\\\\\\\N 66666666666666 Alcohols S e n O t e K s e d Y .n e d N 5 _ - Esters . ___=\\\\\\\\ Storage duration (months) _ . . . . 0 0 O O 1 50: 4o- AEaav cazobcmocoo 191 Figure 18. Total esters, aldehydes, alcohols and ketones obtained by Likens-Nikerson extraction technique. Apples were stored for six months in low ethylene CA ((1 PPM) at 3.3°C and subsequently for 2 days of ripening at 22°C. Numbers are the average of three replicates. 192 .\\\‘ '///. NW7 _anlu// E=NM66 .6664 666 s =N|I666 =_N|66666/6,. 2 . 7666 ___Nn.l"|.l6666 7/// Esters - Ketones — Alcohols - Aldehydes =N EN EN EN __ “more: ==N\\\\\\\\\\\\\\\\\\\\\. :85: EN 30- d 4 q . 0 0 0 2 .| 9:93 cozobcoocoo 666 0 Storage duration (months) 193 Figure 19. Total esters, aldehydes, alcohols and ketones obtained by Likens-Nikerson extraction technique. Apples were stored for six months in low ethylene CA (<1 PPM) at 3.3°C and subsequently for 7 days of ripening at 22‘C. Numbers are the average of three replicates. 194 -- Ketones - Alcohols 'III. Nm //// .\\\\ — Aldehydes u NW6 _ __Nm6 ___N 6 . \N 66 EN , 666 __N 66 ___N ,6 6 ____N\\ 666 ____Nm66 __ amQZOI 6666666666,. .66666666 _ 628: jNi‘ . 50- 4o- . . . O O 3 2 A89; cozobcoocoo 10- O 6 1 Storage duration (months) 195 Figure 20. Total esters, aldehydes, alcohols and ketones obtained by Likens-Nikerson extraction technique. Apples were stored for six months in low ethylene CA (<1 PPM) at 3.3‘C and subsequently for 14 days of ripening at 22°C. Numbers are the average of three replicates. 196 __ ammZOI _ «motor g ====N\\\\\\\\\\\\\\\\\\N s e n O t e K _ _____ _____N\\\\\\\\\\N N\\\\\N ////./ _N N __NM65 m ___Nm.......6s _ __NW65 Alcohols 66664 64 ===N\\\\\\\\\\\\N 63 _=_N\\\\\\\\\\N 666/63 666662 _____N\\\\\\\\\\\\N W==N\\\\\\\\N =.=N\\\\\\\\\N _=_N\\\\\\\\\N 66662 6666 6o 6o 6.. 50w d O 4 - d 0 O 3 2 AEaav cozobcmocoo _ t _ 0 0 Storag: duration (month; 197 and increased thereafter (refer to Chapter 1) until the sixth month of storage. Liu (1985) used two different ethylene levels (10 ul/l and 500 ul/l) in the CA storage of McIntosh apples and found no significant difference in the flesh firmness. He also concluded that low ethylene CA storage is only advantageous for early harvested McIntosh apples, i.e. at the preclimacteric stage. In addition, it has been shown that ethylene removal during CA storage is beneficial for the retention of fruit firmness in McIntosh apples. (Laughed et al., 1973; Liu, 1977, 1978, 1979; Lidster et al., 1983). According to a study conducted by Liu (1985), apples stored in low ethylene CA for seven months had better flesh firmness, normal sweetness and acidity. However, according to a consumer panel these apples were lacking in the full aroma of the ripe McIntosh apple when they were cooked. The findings in this study agree with Liu's investigations. EFFECT OF RIPENING When naturally ripened, low boiling point esters build up to a maximum in a few weeks after postharvest depending on the holding temperature (Dimick et al., 1983). Guadagni et al. (1971) reported that the temperature of ripening is an important factor in the flavor of climacteric fruits. Aroma production is inhibited at 46°C, and at 32‘C the production 198 rate of esters increases with the highest rise at 22 °C. To study the effect of ripening on flavor production, Empire apples were held at 22°C for 2, 7 and 14 days. Generally, apples from all three storage conditions were able to generate volatiles. However, the production of volatiles differed with each storage condition. Apples stored in air were able to generate volatiles to a greater extent than those stored in either low or high ethylene CA. Also, apples stored in either low or high ethylene CA lost their ablity to generate volatiles after four months of storage. Storage duration had its highest effect on apples stored in low ethylene CA. The ester/aldehyde ratios after four months of storage for air, low and high ethylene CA are presented in Table 4. The ester/aldehyde ratios show that apples stored in air were able to produce more esters compared to apples stored in low or high ethylene CA. There does not appear to be a difference in the ester/aldehyde ratios between the two harvest dates. Therefore, the ester/aldehyde ratio is not a good indicatior of the ripening effect, even though it has been used by other researchers. 199 Table 4. Ester/aldehyde ratios at harvest and after four months of storage in air, low and high ethylene CA. The ratio is reported after 2, 7 and 14 days of ripening at 22°C. Harvest I Harvest II 2 7 *TI 2 7 14 (days) (days) HA 0.74 0.91 0.99 0.76 1.03 1.20 Air 2.81 2.73 3.78 2.70 3.80 5.13 HCA 2.07 1.95 2.97 2.80 1.56 1.67 LCA 1.95 2.18 1.85 1.62 1.63 1.70 Harvest I on Oct. 2 Harvest II on Oct. 9 HA- at harvest HCA- high ethylene CA LCA- low ethylene CA 200 REFERENCES Adams, D. 0., and Yang, S. F. 1969. Ethylene biosynthesis: identification of 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proc. Natl. Acad. Sci. 76: 170. Angelini, P., Fross, D. A., Bazinet, M. L., and Merritt, C. Jr. 1967. Methods of isolations and identification of volatile compounds in lipids. J. Amer. Oil Chem. Soc. 44(2): 26. Bangerth, F. 1978. The effect of substitiuted amino acid on ethylene biosynthesis, respiration, ripening and preharvest drop of apple fruits. J. Amer. Soc. Hort. Sci. 103: 401. Bemelmans, J. M. 1978. Review of isolation and concentration techniques. In "Progress in Flavour Research," D. G. Land and H. E. Nursten (Ed.), p. 79. Applied Science Publishers Ltd., London. Burg, S. P., and Burg, E. A. 1966. Fruit storage at subatmospheric pressures. Science 153: 314. Dimick, P. S., Hoskin, J. C., and Acree, T. E. 1983. Review of apple flavor-state of art. CRC Crit. Rev. Food Sci. Nutr. 181: 387. Fidler, J. C., and North, C. J. 1969. Production of volatile organic compounds by apples. J. Sci. Fd. Agric. 20: 521. Fidler, J. C., and North, C. J. 1971. The effect of periods of an anaerobiosis on the storage of apples. J. Hort. Sci. 46: 213. Flath, R. A., Black, D. R., Guadagni, D. G., McFadden, W. H., and Schultz, T. H. 1967. Identification and organoleptic evaluation of compounds in apple essence. J. Agric. Fd. Chem. 15: 29. Forsyth, F. R., Eaves, C. A., and Lightfoot, H. J. 1969. Sensory quality of McIntosh apples as affected by removal of ethylene from controlled atmosphere. Cand. J. Plant Sci. 49: 567. 201 Guadagni, D. G., Bomben, J. L., and Hudson, J. S. 1971. Factors influencing the development of aroma in apple peel. J. Sci. Fd. Agric. 22: 110. Jacob, M. A., Hippler, R., and Luethi, H. R. 1969. Occurrence of hexyl-Z-methylbutyrate in apple aroma. Mitt. Geb. Lebensmittelunter. Hyg. 60(3): 223. Janes, H. W., and Frenkel, C. 1978. Inhibition of ripening processes in pears by inhibitors of cyanide-resistant respirators and by silver. J. Amer. Soc. Hort. Sci. 103(3): 394. Kidd, F., and West, C. 1939. The production of volatiles by apples: Effects of temperature, maturity, technique of estimation, etc. Rept. Food Invest. Bd. (Gt. Brit.), p.136. Kim, I., and Grosch, W. 1979. Partial purification of lipoxygenase from apple. J. Agric. Fd. Chem. 27: 243. Knee, M. 1975. Changes in structural polysaccharides of apple ripening during storage. Facteurs et regulation de la maturation des fruits. Colloq. Intern. CNRS 238: 241. Knee, M. 1980. Physiological responses of apple fruits to oxygen concentration. Ann. App. Biol. 96: 243. Knee, M. and, Hatfield, S. G. 1981. The metabolism of alcohols by apple fruit tissue. Ann. App. Biol. 98: 157. Knee, M. 1982. Fruit softening III. Requirement for oxygen and ph effects. J. Exp. Bot. 33: 1263. Knee, M., Looney, N. E., Hatfield, S. G., and Smith, S. M. 1983. Initiation of rapid ethylene synthesis by apple and pear fruits in relation to storage temperature. J. Exp. Bot. 34: 1207. Knee, M. 1985. Evaluating the practical significance of ethylene in fruit storage. In " Ethylene and Plant Development," G. A. Roberts and J. A. Tucker (Ed.), p. 297. Butterworths, London. Likens, S. T., and Nickerson, G. B. 1964. Detection of certain hop oil constituents in brewing products. Proc. Amer. Soc. Brew. Chem. 5: 13. 202 Lidster, P. D., Lightfoot, H. J., and McRae, K. B. 1983. Fruit quality and respiration of McIntosh apples in response to ethylene, very low oxygen and carbon dioxide storage atmosphere. Scientia Hart. 20: 71. Liu, F. W. 1977. Varietal and maturity differences of apples in response to ethylene in controlled atmosphere storage. J. Amer. Soc. Hort. Sci. 102: 93. Liu, F. W. 1978. Effects of harvest date and ethylene concentration in controlled atmosphere storage on the quality of McIntosh apples. J. Amer. Soc. Hort. Sci. 103: 388. Liu. F. W. 1979. Interaction of diaminozide, harvesting date, ethylene in CA storage on McIntosh apple quality. J. Amer. Soc. Hort. Sci. 104: 599. Liu, F.W. 1985. Low ethylene controlled-atmosphere storage of McIntosh apples. In "Ethylene and Plant Development," T. A. Roberts and G. A. Tucker (Ed.), p. 385. Butterworths, London. Looney, N. E. 1972. Interaction of harvest, maturity, cold storage, and two growth regulators on ripening of 'Bartlett' pears. J. Amer. Soc. Hor. Sci. 97(1): 81. Lougheed, E. C., Franklin, E. W., Miller, S. R., and Proctor, J. T. 1973. Firmness of McIntosh apples as affected by alar and ethylene removal from the storage atmosphere. Can. J. Plant Sci. 53: 317. MacLeod, A. J., and Cave, S. J. 1975. Volatile flavor components of eggs. J. Sci. Fd. Agric. 26: 351. Mehlitz, A., and Gierschner, K. 1961. Beitrag zur qualitasbeurteilung von aromakonzentraten und aromadestillaten aus fruchtsaften. Pro. Sci. Tech. Comm. Int. Fed. Fruit Juice Prod. 3: 51. Merritt, C., Bresnick, S. R., Basinet, M. L., Walsh, J. T., and Angelini, P. 1959. Determination of the volatile components of foodstuffs. Techniques and their application to the studies of irradiated beef. J. Agric. Fd. Chem. 7: 784. Peacock, B. C. 1972. Role of ethylene in the initiation of fruit ripening. Queensland J. Agric. Anim. Sci. 292: 137. 203 Tressl, R., Holzer, M., and Apetz, M. 1975. Biogenesis of volatiles in fruit and vegetables. In "Pro.Int. Aroma Research", p. 41. Pudoc., Wageningen, The Netherlands. Van Straten, S., De Vijer, Fl., De Beauveser, J. C., and Visscher, C. A. 1977. " Volatile Components in Food". Cent. Insti. Nutr. and Food Res., TNO Zeist, The Netherlands. SUMMARY AND CONCLUSIONS The purpose of this study was to investigate the flavor profile of Empire apples as influenced by harvest maturity, storage duration, low and high ethylene controlled atmospheres (CA), and ripening. Empire apples were harvested on Oct. 2 and Oct. 9, 1984. Apples harvested on Oct. 2 were preclimacteric, judging from their internal ethylene concentration of (0.097 PPM and flesh firmness of 18.0 lbs-force. Apples harvested on Oct. 9 were just at the beginning of their climacteric rise with an internal ethylene concentration of 0.226 PPM and flesh firmness of 17.8 lbs-force. The l-amino cyclopropane 1-carboxylic acid (ACC) concentration, malic enzyme activity, protein content, flesh firmness and water soluble poyluronide content (WSP) were determined for both harvests during five months of storage in low (<1 PPM) and high ethylene (>100 PPM) contrdlled atmospheres (3% 002, 2% 02 and a relative humidity of 98%), and air storage at 3.3°C. Apples were ripened at 22’C for 2, 7 and 14 days. The malic enzyme activity, protein synthesis, WSP and ACC contents for apples stored in air were higher compared to high and low ethylene CA storage . Fruits stored in air for five months lost their flesh 204 205 firmness very quickly. Late harvested fruits were softer after five months of storage in all treatments with a ripening period of 7 and 14 days. CA storage suppressed the rate of protein accumulation. The ACC content of apples was very low at harvest. Fruits stored in air at 3.3°C acummulated ACC in a linear manner from the beginning of the storage period. The malic enzyme activity showed the same trend as the protein content. The WSP content of apples stored from the first harvest with no ripening period, increased for both low and high ethylene CA. Apples stored in air had a sharp increase in WSP content after one month and then declined. The same pattern was seen for flesh firmness, which had a sharp decline until the second month and remained unchanged thereafter. Flavor profile changes during storage and ripening were determined using dynamic headspace, vacuum distillation and Likens-Nickerson techniques. Identification and confirmation were determined using capillary gas chromatography (GC) and mass spectrometry (MS). Dynamic headspace analysis was conducted monthly on apples harvested on Oct. 2 for the three storage conditions for a duration of six months followed by ripening at 22‘C for 2, 7 and 14 days. Forty-nine compounds were identified using dynamic headspace technique. Compounds identified included 24 esters, 13 alcohols, 7 aldehydes and 2 ketones. The most dominant 206 components identified were methyl 2-methyl butanoate, ethyl 2- methyl butanoate, butanal, 2-methyl butanal, hexanal, butanol 2-pentanol and 3-hexanol. The concentrations of volatile compounds were higher for apples stored in air compared to those stored in high ehtylene CA. Low ethylene CA greatly reduced the total concentration of volatiles. Apples had their highest levels of volatiles after four months of storage, which relates to the climacteric rise in respiration ( high ACC content ). The concentration of volatiles decreased thereafter. Ripening at 22°C increased the ability of apples stored in all three storage conditions to generate volatiles. However apples stored in low ethylene CA lost much of their ability to generate volatiles, especially after four months. To investigate the effect of harvest maturity on the flavor profile of Empire apples, two harvest dates were used. Vacuum and Likens-Nickerson distillation were used to determine the flavor profile and changes in flavor due to maturity at harvest and storage conditions. Fifty-eight compounds were identified by GC-MS in extracts obtained by using vacuum distillation. The most dominant compounds were esters of acetic and butanoic acids. Seventy-seven compounds were identified from the extract obtained by using the Likens-Nickerson technique. Most compounds identified in this work have been found in in other 207 apple varieties. Methyl propyl Z-methyl butanoate, 2-methyl propyl hexanoate, butyl 3-methyl butanoate and methyl 2-butyl heptanoate were identified in extracts obtained by vacuum distillation for the first time in Empire apples. 2-Methyl 2-methylbutyl propionate, 3-methy1 butyl propionate, methyl propyl 2-methyl butanoate, 2-methyl propyl hexanoate, 2-methy1 butyl hexanoate, 2-methyl hexanoate, ethyl 1-methyl hexanoate, 2-buty1 2-octanal, 2-cyclo hexenal, isooctanol, 4-methyl 4-octanol, 7-methyl 4-octanol, 6-ethy1 undecane, 2-methyl pentatonic acid, 1,4 dimethyl benzene, and ethyl propyl benzene were identified in the extract obtained using the Likens-Nickerson technique. Generally apples stored in air had the highest concentration of volatiles during six months of storage. Apples harvested on Oct. 2 had the highest volatiles after four months of storage in all three storage conditions. Apples harvested on Oct. 9 reached their climacteric rise after three months of storage in all three storage conditions. The concentration of volatiles after four months decreased to a level lower than at harvest. Ripening at 22°C for 2, 7 and 14 days greatly enhanced the level of volatiles for all three storage conditions. However, for apples stored in low ethylene CA , ripening did not have a significant effect on the generation of volatile compounds. 208 A low ethylene CA storage condition is beneficial if apples are harvested at their preclimacteric stage. Studies on ethylene removal from CA storage need to be carried out for extended periods with various ethylene concentrations to fully confirm the biochemical changes outlined above. APPENDICES APPENDIX A DATA FOR CHAPTER 1 209 Table 1. Chemical analysis of Empire apples after three months of storage in air, low and high ethylene CA and after subsequent ripening at 22°C for 0, 7 and 14 days. Numbers are the average of three replicates. Harvest Ash Moisture Juice(ml/g) pH Acidity Oct. 2/84 Air 0-D .18 86.97 .65 3.3 3.15 LCA 0-D .19 86.84 076 302 4.96 HCA 0-0 .16 90.03 .72 3.2 4.42 Air 7‘D .18 90048 067 306 2075 LCA 7-D .16 89.83 .72 3.3 3.15 HCA 7"D .21 89005 071 304 3035 Air 14-D .18 89.57 .65 3.2 3.02 LCA 14-D .17 88.24 .65 3.4 3.82 HCA 14-D .14 87.78 .63 3.3 3.75 Oct. 9/84 Air 0'1) .18 88090 066 3.5 3008 HCA O-D 016 83036 070 303 3082 Air 7-D .14 88.48 .67 3.2 3.55 LCA 7"D .19 87044 .70 3.1 3029 Air 14-D .14 88.58 .66 3.6 2.61 LCA 14‘D .13 88009 068 303 3068 HCA 14-D .13 89.44 .69 3.3 3.95 LCA-low ethylene controlled atmosphere( 3% C02; 2% 02 and <1 PPM ethylene). HCA-high ethylene controlled atmosphere( 3% C02; 2% 02 and >100 PPM ethylene). O-D, zero daY; 7-D, seven ripening at 22 °C. days and 14-D, fourteen days of APPENDIX B DATA FOR CHAPTER 2 210 Table 1. Volatiles in headspace of Empire apples at harvest. Volatile Ethyl formate Methyl acetate Butanal 2-Methyl butyraldehyde Isovaleraldehyde Propyl formate Ethyl propionate Propyl acetate Valeraldehyde Methyl-Z-methyl butyrate Butyl fromate Ethyl butyrate Ethyl-Z-methyl butyrate Butyl acetate Amyl acetate Isoamyl acetate 2-Methyl-1-propanol 1-Butanol 3-Methyl-1-butanol Heptanal Ethyl hexanoate Amyl butyrate Cis-3-hexen-1-ol Phenyl acetaldeyhde Ethyl phenyl acetate Total esters Total aldehydes Total alcohols Substances are given in order of elution from GC column. Concentration (PPM) Days of Ripening .484 .323 .380 .138 00006000 blxl . 55 0.052 0.873 0.053 0.997 0.065 0.979 0.470 0.306 4.320 0.355 2.649 6.542 1.381 ---ns ---ns 0.759 2.497 0.339‘ ---ns 0.075 0.034 ---ns 0.046 17.607 2.568 3.256 211 Table 2. Volatiles in headspace of Empire apples after one month of storage at 3.3°C in air. Concentration (PPM) Days of Ripening Volatile 2 _7_ Methyl Acetate 0.091 0.106 Ethyle Acetate 0.143 0.048 2-Methylbutyraldehyde 0.118 0.288 Valeraldehyde 0.033 0.043 Methyl butyrate 0.207 0.209 Methyl 2-methylbutyrate 0.199 0.307 Butyl format 0.049 0.066 Ethyl butyrate 0.044 0.057 Ethyl 2-methylbutyrate 0.125 1.817 2-Pentanone 0.032 0.037 Butyl acetate 0.043 0.223 2-Methyl-1-propanol 0.022 0.028 Amly acetate 0.059 0.165 Isoamyl acetate 0.031 0.042 2-Pentanol 0.065 0.123 3-Heptanone 0.020 0.038 1-Butanol 0.060 0.877 3-Hexanol 0.487 0.128 2-Methy1-1-butanol 0.021 0.025 Z-Hexanol 0.046 0.065 1-Pentanol 0.079 0.139 Hexyl acetate 0.066 0.077 Trans-3-hexen-1-ol 0.058 0.058 Cis-3-hexen-1-ol 0.040 0.160 Phenyl acetaldehyde 0.144 0.150 Ethyl phenyl acetate 0.155 0.092 Nonanoic acid 0.320 0.209 Total esters 1.212 3.209 Total aldehydes 0.295 0.481 Total alcohols 0.878 1.603 Total ketones 0.052 0.075 Substances are given in order of elution from GC column. 212 Table 3. Volatiles in headspace of Empire apples after two months of storage in air at 3.3’0. Volatile Ethyl formate Methyl acetate Butanal Ethyl acetate 2-Methyl butyraldehyde Propyl formate Ethyl propionate Propyl acetate Valeraldehyde Methyl butyrate Methyl-Z-methyl butyrate Butyl formate Ethyl butyrate Ethyl-Z-methyl butyrate 2-Pentanone Butyl acetate 2-Heptanone 2-Methyl-2-propanol 2-Methyl-2-pentanol Amyl acetate 3-Pentanol Isoamyl acetate 2-Pentanol Heptanal 1-Pentanol Cis-3-hexen-1-ol Phenyl acetaldehyde Ethyl phenyl acetate Total esters Total aldehydes Total alcohols Total ketones Substances are given in order of elution from CC column. Concentration (PPM) Days of Ripening N 846 532 409 058 O O O N U! an. N 0.0454 1.7898 0.0604 0.0544 5.3334 2.9294 0.6323 0.1133 1 7 1 0 l 162 639 212 546 150 745 738 175 O O —i (D N O GNU-‘N-‘NOb OOOOOO-‘UIOO O \l (a) \l b U! U) \l k —c 0.0479 0.2031 10.048 0.0458 0.4174 0.1333 0.4013 0.0465 0.0648 0.0645 1.8888 0.3393 0.0603 0.1662 0.0934 0.1897 0.1095 6.455 .5403 1112 1791 H 0.5343 0.1593 10.345 3.7063 4.6780 0.5432 0.6896 1.6306 1.3532 1.6851 8.4500 0.0592 0.2305 20.297 0.0518 0.6836 0.1027 0.5559 0.0510 0.0598 0.0707 2.7957 0.4999 0.0646 0.1310 0.0548 0.1761 0.0958 41.620 16.617 1.3633 0.1543 213 Table 4. Volatiles in headspace of Empire apples after three months of storage at 3.3‘C in air. Volatile Methyl acetate Butanal 2-Methy1butyra1dehyde Propyl formate Ethyl propionate Methyl-Z-methyl butyrate Butyl formate Ethyl butyrate Ethyl-Z-methyl butyrate Butyl acetate 2-Methy1-1-Propanol 2-Pentanol Hexyl acetate Cis-3-hexen-1-ol Ethyl phenyl acetate Nonanoic acid Total esters Total aldehydes Total alcohols Substances are given in order of elution from CC column. Concentration (PPM) Days of Ripening 0.0275 0.0438 0.1094 0.0584 0.1439 0.3090 1% O 0 hence ONO 75 452 153 721 075 226 471 215 569 607 824 bNo—LN‘ -‘ U 4‘ N O O O O O O O ddONoo—DmdmN—‘U‘d-fiN OOCOOOOCOOOOOOOC 0 3.6133 0.2877 0.1266 214 Table 5. Volatiles in headspace of Empire apples after four months in air at 3.3°C. Concentration (PPM) Days of Ripening Volatile 2' Z, 14 Ethyl acetate 0.0594 0.0904 0.1079 2-Methyl butyraldehyde 0.0694 0.0803 0.0944 Ethyl propionate 1.4505 2.1740 6.1740 Propyl acetate 0.1301 0.2279 0.2575 Valeraldehyde 0.0680 0.0829 0.1100 Methyl butyrate 0.1188 0.2292 0.1333 Methyl-Z-methyl butyrate 0.1658 2.6844 6.6338 Ethyl butyrate 0.9696 1.9886 2.2985 Ethyl-Z-metgyl butyrate 1.3888 9.9402 22.196 2-Pentanone 1.2086 4.0763 4.8770 Butyl acetate 0.6461 4.1960 4.8000 Hexanal 0.7600 28.871 41.410 ‘ 2-Methyl-1-propanol 1.8830 3.1675 3.4344 2-Methyl-1-pentanol 0.0614 0.0871 0.1327 Amyl acetate 0.9203 3.3905 4.5923 3-Pentanol 0.4836 0.7938 0.9903 2-Pentanol 1.2414 1.5294 4.7983 Heptanal 0.0878 0.1299 0.3268 3-Methyl-1-butanol 0.2359 0.3415 2.5916 3-Hexanol 0.0719 0.0825 0.2265 2-Hexena1 0.0846 0.1182 0.1413 2-Hexanol 0.1146 0.2002 0.3627 1-Pentanol 0.1342 0.1634 0.2677 Cis-3-hexen-1-ol 0.1035 0.1053 0.1620 Nonanoic acid 0.1828 0.1932 0.2120 Total esters 5.8489 24.9212 47.1933 Total aldehydes 1.0698 29.2823 42.0825 Total alcohols 4.3295 6.4707 12.9662 Total ketones 1.2086 4.0763 4.8770 Substances are given in order of elution from CC column. Table 6. Volatiles in headspace of Empire apples after 215 five months of storage at 3.3°C air. Volatile Methyl acetate Butanal Ethyl acetate Propyl formate Ethyl propionate Propyl acetate Valeraldehyde Methyl butyrate Methyl-Z-methyl butyrate Butyl formate Ethyl butyrate Ethyl-Z-methyl butyrate 2-Pentanone Butyl acetate 2-Methyl-1-propanol Amyl acetate Isoamyl acetate 2-Pentanol Heptanal 1-Pentanol Cis-3-hexen-1-ol Phenyl acetaldehyde Ethyl phenyl acetate Nonanoic acid Totla esters Total aldehydes Total alcohols Total ketones Substances are given in order of elution from 00 column. Concentration (PPM) Days of Ripening 2 0.0412 0.0459 0.0481 0.0191 0.0681 0.0771 0.0501 0.0458 0.4398 0.0783 0.0846 0.0747 0.0391 0.0601 0.0405 0.0599 0.8899 0.0398 0.0524 0.2640 0.0414 0.0247 0.0106 0.0212 1.9973 0.1731 0.3857 0.0391 cos. c U \I U 0.0376 0.0236 0.0105 0.0205 0.3139 0.6267 0.0567 0.1844 1.3653 0.3617 0.2152 0.0924 0.0529 0.0463 0.1785 0.1957 ----ns ----ns ----ns 0.0084 0.0306 3.2052 1.5335 0.4717 0.1226 6.0784 1.8178 0.2709 0.3617 Table 7. Volatiles in headspace of Empire apples after six 216 months of storage in air at 3.3°C. Volatile Methyl acetate Ethyl acetate Propyl formate Methyl butyrate Methyl-Z-methyl butyrate Ethyl-Z-methyl butyrate Butyl acetate 2-Hexanone 2-Methyl-1-propanol Isoamyl acetate 2-Pentanol Heptanal Cis-3-hexen-1-ol Phenyl acetaldehyde Ethyl phenyl acetate Nonanoic acid Total esters Total aldehydes Total alcohols Total ketones Substances are given in order of elution from CC column. Concentration (PPM) Days of Ripening ho 0.035 0.043 0.059 0.054 0.270 0.035 0.046 0.072 0.028 0.311 0.034 0.046 217 Table 8. Volatiles in headspace of Empire apples after one month of storage at 3% 602/ 2% 02 at 3.3°C with <1 PPM ethylene. Volatile Methyl acetate Ethyl acetate Propyl acetate Ethyl propionate Valeraldehyde Methyl 2-methylbutyrate Ethyl butyrate Ethyl 2-methyl butyrate 2-Pentanone Butyl acetate Z-Methyl-l-propanol Amyl acetate Isoamly acetate 2-Pentanol 3-Heptanone l-Butanol 3-Methyl-1-butanol 2-Hexanol 2-Hexenal 2-Methyl-1-butanol Ethyl hexanoate 2-Hexanol Hexyl acetate Amyl butyrate Cis-3-hexen-1-ol Ethyl phenyl acetate Nonanoic acid Total esters Total aldehydes Total alcohols Total ketones Substances are given in order of elution from CC column. Concentration (PPM) Days of Ripening _2 0.029 0.042 0.039 0.038 0.037 0.168 0.036 0.023 0.029 0.039 0.067 0.090 0.025 0.060 0.025 0.077 0.053 0.091 0.037 0.031 0.036 0.034 0.114 0.064 0.059 0.095 0.183 0.839 0.074 0.438 0.054 _7_ 0.034 0.050 0.046 0.039 0.040 0.252 0.042 0.027 0.030 0.043 0.085 0.169 0.033 0.082 0.033 0.106 0.063 0.128 0.047 0.035 0.038 0.056 0.209 0.076 0.166 0.095 0.190 1.153 0.087 0.721 0.063 14 0.048 0.262 0.082 0.036 0.145 0.456 0.048 0.086 0.032 0.067 0.087 0.196 0.038 0.098 0.040 0.123 0.069 0.164 0.048 0.041 0.039 0.065 0.277 0.084 0.226 0.095 0.196 coo- . O coo-u» NVW‘ muwoo 218 Table 9. Volatiles in headspace of Empire apples after two months of storage with 3% C02/ 2% 02 and (1 PPM ethylene at 3.3°C. Concentration (PPM) Days of Ripening Substances are given in order of elution from CC column. Volatile 2 _Z _1_4 Ethyl formate .0.0416 0.0519 0.0574 Butanal 0.1055 0.1744 0.4149 Ethyl acetate 0.0424 0.0513 0.1770 2-Methyl butyraldehyde 0.0354 0.0398 0.0402 Propyl formate 0.0300 0.0579 0.0848 Ethyl propionate 0.0551 0.0829 0.3436 Propyl acetate 0.0465 0.0502 0.0630 Methyl-Z-methylbutyrate 0.2127 0.3835 1.4441 Ethyl butyrate 0.1473 0.1134 0.8564 Ethyl-1-methyl butyrate 0.0867 0.0671 0.4245 2-Pentanone 0.0465 0.0532 0.0576 Butyl acetate 0.2234 0.0619 0.1611 2-Methyl-2-pentanol 0.2911 0.5837 0.3693 Ethyl hexanoate 0.0758 0.1421 0.9400 Cis-3-hexen-1-ol 0.2186 0.1580 0.0816 Ethyl phenyl acetate 0.1236 0.1033 0.1025 Nonanoic acid 0.2360 0.2140 0.2040 Total esters 1.0851 1.1655 4.6544 Total aldehydes 0.1409 0.2137 0.4451 Total alcohols 0.5099 0.7417 0.4509 Total ketones 0.0465 0.0532 0.0576 219 Table 10. Volatiles in headspace of Empire apples after three months of storage with 3% C02/ 2% 02 and <1 PPM ethylene at 3.3‘C. Volatile Methyl acetate Butanal Ethyl acetate Z-Methyl butyraldehyde Isovaleraldehyde Propyl formate Ethyl propionate Valeraldehyde Methyl-Z-methyl butyrate Butyl formate Ethyl butyrate Ethyl-Z-methyl butyrate Butyl acetate 2-Methy1-1-pentanol Isoamy acetate 2-Pentanol Heptanal Hexyl acetate Cis-3-hexen-1-ol Diethyl succinate Ethyl phenyl acetate Nonanoic acid Total esters Total aldehydes Total alcohols Substances are given in order of elution from CC column. Concentration (PPM) Days of Ripening 0.0779 0.2329 0.8806 0.0579 0.0279 0.0543 0.0534 0.1967 0.0380 0.0449 0.0845 0.1885 0.0804 0.1683 0.1836 1.0950 0.4387 0.2799 1‘1 447 617 959 798 525 533 071 O O -uo an» NH” O dd—ddoduod—~u-abo0aoo 823 878 173 015 729 241 527 438 639 509 180 034 920 OOOOOOOCCOCOOOOOOOOOOO 0 2.9175 0.4666 0.3765 lg 0.0693 ----ns 0.4133 0.1520 0.0438 0.9888 0.4932 0.1529 0.1749 0.7225 0.3263 0.7295 0.3566 0.0832 0.4058 0.1682 0.4934 0.1483 0.0766 0.1498 0.1009 0.2118 5.0792 0.8421 0.3280 220 Table 11. Volatiles in headspace of Empire apples after four months of storage with (1 PPM ethylene and 3% C02/ 2% 02 at 3.3°C. Volatile Methyl acetate Butanal 2-Methyl butyraldehyde Propyl formate Ethyl propionate Propyl acetate Methyl butyrate Methyl-Z-methyl butyrate Butyl formate Ethyl-Z-methyl butyrate 2-Pentanone Butyl acetate 2-Methyl-1-propanol Amyl acetate 3-Pentanol Isoamyl acetate 2-Pentanol Heptanal Cis-3-hexen-1-ol Phenyl acetaldehyde Ethyl pheny acetate Nonanoic acid Total esters Total aldehydes Total alcohols Total ketones Substances are given in order of elution from CC column. Concentration (PPM) Days of Ripening 2 0.0405 0.1391 0.0324 0.0528 0.0386 0.6774 0.0385 0.3394 0.0550 0.0517 0.0301 0.0524 0.0767 0.0368 0.0531 0.4506 0.0376 0.1920 0.0409 0.2348 0.1106 0.2398 1.9443 0.5983 0.2083 0.0301 .7. 0.0434 0.4044 0.0469 0.0330 0.0580 0.0611 0.0704 1.0481 0.0629 0.8879 0.4145 0.1568 0.0783 0.0312 0.0307 0.7381 0.0640 0.1258 0.0314 0.1920 0.0994 0.2202 3.2903 0.7691 0.2044 0.4145 ‘13 0.1060 2.9982 0.0444 0.0708 0.0944 0.8013 0.1143 2.4818 0.0696 2.5167 0.7553 0.3009 0.1146 0.0275 0.0621 1.6935 0.1358 0.1442 0.0295 0.1760 0.0949 0.2032 8.3713 3.3628 0.3420 0.7553 Table 12. Volatiles in headspace of Empire apples after five months 221 of storage with (1 PMM ethylene and 3% C02/ 2% 02 at 3.3%. Volatile Ethyl acetate Ethyl propionate Methyl butyrate 2-Methyl methylbutyrate Ethyl butyrate Ethly-Z-methyl butyrate Butyl acetate Hexanal 2-Methyl-1-propanol 2-Mehtyl-1-pentanol Amyl acetate 2-Pentanol 1-Pentanol Cis-3-hexen-1-ol Total esters Total aldehydes Total alcohols Sustances are given in order of elution from CC column. Concentration (PPM) Days of Ripening 1.042 0.045 0.327 1 0.283 0.054 0.359 0.244 0.046 0.504 0.263 0.208 0.075 0.102 0.880 0.178 0.186 0.144 2.633 0.206 0.685 4.304 0.215 0.401 Table 13. Volatiles in headspace of Empire apples after six 222 months of storage with <1 PPM ethylene and 3% C02/ 2% 02 at 3.3‘C. Volatile Methyl acetate Butanal 2-Mehtyl butyraldehyde Propyl formate Ethyl propionate Propyl acetate Valeraldehyde Methyl butyrate Methyl-Z-methyl butyrate Butyl butyrate Ethyl-Z-methyl butyrate Butyl acetate Isoamyl acetate Ethyl phenyl acetate Total esters Total aldehydes Substances are given in orde of elution from CC column. Concentration (PPM) Days of Ripening No 0.034 0.088 0.033 0.051 0.065 0.046 0.041 0.053 0.184 0.082 0.034 0.036 0.025 0.089 bu 0.036 ---ns ---ns 0.062 ---ns 0.054 ---ns 0.055 0.354 ---ns 0.053 0.045 0.029 0.086 0.774 ---ns 14 0.048 ---ns ---ns 0.062 ---ns 0.062 ---ns 0.068 0.740 ---ns 0.063 0.057 0.035 0.083 1.218 ---ns 223 Table 14. Volatiles in heahsgace of Empire apples after one month of storage with 3% C0 / 2% 02 and >100 PPM ethylene at 3.3‘C. Concentration (PPM) Days of Ripening Volatile _2 1 fl Methyl acetate 0.0575 0.0669 0.1063 Ethyl acetate 0.0858 0.1405 0.3452 2-Methyl butylraldehyde 0.0725 0.1291 4.6456 Valeraldehyde 0.0321 0.0394 0.0496 Methyl butyrate 0.0813 0.1035 0.2309 Butyl formate 0.0395 0.0460 0.0500 Methyl-Z-methybutyrate 0.1450 0.2003 0.3626 Ethyl butyrate 0.0379 0.0465 0.0811 Ethyl-Z-methyl butyrate 0.0775 0.2709 0.4335 2-Pentanone 0.0314 0.0331 0.0349 Butyl acetate 0.0396 0.0551 0.1835 Amyl acetate 0.0426 0.0946 0.1225 Isoamyl acetate 0.0249 0.0314 0.0514 2-Pentanol 0.0651 0.1333 0.1281 3-Heptanone 0.0248 0.0281 0.0330 1-Butanol 0.0695 0.0887 0.1048 2-Methyl-1-butanol 0.0251 0.0346 0.0391 2-Hexanol 0.0613 0.0902 0.1145 Ethyl Hexanoate 0.0346 0.0360 0.0380 Total esters 0.666 1.0192 2.004 Total aldehydes 0.1046 0.2731 4.6952 Total alcohols 0.2209 0.3468 0.3865 Total ketones 0.0562 0.0612 0.0679 Substances are given in order of elution from CC column. 224 Table 15. Volatiles in headspace of Empire apples after two months of storage with 3% C02/ 2% 02 and >100 PPM at 3.3°C. Concentration (PPM) Days of Ripening Volatile _2_ 1 L4 Methyl acetate ----- ns 0.2991 ----ns Ethyl acetate 0.1405 0.5126 1.1157 2-Methyl butyraldehyde 0.5209 0.8929 1.6558 Propyl formate 0.0218 0.0266 0.0446 Ethyl propionate 0.0799 0.0838 0.6986 Propyl acetate 0.0832 0.1086 0.1266 Valeraldehyde 0.0535 0.0649 0.0883 Methyl butyrate 0.0648 0.1053 0.4647 Methyl-Z-methyl butyrate 0.1389 0.1713 0.4966 Ethyl butyrate 0.0838 0.1527 0.1693 Ethyl-Z-methyl butyrate 0.1584 0.4178 0.8160 2-Pentanone 0.0319 0.0347 0.0387 Butyl acetate 0.0441 0.0541 0.0625 2-Heptanone 0.0459 0.0505 0.0542 2-Methyl-2-pentanol 0.0439 0.0486 0.0719 Isoamyl acetate 0.1447 0.1880 0.6399 1-Pentanol 0.1006 0.0806 0.0730 Cis-3-hexen-1-ol 0.0272 0.0256 0.0247 Phenyl acetaldehyde 0.1455 0.1461 0.1425 Ethyl phenyl acetate 0.0892 0.0869 0.0859 Total esters 1.0493 2.2068 5.1600 Total aldehydes 0.7199 1.1039 1.8866 Total alcohols 0.1717 0.1548 0.1696 Total ketones 0.0778 0.0852 0.0929 Substances are given in order of elution from CC column. Table 16. Volatiles in headspace of Empire apples after three months 225 storage with >100 PMM and 3% 002/ 2% 02 at 3.3‘C. Volatile Methyl acetate Butanal Ethyl acetate 2-Methyl butyraldehyde Isoveraldehyde Propyl formate Ethyl propionate Valeraldehyde Methyl-Z-methyl butyrate Butyl formate Ethyl butyrate Ethyl-Z-methyl butyrate Butyl acetate 2-Methyl-1-pentanol Isoamyl acetate 2-Pentanol Heptanal Hexyl acetate Cis-3-hexen-1-ol Diethyl succinate Ethyl phenyl acetate Nonanoic acid Total esters Total aldehydes Total alcohols Substances are given in order of elution from CC column. Concentration (PPM) Days of Ripening 0.0403 0.0544 0.0943 0.1932 0.0704 0.1743 0.1834 2.1103 0.4774 0.4677 1 0.0431 0.0874 0.2235 0.0856 0.0574 0.5349 0.1237 0.2365 0.2365 0.7564 0.2179 0.1349 0.1294 0.0844 0.4021 0.1566 0.0565 0.1645 0.1604 0.0985 0.1203 0.1875 2.9402 0.5234 0.6469 0.4397 0.8753 0.4321 0.8749 0.5309 0.1758 0.5481 5.4632 1.4297 1.5042 226 Table 17. Volatiles in headspace of Empire apples after four months of storage with >100 PPM ethylene and 3% C02/ 2 % 02 at 3.3°C. Volatile Ethyl propionate Propyl acetate Valeraldehyde Methyl butyrate Methyl-Z-methyl butyrate Butyl formate Ethyl butyrate Ethyl-Z-methyl butyrate 2-Heptanone Butyl acetate Hexanal 2-Methyl-1-propanol 2-Methyl-1-pentanol Amyl acetate 2-Pentanol Heptanal 3-Methyl-1-butanol 3-Hexanol 2-Hexenal 2-Hexanol 1-Pentanol Cis-3-hexen-1-ol Nonanoic acid Total esters Total aldehydes Total alcohols Total ketones Substances are given in order of elution from CC column. Concentration (PPM) Days of Ripening 0.00 00.000. 0‘ b b ‘0 . . d‘dddd 4.0014 1.0494 2.6261 1.1802 1 0 0 0 1 0.5876 0 8 3 3 0.0839 2.6474 2.3231 0.2364 1.0589 0.2033 0.3112 0.2425 0.2631 0.1614 0.2036 18.955 3.8271 4.6125 3.3637 3.9907 3.2207 0.5817 2.4934 0.6105 0.3516 0.3172 0.3359 0.2770 0.2130 32.044 4.4349 7.7179 4.8049 227 Table 18. Voltiles in headspace of Empire apples after five months of storage with >100 PPM ethylene and 3% C02 / 2% 02 at 3.3'C. Volatile Methyl acetate Butanal Ethyl acetate 2-Methyl butyraldehyde Isovaleraldehyde Propyl formate Ethyl propionate Valeraldehyde Methyl butyrate Butyl formate Ethyl butyrate Ethyl-Z-methyl butyrate Butyl acetate Hexanal Amyl acetate 3-Heptanone Hexyl acetate Cis-3-hexen-1-ol Ethyl pheny acetate Nonanoic acid Total esters Total aldehydes Total alcohols Total ketones Substances are given in order of elution from CC column. Concentration (PPM) Days of Ripening 2 0.0352 -~--ns 0.0679 0.0363 0.0814 0.0524 ----ns 0.0352 ----ns 0.0235 0.0672 0.0835 0.0578 ----ns 0.8258 0.0262 0.2314 0.1440 0.1000 0.2200 1.5799 0.1529 0.1440 0.0262 1 0.0698 0.1121 .3850 .0933 2.0647 0.2489 0.0869 0.1029 1— 0.1612 0.0509 1.0367 0.0432 ----ns 1.0868 0.1191 0.9950 0.1853 0.0153 0.6394 0.6515 0.5096 0.1418 0.2468 0.3464 0.1265 0.0517 0.0936 0.1841 4.8718 1.2309 0.0517 0.3464 Table 19. Volatiles in headspace of Empire apples after six 228 months of storage with >100 PPM ethylene and 3% C02/ 2% 02 at 3.3°C. Volatile Ethyl acetate Ethyl propionate Propyl acetate Methyl-Z-methyl butyrate Ethyl-Z-methyl butyrate 2-Pentanone Butyl acetate Hexanal 3-Hexanol Amyl butyrate Total esters Total aldehydes Total alcohols Total ketones Substances are given in order of elution from CC column. Concentration (PPM) Days of Ripening 1% 0.048 0.037 0.044 0.419 0.046 0.046 0.062 0.083 0.052 0.053 0.709 0.083 0.052 0.046 Table 20. Computer program to calculate the concentration of 50 C 100 200 300 400 500 510 520 600 650 700 750 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2750 2800 2900 3000 3100 3150 3160 3200 3300 3350 3360 3362 A(1)-0.1414103E+12 A(2)-1.461985E+11 A(4)-1.72047E+11 volatiles LS PRINT "PROGRAM TO CALCULATE CONCENTRATION (PPM) FROM GLC DATA" PRINT PRINT PRINT PRINT PRINT:PRINT:PRINT:PRI 229a BY CARLOS A. LEVER NT PRINT "TO FINISH TYPE CTRL-C":PRINT:PRINT DIM A(100),B(100),C(1 A(3)-0.1130932E+13 A(5)-2.725919E+11 A(6)-8.575977E+10 A(7)-1.96957E+11 A(8)-8.671341E+10 A(9)-1.490259E+11 A(10)-1.538565E+11 A(11)-9.3835E+10 A(12)-1.477107E+11 A(13)-1.598615E+11 A(14)-9.330773E+10 A(15)-1.315066E+11 A(16)-2.113207E+11 A(17)-1.608715E+11 A(18)-2.37549E+11 A(19)-8.302558E+10 A(20)-1.720478E+11 A(21)-7.205701E+10 A(22)-1.148997E+11 A(23)-1.215056E+11 A(24)-0.8671341E+11 A(25)-.232845400E+12 A(26)-.271559500E+11 A(27)-.149025900E+12 A(28)-.728870000E+11 A(29)-0.2113207E+12 A(30)-0.1598615E+12 A(31)-.1074385E+11 : A(32)-.110675400E+12 A(33)-.698408600E+11 A(34)-.290235000E+12 A(35)=0.214888E+12 00) :B(1)-0.7123926 :B(2)-.717897 :B(3)-0.845261 :B(4)-.7387753 :B(5)-.7028931# :B(6)-.6828581 :B(7)-.729167 :B(8)-.6763251 :B(9)-.7148961 :B(10)-.7219842 :B(11)-.7140636 :B(12)-.7153183 :B(13)-.7174808 :B(14)-.6931795 :B(15)-.724546 :B(16)-.7202132 :B(17)-.7142311 :B(18)-.7425603 :B(19)-.6732945 :B(20)-.7387753 :B(21)-.7115752 :B(22)-.7136509 :B(23)-.7102897 :C(1)--82627.53 :C(2)--104555.3 :C(3)--168244.8 :C(4)--48988.23 :C(5)--2446471 :C(6)--151600.2 :C(7)--87181.65 :C(8)--1523721 :C(9)--126781.5# :C(10)--82466.55 :C(11)--82002.66 :C(12)--88316.21 :C(13)--93161.52 :C(14)--121632.8 :C(15)--59790.57 :C(16)--1742791 :C(17)--118039.6 :C(18)--55313.89 :C(19)--159726 :C(20)--48988.23 :C(21)--53711.77 :C(22)--94850.59 :C(23)--106304.9 :B(24)-0.6763251 :C(24)--152372.0 :B(25)-.706081300 :C(25)--239601.50 :B(26)=.700748000 :C(26)- -40082.0500 :B(27)-.714896100 :C(27)--126780.500 :B(28)-.709095800 :C(28)--71370.2600 :B(29)-0.7202132 :C(29)--174279.0 :B(30)-0.7174807 :C(30)--93161.52 B(31)-0.559670200 :C(31)--458147.70 :B(32)-.714872800 :C(32)--83231.1900 :B(33)-.693365000 :C(33)--221880.900 :B(34)-.72136710 :C(34)--184647.200 :B(35)-0.7447405 :C(35)--51749.05 Table 20 (cont.). Computer 3364 3366 3368 3370 3380 3390 3400 3430 3440 3450 3460 3470 3500 10000 10100 10400 10450 10500 10600 10700 10800 of volatiles. A(36)-0.1148997E+12 A(37)-0.5398271E+10 A(38)-0.1186136E+12 A(39)-0.4661466E+11 A(40)-0.556913E+11 A(41)-0.1175185E+12 A(42)-.914349600E+10 A(43)-0.9143496E+10 A(44)-0.1315066E+12 A(45)-0.2107108E+12 A(46)-0.2046436E+12 A(47)-0.9082665E+10 A(48)'.698408600E+11 229b program to calculate the concentration :B(36)-0.71365090 :B(37)-0.6038451 :B(38)-0.7283645 :B(39)-0.7099918 :B(40)-0.6931186 :B(41)-0.7127306 :B(42)-.506734100 :B(43)-0.5067341 :B(44)-0.724546 :B(45)-0.743259 :C(36)--94850.59 :C(37)--37434.39 :C(38)--38507.46 :C(39)--37079.3 :C(40)--86318.3 :C(41)--101726 :C(42)--716527.600 :C(43)--716527.6 :C(44)--59790.57 :C(45)--52272.02 :B(46)-0.75901150 :C(46)--49285.11 :B(47)-0.5457443 :C(47)--321809.2 :B(48)-.693365000 :C(48)--221880.900 PRINT "INPUT STANDARD NUMBER (1-48) AND AREA VALUE AS (N,V)" INPUT N,AREA CON-((AREA-C(N))/A(N))°(1/B(N)):CONC-CON*1E+06 PRINT PRINT "STD No.";N;";";"AREA-";AREA;";";"CONCENTRATION-";CONC; LPRINT "STD No.";N;";";"AREA-";AREA;";";"CONCENTRATION-";CONC; CONC-O:AREA-0:PRINT:PRINT:GOTO 10000 END 230 Table 21. Linear regressions for the standards used for the quantitation analyses. Standards Ethyl Formate Methyl Acetate Butanal Ethyl Acetate 2-Methylbutyraldehyde Isovaleraldehyde Propyl Formate Ethyl Propionate Propryl Acetate Valeraldehyde Methyl Butryrate Methyl-Z-methylbutyrate Ethyl butyrate Ethyl-Z-methylbutyrate 2-Pentanone Butyl Acetate Hexanal 2-Methy1-1-propanol 2-Methyl-1-pentanol 3-Pentanol 2-Pentanol 3-Heptanone I-Butanol 2-Heptanone Heptanal 3-methyl-1-butanol 3-Hexanol 2-Hexenal 2-Methyl-1-butanol Ethyl hexanoate 2-Hexanol 1-Pentanol Hexyl acetate Amyl butyrate Trans-3-hexen-1-01 1-Hexenol cis-3-hexen-1-O1 2-methyl propanol Butanoic Acid Nonanoic acid an .5569133+11 .72057E+11 .5398271E+10 .7288700E+11 .1461985E+12 .93835E+11 .1186136E+12 .1215056E+12 .8575977E+11 .1148997E+12 .1148997E+12 .1130932E+13 .1106754E+12 .1414103E+12 .2902350E+12 .2328454E+12 .1720478E+12 .1720478E+12 .9330773E+11 .1315066E+12 .1608715E+12 .214888E+13 .4661466E+11 .2113207E+12 .2113207E+12 .1490259E+12 .1492590E+12 .1967570E+12 .2375495E+12 .2725919E+12 .1538565E+12 .8671341E+11 .8302558E+11 .8671341+11 .1598615E+12 .1598615E+12 .1477107E+12 .2715595E+11 .6984086E+11 .10743850E+11 3522 BS3) .6931186 -86318.3 .711575200 -53711.77 .6038451 ~37434.39 07090958 -71370026 .717897 -104555.3 .7140636 -82002.66 .7283645 -38507.46 .7102897 -106304.9 .6828581 -151600.2 .7136509 -94850.59 .7136509 -94850.59 .845261 -168244.8 .7148728 -83231.19 .7123926 -82627.53 .7213671 -184647.2 .7060813 -239601.5 .7387753 -48988.23 07387753 -48988023 .6931795 -121632.8 .724546 -59790.57 .7142311 -118039.6 .7447405 -51749.05 .7099918 -37079.3 .7202132 -174279 .7202132 -174279 .7148961 -126780.5 .7148961 -126780.5 .7291670 -87181.65 .7425603 -55313.89 .7028931 -244647.0 .7219842 -82466.55 .6763251 -152372.0 .6732945 -159726.0 .6763251 -152372 .7174807 -93161.52 .7174807 -93161.52 .7153183 -88316.21 .7007480 -40082.05 .6933650 -221880.9 .5596702 -458147.7 fig .994 .993 . 987 .987 .988 .988 .987 .987 .989 .987 .987 .985 .988 .987 .988 .985 .988 .988 .987 .988 .987 .987 .986 .986 .986 .987 .987 .988 .988 .987 .986 .986 .986 .986 .987 .987 .987 .984 .971 .995 Table 22. Retention times for the standards using bonded, 231a non-bonded split and splitless injection with 50 and 60 m Carbowax 20 M capillary columns. Name Ethyl Formate Methyl Acetate Butanal Ethyl Acetate 2-Methylbutyraldehyde Isovaleraldehyde Propyl Formate Ethyl Propionate Propyl Acetate Valeraldehyde Methyl Butyrate 3-Pentanone Methyl-Z-methylbutyrate Butyl Formate Ethyl Butyrate Ethyl-Z-methylbutyrate 2-Pentanone Butyl Acetate 2-Hexanone Hexanal 2-Methyl-1-Propanol 2-Methyl-1-pentanol Anyl Acetate 3-Pentanol Isoamyl Acetate 4-Heptanone 2-Pentanol 3-Heptanone 1-Butanol 2-Heptanone Heptanal 3-methyl-1-butanol Bonded Split 60 E 5.94 6.73 7.48 7.86 8.80 9.01 o o o o o \O U ddddddddd‘d \omebNNNNd-Hom O mONVOQUOb-Mko Splitless '05 c: 13 ubww-no-‘wk-Momsooooasnaxooos UNNNOpNONwww-‘U‘Udmflmo \OQONO‘kaNNd-‘COGQNO‘U‘U dddddddddd‘dd Non-bonded '0‘ c: 18 o o o o o o o o wUVNNVUbVomU‘NNN-‘QN‘O-‘d mmVGO\MN-‘C\O\D\O\OG\INO\O\UUIUI . uwNwoooooowbmJ-‘Obnooouoosoo ddddddddd Split Splitless 'UI c: 18 00000000000000 COGU'Ok9UNOODMQVNVO‘O‘U‘U‘WU‘kbb1§UUUWNN . OUIVObw-‘UQCNUIflM-‘N-‘w-‘QQO‘UICUIO-‘NO‘NOb Ndddddddddd Table 22 (cont.).Retention times for the standards using bonded, 231b non-bonded split and splitless injection with 50 and 60 m Carbowax 20 M capillary columns. Name 3-Hexanol 2-Hexenal 2-Methyl-1-butanol Trans-Z-Hexenal Ethyl Hexanoate Z-Hexanol 1-Pentanol Hexyl Acetate Amyl Butyrate N-Octanol Propanoic Acid Trans-3-Hexen-1-01 1-Hexenol Cis-3-Hexen-1-01 2-Fura1dehyde Benzaldehyde 1-0ctanol Butanoic Acid Phenyl-acetaldehyde Undecrylenic aldehyde Diethyl Succinate 1-Nonanol Ethyl Phenyl Acetate Nonanoic Acid Bonded Split §Q|m 32.64 33.55 34.38 34.58 34.82 36.10 37.28 38.76 39.42 40.26 43.00 45.66 46.49 48.05 52.34 54.45 56.37 61.28 63.89 66.34 68.21 69.56 74.95 99.33 Splitless Split 922 30.48 30.71 31.97 32.41 32.95 34.35 35.00 36.72 38.35 40.12 40.85 41.65 43.14 54.00 56.32 56.78 58.76 62.08 64.84 66.58 68.95 69.38 69.95 92.71 Non-bonded Splitless .622 22 28.75 23.83 29.09 24.34 29.28 26.04 29.94 26.15 30.95 26.27 32.98 30.72 33.97 32.47 33.97 32.98 33.97 32.98 36.25 34.45 41.94 38.92 42.02 41.36 42.02 42.23 43.50 45.30 49.70 47.46 53.45 48.23 57.10 52.12 60.48 55.80 60.81 56.23 63.04 58.34 63.30 59.23 63.76 63.54 69.31 69.55 92.06 74.52 APPENDIX C DATA FOR CHAPTER 3 232 Figure 1. Mass spectra of methyl propyl 2-methyl butanoate. zmaznnxmuanz c m.u warm n“ ansumbza r2 _OIow ammo ouumoumm 1 coax" ”comma 01 V 1.. (I 5» 233a w I'Jl 01 n m&. mu Om!rtz!@@ omlrfimlmm worzupmmpspmnmI—mwbipwmw } -L :- _— b -D 50 mo 1— q» .. p33 .. him... 5. .4 s. - LP? .5 —q P n L L b PanQ-L bib “Mupflfi .— - .. rumflin . rum, 1 (I _14. dfllqdfiddd Id d—d‘ ..lldd dd 1.11111d dill-1. ‘1. 111- ‘d ddld‘ "NC ~50 an pQC NOD ...mmznn.....m.. H DEE C N . ....» flmrm nu UfimumDZA r2 ~Q1Qm GENO chino membmi. trummornm MVEKZHI mvmnqmr III. 1mg anmfimfi fimIanM 2:“ Hum mnu mm MEADZDHD DOHO. M n ma. mu cwlbtmlfim omlnnmlmm Xmarir . p :mqrcrflnafi WODZuthpapmmmlhmmaamem ”003" Hemmmm £4 aumuufl.m . Eu 4...... m...» 1 mm mun” > — -— nbfi b» L b>>F _. hslpp bp—b— “1*“ Hmwnlbg “mm 6 [1 L “003“ Mme antler Mame 1 4» (A «m 1 ~09 . .. .....l .V I .Mslfi J.N. 11.: L _. a.» __ r= _. :6 an... Eu 5m .6 8263382 3 . 3 1 2 “OH . mm 1 n — .... ......— o. ...-.o- .. .. . -.—— ... 6.? *.H.. . . .. .Hro-w-oruw. . . .H'ww. . ..... . o o o. o o. . go me @0 p90 “No —&Q pmo ll ll—IJI-l-«JI.—I4|~I-1-I.—l~l-14l~1—lfi114141_ld.xqu|fi 411%.aj.141141d141.fiuaJ|—1-1_11JJ|3 .ficqlql4.4..-l—1Jl1aI—l.-l]:14lfi 4401.....«1‘1141—1114341 awe N00 234 Figure 2. Mass spectra of ethyl 1-methyl hexanoate. 1. n ma. Du lerfizlmm ZNEZDORmHUEE C 0.. L warm n" a mumnzi omunnmnmm F2 walom ODMO O~IN01mm 1 .nnzuuuumauuumuuwrwsuwuu ... I... . Hoax" Humpm Ed flupuwo. mm ppw 1.1 fl ah .J a 5 3 2 ma 1 Up um 81 _4m ‘fa mu . com - mu d a _ m m —— b_ b—_ h— hP —_ b P u uh— uh 1P Pn—b ——rL—1 - 5 FW“.- “b r. bu - s» >1 bP bb P. h —-u._q..d «dd uqu—du qfiqqud .qqqficu —ud-q—1-uuu—JdluqluufiqJ-ddu-u1ouq-.ddqqdu_ 80 m0 @0 ~00 um p50 "mo pQO NOD :mNZDoxmaubm c M.“ n ma. mu lebtmlmm nnrm r“ opmucrzn om1pp31mm r2 _o1om name ou1moumm rammnmu. PP). ~00 Hmo HAG Hmo pmG moo NNG on m0 211-lb meow 3:1 I; o rwdw mHu dd.w mp Poexu.mo 4; m4 .Lmflpb.b mopz.mpmm«mpmm1m1mm«mmmm 3. mm L a; w 4__ pm ppm ”A; Aaoxu mwa.1 1 .1 1 mopz.r 0mm. av mm .em ”Nu ppm “A. ooznanmoz .4 14“ mm Ham 1 . L311 14.141.111.111 131114141. 141.141.114.144. 43143111331111; woo Hmo who mmo on 242 Figure 6. Mass spectra of ethyl 2-methy1 butanoate. 2#3a momzumomummmlmmuummo ma smonmw.0 J ‘ rtORI abbdm pmmw mm .2 ppm ' ’1 ’ P 11“.‘1‘.1“‘1“‘“‘1“|¢‘1“4‘“{“““-“1‘l mrQ NAG “mo moo —’D1D-——_>b1>bbt>—bth>b i1441~4u111 11111111111111114 m0 HAO Hma JP 243b OQIHAON 381 pus mHa mp wCADZO Hoax. m11qm ma .mcumw.m mopz.momummm1mmpummo Hem mm 1 41 ppm . F__ -._r . r _. fimo Hoax. “mm mopz.r mama _ mm 2 m a; Rpm - __. ., 01W 1 _r r awe ooznpmHmoz A 4 mm m0 poo Hms 9A0 Hmo Hma mac me N‘s 244 Figure 7. Mass spectra of 2-methy1 pentanoic acid. 245a up mg. 1UO“OL.m om. mODZIHwomnpwwmlpAomnpbmm om 8“ Sm . . . . . . .. pom. pom ppo ppm “ma. 2’+5b .Omlpmom 381 ppm mHu dm Um 4 Aoox. pmqoup ma .uonor.m moD21puomupummupaomupamm .qw _ mm 2: t l Hoax. mmm 11 . mop21r mumu . d3 . mg L? , # mm oosnanmoz .Imum mm Hog 1...... .._. .....:. ........ . . ...... If4443§§1§1.1141.4114.1411.11414441‘.414<fi4141.1414< we H00 umo HAG pmo pmo NOQ mmo NAG 246 Figure 8. Mass spectra of heptyl heptanoate. 2Q7a . nipqm m4 1puo®.pm.m lieu “UH mm HAw p®® fimQ HAG mQ pmd Hdm Hmm pmo monzamommnmmbmlmmmAum©GQ ”mo mom mum moo mwo mum on 21+7b we. mwm mH. 4r I . coon. mrcqm m4 .ptootum.m mopz.mcwmumosm1mmmr.muoo ”6 Z; . 1.‘u mm 1 mm 2.. 1.___.:J. ...._ ,.1._r1.LL. 15m mmm :3 “mm “mm mum mum . Hoax. mmm . mopz.rpmouu as ow. ppm “up 4 w mm A _. :.____. .._.. Tr... |1_PIII11W .U HMN H41.» Hmm 900 mmm ooznpmHmoz a i we ”mu ”mu 1 ——O—o. ......o——o-o-ou ....- —.-o_.cooooooo-.o——. Hmm pom mum mum 248 Figure 9. Mass spectra of 2-buty1 2-octana1. ominfiflsmfi zmaznoxm_onz c m.u . n mg. mg n_rm nu cumunge owunnmumm r: “ouom nzwo ou1uoumm ao_xu a ma nauumm.w mnnznwwumawmnuuwuooamuuw ea a a a “..., _# n 2 u. p can you _&0 “mo to me we ~00 “no 2m2220imaorz. C1r.( n m3. mu Dmsnfiasmm fiurm bu 01m.mDZ& lebvximm P2 palom GENO OpINOImm Fummbmz. :F. munrnm manZHI wfimfi 433 Pmmmrn< 11 0—.INNU ZEH awn mun ME DOANZEF. N mca