. ‘ I I ~I' ATE l- ’Ilill’illllum 293 ERSITY LIBRARIES Will!!!IIIHIUllHlWl 1561 9095 ll 4. LIBRARY Mlchigan State University This is to certify that the thesis entitled USE OF 2-NONANONE VAPOR TO CONTROL DECAY IN MODIFIED-ATMOSPHERE PACKAGES OF "PRECUT" APPLE SLICES presented by Rujida Leepipattanawit has been accepted towards fulfillment of the requirements for M.S. degree infinkaginL ‘2’" M a , V Major professor D t 8/21/4 é DY‘. Ruben Hernandez a e . 0.7639 MS U is an Affirmative Action/Equal Opportunity Institution - ‘— -——— .~ * ——‘—.—— - ——-'——.-——v—" - — PLACE IN RETURN BOX to remove thie checkout from your record. TO AVOID FINES return on or More dete due. DATE DUE DATE DUE DATE DUE ‘ USE OF 2-NONANONE VAPOR TO CONTROL DECAY IN MODIFIED-ATMOSPHERE PACKAGES OF "PRECUT" APPLE SLICES By Rujida Leepipattanawit A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE School of Packaging 1996 ABSTRACT USE OF 2-NONANONE VAPOR TO CONTROL DECAY IN MODIFIED-ATMOSPHERE PACKAGES OF ’PRECUT’APLLE SLICES By Rujida leepipattanawit A flow-through vapor exposure system was developed to test the effect of the natural volatile 2-nonanone on the decay of apple slices. 2-Nonanone vaporized in the system was continuously delivered to apple slices and PDA inoculated with Penicillium expansum Link. and Botrytis cinerea Pers. The vapor concentration in the system was determined by Gas Chromatography. Treatments with 2-nonanone vapor at 133 to 303 p1 of 2-nonanone vapor per liter of air at 23°C retarded the growth on PDA. At a concentration of 303 til-liter", the fungal growth was completely suppressed. However, the fungus was not killed as it regrew when transfered to 2-nonanone free air. No lesion of P. expansum and B. cinerea developed on apple slices treated with 290 and 222 pl°liter‘(respectively). P. expansum was more susceptible to 2-nonanone at 5°C than at 23°C. The growth rate of P. expansum at 5°C was approximately 4 times less than that at 23°C. Approximately 50 ul-literl supressed P. expamsum on both agar and apple slices. Treatment with 100 til-literl at 5°C apparently killed P. expansum since no re-growth was detected after transfered to air. Physiological damage was found on the skin of the apple slices after 24 hr treatment at 23°C and 6 hr treatment at 5°C. To advanced education iii ACKNOWLEDGMENTS I would like to take this opportunity to thank my major advisor Dr. Ruben Hernandez for his time, effort, and guidance. I also would like to express my deepest appreciation to Dr. Randolph M. Beaudry for his patient training, financial support, and understanding through out this research. My sincere thanks go to all members of my family, my dad, mom, sisters and brothers. I appreciate the warm hospitality of the post harvest group (Jun, Fan, Silvada, Stacie, Weimin, Rufino, Nazir, Cathy, Wang, TJ, and other). I thank all of my friends (Kok, Pang, Fon Aud, and others) for their help and encouragement. Special thank to Duke for his care and support. I wish to thank Crop & Food Bioprocessing Center/Research Excellence Fund (CFBC/REF) and Center for Food & Pharmaceutical Packaging Research (CFPPR) for their financial support. iv TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION CHAPTER 1 Literature Review CHAPTER 2 Use of 2-nonanone vapor to control decay in modified-atmosphere packages of ’precut’ apple slices. 1. Abstract 2. Materials and Methods 3. Results 4. Discussion 6. References CHAPTER 3 Method development A. Bioassay B. Gas analysis C. Biological Technique CHAPTER 4 Conclusion APPENDICES Appendix 1 Appendix 2 BIBLIOGRAPHY page vi viii 17 18 21 27 3O 35 49 52 54 66 68 7O 76 LIST OF TABLES Table Chapter 2 1 Effect of 2-nonanone treatment on growth rate of Penicillium expansum on PDA held for 7 days at 23°C. 2 Effect of 2-nonanone on Penicillium expansum growth on inoculated apple slices held for 7 days at 23°C. 3 Effect of 2-nonanone treatment on growth rate of Botrytis cinerea on PDA held for 7 days at 23°C. 4 Effect of 2-nonanone on Botrytis cinerea growth on inoculated slices held for 7 days at 23°C. 5 Effect of 2-nonanone treatment on growth rate of Penicillium expansum and Botrytis cinerea on PDA held for 30 days at 5°C. 6 Effect of 2—nonanone on decay of apple slices inoculated with Penicillium expansum and Botrytis cinerea held for 30 days at 5°C. Chapter 3 1 Comparison of the accuracy between saturated headspace method (1) and unsaturated headspace method (2) in generating of the standard calibration curve. 2 Comparison of GC-response between using stainless steel needle and glass-column needle (PB-l). vi page 37 38 39 4O 41 42 57 58 LIST OF TABLES (cont.) Preliminary test of the effect of 2-nonanone on Penicillium expansum growth rate at 23°C. 59 Comparison of the fungal colony diameter on agar inoculated with different concentrations and volumes of spore suspension held 6 days at 23°C. 60 vii LIST OF FIGURES Figure Chapter 2 1 Flow-through vapor exposure system. 2 Calibration curve for 2—nonanone (the concentration of 2-nonanone was estimated from Perry’s Chemical Engineering Handbook). 3 Fungal growth on inoculated petri dishes in air as affected by RH. 4 Effect of 2-nonanone treatment on growth rate of Penicillium cinerea at 5°C and 23°C (more than 90%RH). 5 Effect of 2-nonanone treatment on the growth rate of Botrytis cuinerea at 23°C. Chapter 3 1 Calibration curve for 2-nonanone (the concentration of 2-nonanone was estimated from Perry’s Chemical Engineering Handbook). 2 Variation in GC-response when using different injection volumes. 3 Variation in GC-response in response to the number of times the plunger was pumped. 4 Effect of 2-nonanone treatment on growth rate of Bonytis cinerea at 23°C when fungi were not directly in contact with 2-nonanone vapor (fungi were covered by a loose fitting petri lid). viii page 43 44 45 46 47 61 62 63 LIST OF FIGURES (cont.) Appendix 2 1 Vapor pressure of 2-nonanone as a funtion of temperature. 2 Growth of Penicillium expansum in air and exposed by 2-nonanone at various concentrations at 23°C. 3 Growth of Botrytis cinerea in air and exposed by 2-nonanone at various concentrations at 23°C. 4 Growth of Penicillium expansum in air and exposed by 2-nonanone at an average of 26,ul-literl at 5°C. ix 72 73 74 75 INTRODUCTION The potential market for apple (malus) slices is expected to be large. Institutional users of fresh fruit, such as airline companies, hotels, schools, hospitals, and household consumers utilize fresh slices for fresh pie filling, dessert packs, and salad bar offerings. While other lightly processed fruits, such as pineapples, melons, cantaloupes, are becoming ever more successful, the commercialization of slice apples has been limited by several problems including moisture loss, texture change, browning and microbial decay. A major concern is that decay will occur within the required 14 day shipping and marketing period. The use of natural volatiles to retard fungal decay on apple slices is now under consideration as a new approach to control the decay problem. A number of natural compounds including aldehydes, ketones, and acids are able to minimize decay on whole fruits. Literature does not report any study on the effect of the natural volatiles on slice fruits. Our goal, therefore, was to develop a bioassay system and evaluate a likely volatile compound for its effect on decay of sliced apple. The bioassay involved the construction of a flow-through vapor exposure system in which vapor at various concentrations was continuously generated and delivered to the treatment chamber. This flow-through system simulated a real product package system at steady-state in that 02 and C02 were held constant. 2 This study selected 2-nonanone as a natural volatile with low mammal toxicity, pleasant odor, resistance to rapid decomposition. The effect of 2-nonanone on the growth of Penicillium axpansum Link and Bonytis cinerea Pers. on apple slices and PDA media was evaluated. The effect of temperature on fungal sensitivity to 2- nonanone was also examined. A gas chromatography analysis technique to measure the vapor concentration in the headspace of the test chamber was developed and standardized. CHAPTER 1 LITERATURE REVIEW Lightly processed fruits. In recent years, market demand for lightly processed fruits and vegetables (also known as minimally- , partially-, fresh-, and preprepared-processed products) has rapidly increased (Cook, 1992; Hoag, 1995; Schlimme, 1995). Industry growth has been consumer driven, because conventionally processed products such as canned, frozen, dried or brined foods have not effectively satisfied consumer need. Most consumers prefer fresh food to processed products, but demand convenience and improved nutritional quality. Recent trends in health awareness, have also lead consumers to shift from canned to the fresh-like or natural product, these trends have resulted in about a 10 percent rise in total fruit consumption between 1978 and 1988 (Cook, 1992). Light processing, such as washing, sorting, trimming, peeling, slicing, or chopping does not affect most fresh-like quality attributes and increases convenience and time savings. The most crucial problem for lightly processed products is undesirable physiological changes. The cut surface of fruits and vegetables causes a loss of cellular integrity, destroying compartmentation of enzymes and substrates. As a result, browning and secondary metabolite formation rapidly occur. In addition, senescence and off-flavors may be enhanced (Burns, 1995), and significant contamination of fungi and bacteria might occur on the cut surface. Post-processing problems such as those described need to be diminished for most fruit products. Several approaches have been used to solve these problems including: selecting cultivars able to maintain product quality during light processing; temperature and humidity control; chemical additives; edible coatings; and modified 5 atmospheres. Each commodity will have its own particular post-processing problem calling for commodity specific preservative treatments. Consumer acceptance of fresh-cut fruits appears to be high. According to a recent survey, 52 percent of households reported they would purchase one or more fruits more frequently if they were more convenient to prepare (Hoag, 1995). Growth of the fresh-cut fruit market has been predicted to be massive, similar to that for cut salads and vegetables. Some precut fruits such as pineapples, melons, and cantaloupes, have already entered the pre-cut market and have proven to be successful. While precut apples are not yet readily available, demand is expected to increase rapidly once an acceptable product is developed (Hoag, 1995). Institutional users such as airlines, hotels, schools, and hospitals have shown interest in this type of product. In addition, there is a number of potential markets for fresh pie fillings, dessert packs, and salad bar offerings. Problems in development of precut apples. The development of precut apple products has been limited by several problems including browning, moisture loss, texture change, and decay. Control measures for each of these problems exist. The difficulty lies in combining treatments successfully such that the product is treated with some chemicals, before packed in the MAP. Browning. Browning in apples begins immediately after cutting the fruit skin (Nicoli, 1993). Disruption of cells at the cut surface leads to discoloration by allowing phenolic substrates and the enzyme polyphenol oxidase (PPO) to come into contact (Brecht, 1995). A number of chemical treatments have been suggested to reduce browning from slicing, most of which are antioxidants although some act a PPO itself. Ca(OCl)2 is more effective than NaOCl in reducing browning on apples (Brecht, 1995). Other chemicals used for reducing browning include ascorbic acid, chlorine, meta-bisulfite and 4-hexylresorcinol. Meta-bisulfite and 4-hexylresorcinol is more effective than the others noted. Recently, an edible coating derived from seaweed, has been shown to have potential to reduce respiration of apple slices. Alginic acid from seaweed preserves apple slices up to 8 days without browning, which is significantly better than the 2-3 days provided by salts of vitamin C (Stephens, 1994). A modified atmosphere consisting of 1-3% oxygen and 3-5% carbon dioxide is able to retard browning and maintain quality of whole apples (Barrett, 1989). However, while modified atmosphere (1% Oz and 5-20% C02) significantly reduce browning relative to air control, the level of browning is still objectionable (Iakakul, 1994). Low oxygen (0.5%) and concentration of carbon dioxide greater than 20% is able to control browning but will likely damage the fruit as well. Temperature also affects rate of fruit discoloration. As temperature decreases, oxidation takes place at reduced rates. Water loss. Water loss is a primary source of deterioration in apples causing weight loss, a decline in appearance quality due to shrivelling, and texture change due to a loss of crispness and juiciness. Water loss is a result of a vapor gradient between saturated internal atmosphere within the intercellular spaces and the less saturated external atmosphere. Water vapor migrates largely through surface opening or injuries, being influenced by both internal and external factors. Internal factors include morphological and anatomical characteristics of the produce, surface-to-volume ratio, surface injuries or cutting, maturity stage, etc. External factors include temperature, relative humidity, air movement, and atmospheric pressure. Slicing of apple fruit causes more surface injuries and higher surface to volume ratio and allowing more air to come into contact with the apple tissue. Surface coatings or waxes have been used to prevent water loss for some products. However, consumers seem to reject the use of coating materials that affect appearance or texture. Consumer preference and numerous technical difficulties with edible coatings has resulted in the common use of packages comprised of high water barrier films to reduce water loss. The flexible polymeric films used for this purpose often have perforations to minimize potential for development of hypoxic conditions within the package atmosphere. Polyvinyl chloride (PVC) is used primarily for over-wrapping, and polypropylene (PP) and polyethylene (PE) bags are widely used for lightly processed products. Multi-layered films often used for this propose are composed of ethylene vinyl acetate (EVA). EVA provides a benefit of organic barrier to the package system. Texture change. Texture change is associated with senescence and loss in fruit firmness. Increasing the calcium content of the fruits can reduce the amount and spwd of texture change and furthermore, is able to reduce susceptibility to physiological disorders (Kader, 1992). However calcium treatments can affect flavor, giving a salty taste to the product (Hanson et al.,l993). Microbial decay. The skin of fruits and vegetables acts as natural barrier to microorganisms and pests and protects against physical damage. However, light processing generates an entry port for microorganisms by exposing unprotected tissue. For sliced apples and other products, fungal contamination may occur during the harvesting or processing operation. When most organisms attack the fruit, they induce the following effects on the host including physical injury, physiological breakdown, texture change, and off flavor. Importantly, even a small lesion results in a complete loss of value so the tolerance limit for decay in any fresh product is zero. The primary cause of spoilage in whole apples is Penicillium expansum Link and Bottytis cinerea Pers. (Singliton, 1987; Snowdon, 1990). Blue mold rot caused by P. expansum is very destructive to apple fruits. The symptoms are soft, watery, light brown lesions, which undergo rapid enlargement at temperatures between 20 to 25°C. As the lesion grows, the decay portion can be easily separated from the surrounding sound tissue. It then becomes pale blue as sporulation occurs. P. expansum is generally considered be a wound parasite and infection commonly 9 follows rough handling and washing procedures after harvest. Infection can occur even at 0°C, although the decay proceeds slowly at cold storage temperatures. Gray mold rot caused by B. cinerea is light to dark brown in color. Gray mold lesions are characteristically less soft than those of blue mold and have a well— defined margin, but rotted tissue cannot be neatly separated from the surrounding healthy tissue. Lesions become covered with gray sporulating mycelium at optimum humidity. Under humid conditions, infection often occurs via wounds sustained during harvesting and handling. Rotting can proceed even at -1°C. Decay can spread to adjacent healthy fruit at this low temperature, but its growth rate is very low (Kiss, 1984; Sommer et al., 1992). The temperature range optimal for growth is 22—25C. B. cinerea can infect the sepals of apple flowers and live in a latent condition inside the tissue until fruit maturation (Tronsmo, 1977). It is unable to grow in unripe fruit, but will begin to grow as fruit mature. B. cinerea is a facultative parasite on several vegetables and fruits, such as strawberry, raspberry, blueberry, grape and apple. Interestingly, human pathogenesis by B. cinerea has also been reported (Beaumont et al., 1985). The number of B.cinerea’s spores on both outside and inside the dwellings has been correlated with the contraction of asthma (chronic coughing). In addition, Botrytis extracts are active in the skin tests in asthmatic patients showing a high sensitization rate comparable to that of Aspergillus filmigatus. Possibilities for controlling decay on cut apples. There are several possibilities for controlling rot of apple slices. Fungal inoculum and activity can be minimized during both preharvest or postharvest. For 10 example, fungicide sprays are usually applied to control fungal infection in the field. However, the use of chemical fungicides was limited by increasing of fungal resistance and chemical residue on the fruits (Eckert and Ogawa, 1988). Penicillium spp. isolates resistant to benomyl increased significantly (Spotts and Cervantes, 1986) as well as B. cinerea Pers. from grapes developed significant resistance to bendimidazoles and dicarboximide (Beever et al., 1989). Preharvest calcium sprays, and postharvest and preharvest calcium drenches (Kader, 1992) used to control bitter pit, also help to confer resistance to fungal infection. For lightly processed products, chlorine, usually in the form of calcium or sodium hypochlorite, is used for reducing fungal decay in many kinds of fruits and vegetables. It is commonly used at the concentration of 50-125 ugliter". For lightly processed products, strict hygiene in both orchard and packinghouse, during handling and light processing, would be critical (Huxsoll, 1989). Other potential techniques (irradiation, chemicals, preservatives, reducing water activity with sugar or salt) have also been used to retard the decay. Gamma radiation has been examined for extension of shelf life of perishable fruits (Thomas, 1986). However, the dosage that controlled microbial activity, was the same as that which damaged the fruits to a level unacceptable by consumers. Temperature control is one of the most important approaches to retard microbial decay. The growth rate of fungi decreases drastically at low temperature. Reducing the temperature (only 1C) effectively increases the lag time and significantly slows the rate of reproduction of microorganisms (Elliot and Michener, 1965; Wills et al.,1989). Low storage temperature also reduces respiration rate and chemical 11 reaction rate, inhibits enzymatic quality loss, and slows down ethylene production, each of which contributes to extending storability. However, psychrophilic microorganisms still grow at high growth rate. Additionally, undesirable physiological changes at low temperature should be considered. Therefore, the use of temperature to control fungal decay has some limits. Controlled atmosphere also has the potential to decrease the fungal growth rate. Rot on strawberry will be reduced when the fruits are stored under low oxygen and high carbon dioxide. B. cinerea on strawberry is dramatically controlled at 0.5% oxygen, but fruits are injured (Couey et al., 1966; Couey and Wells, 1970). Because of the potential for injuriously low 02 levels to be generated in MA packages, there is also a concern of growth of anaerobic bacterium, Clostridium botulinum. Thereby, most of these approaches are still unsatisfactory because of relative effect, toxicity of the chemicals, unacceptable residues, usefulness for complete inhibition of decay, complicated methods, etc. In this research, an emerging technology of the use of natural volatiles from fruits to retard fungal decay on the apple slices has been investigated. The concept is that the compounds produced by fruit, themselves, could reduce decay without adversely affecting fruit flavor. Use of natural volatiles for decay control. Several natural fruit volatiles have been found to retard decay of fresh fruits. Among these biologically active compounds are aldehydes such as acetaldehydes, hexanal, (E)—2-hexenal, nonanal, and benzaldehyde, ketones such as 2-nonanone, and 12 a—pentylpyranone, acids such as acetic acid, benzoic acid and sorbic acid, alcohols such as 1-hexanol, Z-3-hexen-l-ol, benzyl alcohol, and others. Acetaldehyde is a biologically active natural volatile compound that has a minor role in the flavor of fruits and vegetables. This compound is used as a flavoring agent, can be used as a chemical index of fruit ripening (Hayes, 1963), and is approved by EPA as a food additive. However, it was decried as a highly toxic compound, suspected carcinogen, human systemic irritant by inhalation, skin and eye irritant and flammable liquid (Lewis, 1992). Decay of fresh raspberries inoculated with Botrytis cinerea Pers. ex. Fr can be inhibited by acetaldehyde vapor treatment (Prasad and Stadelbacher, 1973; Prasad and Stadelbacher, 1974). Exposing the fruits to acetaldehyde vapor at 0.25% or 0.5% for 70 minutes is as effective in retarding fungi as at 1% or greater concentration. At concentrations of 0.5% and below, off- flavors are not detected. Decay of fresh strawberries, caused by B. cinerea or Rhizopus stonifer Ehr. ex. Fr. is also controlled by fumigation with acetaldehyde vapor (Prasad and Stadelbacher, 1974). Treatments with concentrations of 1%, 4%, and greater for 30, and 60 minutes kills conidia of the fungi. Treatment with 1% acetaldehyde is suggested as a practical control measure because it does not affect the quality of the berries. Stadelbacher and Prasad (1974) also studied the effect of acetaldehyde vapor on decay of apples inoculated with Penicillium expansum (Link) Thom. Inoculated apples and culture media were exposed to acetaldehyde vapor concentrations of 5% for 180 minutes, 1% for 120 minutes, 2% for 60 minutes, and 3% for 30 minutes. Fungal spores treated with acetaldehyde do not germinate in 21 days at 21C on the culture media and fruits. Importantly, treated fruit do not exhibit 13 lenticel or skin injury. Acetaldehyde vapor is highly effective in killing the P. expansum conidia and mycelium on culture media and fruits. Acetaldehyde residues are not detected within 21 day incubation after 8-10 day fumigation. The effects of sixteen volatile compounds occurring naturally in fruits on spore germination and growth of Monilinia fructicola Pers. ex. Fr. and B. cinerea were studied (Wilson et al., 1987). Benzyl acetate, benzyl alcohol, 6-caprolactone, a- decalactone, é-decalactone, 5-octalactone, methylsalicylate and 6-valerolactone inhibited spore germination of both fungi at 1250 pl-liter" . Benzaldehyde vapor entirely suppressed spore germination of B. cinerea at 25 til-literl and M. fructicola at 125 ul'liter‘. Complete inhibition of fungal growth can be obtained by a benzaldehyde, methylsalicylate or ethyl benzoate treatment concentration of 370 [1.1 liter". Chambers (1990) found that benzyl alcohol could control Botrytis cinerea rot on packed grapes during storage, but its efficacy is less than that of 802. Benzyl alcohol was not considered an acceptable treatment because of a physiological disorder that was induced and an off-taste imparted to fruit. Hamilton-Kemp et a1. (1992) and Andersen et a1. (1994) found wounded leaves produce a number of aldehydes, including 6-carbon and 9-carbon compounds via the action of lipoxygenase (Hildebrand, 1989). Of the C-6 and C-9 compounds released from tomato leaf, 5.2 ul-liter‘l of 2-hexenal, 268.7 [ll'litefl of hexanal, 47 ill-literl of 2-nonenal, and 44.8 ul-liter‘l of nonanal completely inhibit hyphal growth of B. cinerea. Alternaria alternata (FR.) Keissl. growth is completely suppressed by 8.3 III-liter“ of 2-nonenal. However, other compounds, including the terpene 14 hydrocarbons 2-carene and limonene, have no significant effect on hyphal growth. Urbasch (1984) found that E—2-hexenal is more effective than hexanal on growth of cultured B. cinerea. She consequently studied the effect of the corresponding unsaturated alcohol, E—2-hexen-1-ol, which was found to inhibit spore germination and fungi growth of B. cinerea. Deng et a1. (1993) studied the effect of 6-carbon aldehydes and alcohols from the lipoxygenase/hydroperoxide pathway on Escherichia coli TBl, Pseudomonas Syringae pv. tabaci Deall, and P. Syringae pv. angulata Deall. They found (E)-2-hexenal vapor completely inhibits proliferation of both P. syringae ( 101 ul-liter") and Escherichia coli TB] (165 til-liter"). Also, 2-hexen-1-ol prevents growth of P. syringae pv. (246.4 pl liter‘) and E. coli (515.2 Ill-liter"). P. syringae pv. angulata is the most sensitive of the organisms tested, being inhibited by as little as 7 III-liter“ (E)—2-hexenal. Unsaturated volatiles showed a greater inhibitory effect than saturated volatiles (Hatanaka, 1987). The essential oils used in medicinal drugs for controlling harmful insects were studied for their antifungal activity against the soil-bome pathogens and the foliar pathogen B. cinerea. Shimoni et a1. (1993) found essential oils extracted from Majorana syriaca (origanum), Satureja thymbra (savory), Micromeria frwicosa (savory), and Salvia trioba (saga), which are aromatic wild plants growing in Israel, to have antifungal properties. Extracts of these herbs were found to inhibit the growth of B. cinerea by 40% and Fusarium oxysporum f. sp. vasinfectum, Macrophomina phaseolina, and Exserohilum turcicum by 100%. Acetic acid was tested for its antifungal activity on apple fruits (Sholberg and Gaunce, 1995). The spores of B. cinerea and P. axpansum do not germinate after 15 treatment with 2.7 and 5.4 mg liter" (1008 and 2016 pl-liter") at 2 and 20C. Use of acetic acid at fungicidal concentration causes no apparent phytotoxic effect on fruit. In our own preliminary work, acetic acid was examined for antifungal properties using apple slices. However, the exposed tissue of apples was damaged at the minimal required concentration for controlling fungi. The cut tissue of apple slices had a moist surface that probably allowed the acid vapor to be easily absorbed, ‘ causing burning with less effective decay control (Sholberg, personal communication). Hinokitiol (B-thujiplicin) extracted from the roots of the Hinoki tree, Hiba arboruitae (Japanese cypress) retarded spore germination and mycelial growth of Botrytis cinerea and Altemaria alternata. It completely inhibited B. cinerea spore germination at concentration of 100 pl-l'l and that of A. altemata at 500 pl-l". Mycelial growth of Bonytis cinerea and Altemaria altemata were inhibited at 250 and 750 pl°l’1 , respectively. Decay on eggplants and red peppers were reduced after dipped in hinokitiol solution of 750 pl-ll (Fallik and Grinberg, 1992) Vaughn et a1. (1993) studied the effect of fifteen volatiles produced by raspberries and strawberries during ripening on fungal decay. They indicated that 5 compounds including benzaldehyde, l-hexanol, E—2-hexenal, Z-3-hexen-1-ol, and 2- nonanone completely inhibited all fungi on fruit at 400 pl liter‘. Of these compounds, benzaldehyde at 40 pl liter“ can completely inhibit A. altemata, B. cinerea and Coletotrichum gloeosporioides spp. The other compounds such as 1- hexanol, E-2-hexenal, and 2-nonanone also inhibit the indicated fungi at 100 pl-liter". 2—Nonanone appears to be particularly promising. It has a low mammalian toxicity and fruity floral odor. It is resistant to rapid decomposition while benzaldehyde, E—2- 16 hexenal and Z-3-hexen-lol are readily oxidized to less volatile compounds. Other natural fruit volatiles such as 2-hexanal, benzaldehyde, acetaldehyde, etc., have strong odor and taste, and some are toxic and were considered by Vaughn et a1. (1993) to be unsuitable. 2-Nonanone, released from encapsulated starch gel was further studied. It was released slowly to raspberry and strawberry fruits stored in film packages. A similar mechanism for chemical release might be used commercially to prevent decay of fruits. Factors that influence the selection of natural volatiles to control fruit decay might be expected to include low plant and mammalian toxicity, a pleasant or unobjectionable odor, resistance to rapid decomposition, commercial availability and adequate volatility (Buckingham, 1994; Lewis, 1992). For these reasons, we selected to examine the suitability of 2-nonanone as a natural volatile for decay control of apple slices. Most reports on use of natural volatiles to control decay were on whole fresh fruits. No prior investigations have been conducted on volatiles to control decay of lightly processed fruits or precut apple slices. In that regard, research in this area would be the first of its kind and much needed. CHAPTER 2 THE USE OF Z-NONANONE VAPOR TO CONTROL DECAY IN MODIFIED-ATMOSPHERE PACKAGES OF SLICED APPLES (Manuscript submitted to ’HortScience’) 17 18 W. A flow-through vapor exposure system was developed to test the effect of the natural volatile 2-nonanone on the decay of apple slices. 2—Nonanone vaporized in the system was continuously delivered to apple slices inoculated with Penicillium expansum Link. and Botrytis cinerea Pers. The vapor concentration in the system was determined by Gas Chromatography. Treatments with 2-nonanone vapor at 133 to 303 pl of 2-nonanone vapor per liter of air at 23°C retarded the growth on PDA. At a concentration of 303 pl-liter", the fungal growth was completely suppressed. However, the fungus was not killed as it regrew when transfered to 2-nonanone free air. No lesion of P. expansum and B. cinerea developed on apple slices treated with 290 and 222 pl-liter‘(respectively). P. expansum was more susceptible to 2-nonanone at 5°C than at 23°C. The growth rate of P. cxpansum at 5°C was approximately 4 times less than that at 23°C. Approximately 50 pl-liter'1 supressed P. expamsum on both agar and apple slices. Treatment with 100 pl-liter’l at 5°C apparently killed P. expansum since no re-growth was detected after transfered to air. Physiological damage was found on the skin of the apple slices after 24 hr treatment at 23°C and 6 hr treatment at 5°C. The demand of lightly processed apple or precut apple is expected to increase rapidly in the 1990’s (Cook, 1992; Hoag, 1995; How, 1991; Schlimme, 1995). Airlines, hotels, schools, and hospitals have shown interest in precut apple products such as fresh pie fillings, dessert packs, and salad bar offerings. The development of retail and institutional precut apple products has been limited by a number of processing—related difficulties which include browning, moisture loss, texture change, 19 and decay. Decay of a sliced product has not received much attention. Browning, moisture loss, and texture change in sliced apples can be controlled or minimized properly by combining modified atmosphere and selected chemical treatments. Although browning can be controlled by using modified atmosphere alone, other techniques using edible coatings, seaweed extract (Stephens, 1994), ascorbic acid, fruit extracts high in ascorbic acid (El-shimi, 1993), and other chemicals have been used together with MAP. On the other hand, moisture loss and respiration rate of cut apples can be controlled by using suitable polymer material (Lakakul, 1994). Texture damage can be reduced by calcium treatment, and proper temperature storage (Kader, 1992). Decay of whole apples can be caused by Penicillium expansum Link and Bonytis cinerea Pers. and a number of other organisms (Singleton, 1987; Snowdon, 1990). Blue mould rot caused by P. expansum is one of the most destructive diseases of post-harvested apples. The symptoms of blue mold attack are soft watery brown spots on the apple surface which undergo rapid enlargement at temperatures between 20°C to 25°C. P. expansum is generally considered to be a wound parasite, and infection commonly follows rough post-harvest handling and washing procedures. Infections can occur even at 0C, although the decay proceeds slowly at cold storage temperatures. B. cinerea causes gray mould rot in apples and is a common pathogen for nearly all stored fruits. Several approaches are used to eliminate decay on entire fruits. Among these are fungicide sprays, calcium, and chlorine sprays, irradiation, reducing water activity with sugar or salt, and temperature control. However, most of these are 20 unsatisfactory for sliced apples. The attention has turned to the use of volative natural compounds to control fungal decay. Natural fruit volatiles have been shown to have potential for retarding or preventing decay of whole and sliced fruit products. Prasad and Stadelbacher (1973), and Prasad and Stadelbacher (1974) indicated that acetaldehyde vapor could be used to retard the decay of fresh strawberries and raspberries caused by both Rhizopus stolonifer Ehr. ex. Fr. and B. cinerea. Complete inhibition of Monilinia fructicola Pers. ex. Fr. and B. cinerea was obtained by benzaldehyde, methylsalicylate and ethyl benzoate at 1250 pl-liter’l (Wilson, 1987). Hamilton-Kemp et al.(1992) found natural volatile compounds such as 2—hexana1, 2- nonenal, and benzaldehyde to have significant inhibitory effects on the growth of fungal hyphae of Alternaria altemata (Fr.) Keissl. and B. cinerea. Vaughn, et al (1993) showed that a number of volatile compounds from raspberry and strawberry fruit have antimicrobial activity. In particular, 2-nonanone was suggested to merit further investigation. Beneficial factors that influence the selection of natural volatiles to control fruit decay, include low plant and mammalian toxicity, pleasant odor, resistance to rapid decomposition, commercial availability, and adequate volatility. A number of natural fruit volatiles such as 2-hexanal, benzaldehyde, acetaldehyde, etc., while effective, have strong odor and taste, and some have an unacceptable level of plant or animal toxicity. 2—Nonanone, however, has a low toxicity to strawberry and raspberry, and has a floral fruity odor. 2-Nonanone or heptyl methyl ketone [ H3C(CH2)COCH3 ] is a natural product found in the algerian oil of rue, altar of rose, clove oil, passion flowers, sorghum, asparagus, tomato, corn, bleu cheese, and beer (Gildemeister, 1913). It is a colorless 21 oil at room temperature with an odor similar to rue. 2—Nonanone was categorized as a medium toxic compound with a LD50 of 400-4000 mg-kg" (Buckingham, 1994; Lewis, 1992). 2-Nonanone is combustible when exposed to heat or flame and reacts with oxidizing materials. When heated to decomposition, it emits acid smoke and irritating flame. The Food and Drug Administration (FDA) has approved 2- Nonanone as a synthetic flavoring substance that is safely used in food. Its equilibrium vapor pressure is approximately 77.26 Pa (800 pl-liter") at 23°C and 21.61 Pa (200 pl-liter“) at 5°C (Perry et al., 1984). Our goal was to determine threshold levels of 2-nonanone needed to completely inhibit P. expansum and B. cinerea growing on general medium, PDA, and apple slices exposed to the volatile. The effect of temperature on fungal sensitivity to 2-nonanone was also examined. Materials and methods A. Material. Plant material. ’Red Delicious’ apple fruits were harvested from the Michigan State University Clarksville Horticultural Experiment Station and stored at 0°C until used. Fruit were removed from the cold room and allowed to equilibrate to ambient temperature (23°C) which took approximately 1 hr. Each fruit was rinsed with tap water, then sliced into eight wedges weighing 20-25 g each, inoculated, and exposed to air in the treatment chambers with 2-nonanone and control chamber without 2- nonanone. The steps of apple slice preparation and inoculation were done under a hood in aseptic technique condition. 22 2-Nonanone. 2-Nonanone (99 % purity) was obtained from Aldrich Chemical Company, Inc.(Saint Louis, MO). It was stored at 4°C until used. Microorganism. Pure strain of Penicillium expansum Link. was obtained in the freeze-dried state from the American Type Culture Collection (Rockville, MD). Botrytis cinerea Pers. on slant-agar tube was kindly obtained from Dr. Rodney Roberts (Pathologist, USDA-ARS) of Wenatchee Tree Fruit Research Station (Wenatchee, WA). Culture media. Potato Dextrose Agar (PDA) was used for supporting the growth of both fungi in the assay system. In addition, V-8 Juice agar was employed for B. cinerea at 5°C to enhance growth. PDA was obtained as finished powder from Difco Laboratories (Detroit, MI) and was mixed with water. V-8 juice agar was prepared according to Handbook of microbiological media (appendix 1). B. Methods. Biological technique. Pure strains of decay organisms Penicillium expansum Link. and Botrytis cinerea Pers. were diluted in sterilized water to obtain a culture solution for preparation of a stable stock culture. The fungal solutions were inoculated on the center of PDA agar and incubated at room temperature (23°C), which is near the 22 2-Nonanone. 2-Nonanone (99 % purity) was obtained from Aldrich Chemical Company, Inc.(Saint Louis, MO). It was stored at 4°C until used. Microorganism. Pure strain of Penicillium axpansum Link. was obtained in the freeze-dried state from the American Type Culture Collection (Rockville, MD). Botrytis cinerea Pers. on slant-agar tube was kindly obtained from Dr. Rodney Roberts (Pathologist, USDA-ARS) of Wenatchee Tree Fruit Research Station (Wenatchee, WA). Culture media. Potato Dextrose Agar (PDA) was used for supporting the growth of both fungi in the assay system. In addition, V-8 Juice agar was employed for B. cinerea at 5°C to enhance growth. PDA was obtained as finished powder from Difco Laboratories (Detroit, MI) and was mixed with water. V-8 juice agar was prepared according to Handbook of microbiological media (appendix 1). B. Methods. Biological technique. Pure strains of decay organisms Penicillium expansum Link. and Bonytis cinerea Pers. were diluted in sterilized water to obtain a culture solution for preparation of a stable stock culture. The fungal solutions were inoculated on the center of PDA agar and incubated at room temperature (23°C), which is near the 23 optimum temperature (25°C) for fungal growth (Snowdon, 1990). After 2 days the single colonies were collected from the petri dishes and transferred to PDA in glass bottles. Inoculated preparation bottles were stored at room temperature for 2-3 days to allow the fungi to cover the surface of PDA. The fungi were then harvested by adding sterilized water to the preparation bottles. A magnetic bar and stirring Fisher Scientific hot plate model 310T (Pittsburgh, PA) were used to elute most fungal spores from the surface of the PDA. The fungal suspension was filtered by strile glass wool to remove the agar and rinsed with sterilized water. The filtrate was homogenized and centrifuged twice at 5000 rpm for 10 min. The fungal culture pallet at the bottom of the suspension was separated from the clear liquid at the top. The culture pallet was suspended in a 20% glycerol aqueous solution to make a stock culture. The spore concentration of the stock culture was determined by directly counting cells with a hemocytometer. In each solution stock, there were 1.2 x 108 cells/m1 of P. expansum and 7.8 x 108 cells/ml of B. cinerea respectively. The stock cultures were stored at -70°C. For inoculating apple slices and agar plates, the stock culture was transferred from ~70°C storage and thawed on an ice bath. Melted stock culture was diluted with sterilized water until its concentration was approximately 10’ cells/ml. A 5 pl aliquot of the fungal solutions was placed on the surface of the apple slice and petri plate samples. Three inoculation sites were made per PDA petri dish, four dishes per desiccator, and two inoculation sites were made per apple slice, two slices per dish and two dishes per desiccator. Petri dishes containing agar were inverted. 24 Bioassay system. A flow—through vapor exposure system (Fig. 1) was constructed. The 9.3-liter glass desiccators and 3 mm-intemal diameter teflon tubing were sterilized and inlet air was filtered by in-line microbial filters (0.45 micron pore size) obtained from Alltech Associates, Inc. (Deerfield, IL). A glass vapor generator containing 2-nonanone was used to create 2-nonanone vapors by bubbling sterilized air through 2-nonanone. The exiting 2-nonanone vapor was mixed with 2-nonanone-free air to reduce the vapor concentration. The concentration of 2-nonanone was controlled by adjusting the flow rates of the air passing through the glass bubbling tube and the 2-nonanone-free air. Flow rates were adjusted with glass microbore capillary tubes in conjunction with a Scientific pressure regulator (South Plainfield, NJ). The concentration of 2-nonanone is here expressed in pl of 2-nonanone vapor per liter of air at 23°C. To generate 2—nonanone concentration higher than 400 pl-liter’l a water bath was used to heat the vapor generator to 50 to 60°C. Air containing 2-nonanone was directed into the desiccator with 3 mm-id flexible teflon tubing, which was impermeable to 2-nonanone vapors. The flow rate through the desiccator was 20 to 25 ml°min". Treatment concentrations of 2-nonanone ranged from 0 to 520 pl-liter" at 23°C and from 0 to 160 pl-liter’l at 5°C. The concentration of 2-nonanone vapor was measured by withdrawing gas sample from the integral glass sampling port fitted with a teflon-lined septum at the outlet of the exposure chamber. Following this, outlet vapors were directed to a fume hood using (1/4 inch) ID flexible tubing. The actual time to achieve the equilibrium or steady state of 2-nonanone concentration at the head space 25 of the treatment chamber was 2-3 days (see appendix 3). The apple slices and petri dishes were then placed in the treatment chamber. The relative humidity (RH) in the chambers was measured by connecting an eletronic hygrometer Indicator Model 15-3001 with a Hygrodynamic, Newport Scientific, Inc. sensor type 4837 KW (Silver spring, MD) to the outlet of the chambers. The RH of the unhumidified experiment was approximately 70% RH. A humid environment was generated in the desiccators, by passing the diluted 2- nonanone vapor through sterilized water contained in the desiccator. The relative humidity in the system was above 90%. 2-Nonanone analysis. Quantification of the 2-nonanone concentration in the treatment chamber was determined by gas chromatographic analysis. Standard concentrations were prepared from the headspace vapor in equilibrium with pure 2-nonanone liquid at 23°C, contained in closed 50 CC glass vials. The vapor concentration was calculated from Perry’s Chemical Engineering Handbook. The relationship between the logarithm of equilibrium vapor pressure and the reciprocal of temperature should be linear in accordance with Clapeyron-Clausius equation (Appendix 2). For our work, a calibration curve of GC response versus 2-nonanone concentrations calculated from the equilibrium vapor pressure (at 3 different temperatures) was constructed. A Carle AGE Gas chromatograph series 400 (Loveland, CO) was equipped with a ionization detector, and 10% DEGS-PS, 80/100 mesh Supelcoport, 3.3 m long, 3 mm OD flame packed column from Supelco (Bellefonte, PA). The column was maintained 26 isotherrnally at 140°C. Helium was used as carrier gas at flow rate of 60 ml-min". Air flow rate was 200 ml'min“ and hydrogen flow rate was 20 ml°min". 2-nonanone showed a retention time of 2.47 min. For the 2-nonanone standard, liquid 2- nonanone (99% purity) was put in 3 glass vials and then surrounded by styroform box to prevent drastic temperature change. These vials were incubated at three different temperature until equilibrium between the liquid and vapor phases was achieved. The vials were fitted with a mininert gas tight septum. A Hamilton gas tight syringe No.1705 (Deerfield, IL) fitted with a stainless steel 22 gauge needle was used to sample vial headspace. The syringe was pumped 10 times and gas sample (50 pl) was removed from the headspace, and directly injected into the gas chromatograph. The desorption time at the GC injection port was 3 sec. 2-Nonnanone was calculated using a standard for each analysis. The 2-Nonanone concentration at the inlet and outlet of the treatment chambers was determined every 4-6 hr at 23°C and 24 hr at 5°C. Each data was an average of 3-5 injections. The concentrations at the outlet port and at the headspace of the test chamber were approximately the same. Antifungal assessments. Inoculated apple slices and petri plates were exposed to treatments for 7-10 days at 23°C and 30 days at 5°C. For the control condition, inoculated apple slices and petri plates were incubated in 2-nonanone—free air. Growth of fungi (diameter of the colonies) was monitored every 24 hr from outside the desiccator. Colony diameter (mm) was measured using a scale marked on the bottom of the inverted clear 27 glass petri dishes. Because of the difficulty of measuring the change of the colony diameter on the surface of the apple slices, only the presence indicated with a ’+’ and absence indicated with ’-’ of lesion growth was noted. Results The standard calibration curve. The relationship between the GC response and 2-nonanone concentration was found to be linear (Fig. 2). The GC-response was linear with 2-nonanone concentration in the air according to the following equation Y =0.08083X + 0.9957 where X is the 2-nonanone concentration (pl-liter") and Y is the GC response in thousands. The coefficient of determination (r2) was 0.999. Standard error was less than 10%. Effect of humidity on the fungal growth. At 90% RH in control chamber (without 2-nonanone), the growth rates of P. expansum and B. cinerea were 0.26 and 0.42 mm-h", respectively as shown in figure 3. Under low RH (70%) the growth rates were similar, 0.27 and 0.48 mm-h“, respectively. The growth of Penicillium expansum under 2-nonanone treatment at 23°C. P. axpansum growth on PDA media was completely stopped at vapor concentration of 303 pl-liter" and its growth rate was reduced relative to air controls 28 by lower concentrations of 2-nonanone as shown in table 1 and figure 4. Fungal growth was 50% inhibited at a concentration of approximately 156 pl-liter". The growth rate of P. expansum was inversely proportional to the 2-nonanone concentration in the headspace as indicated by following equation Y = 0.246192 — 0.000787548 X where X is the 2-nonanone concentration (pl-liter") and Y is the growth rate (mm-h“). The coefficient of determination (r2) was 0.916. Standard error was less than 10%. The inoculated apple slices in the control chamber (without 2-nonanone) started to decay after 28.5 to 35 hr. Fungi inoculated on the fruit slices did not grow when treated with 2-nonanone at 303 pl-liter’l (Table 2). However, fungi grew again after slices were removed from the test chamber. At lower concentrations (133 to 290 pl-liter") the fungi were able to grow on the apple slices, however decay was not noted until 48 hr after inoculation. The growth of Botrytis cinerea under 2-nonanone treatment at 23°C. The growth rate of B. cinerea inoculated on PDA petri dish was decreased by 2-nonanone treatment in a manner similar to that for P. expansum (Table 3 and Figure 5). B. cinerea was completed controlled by 2-nonanone at 303 pl-liter’l and was vulnerable to lower concentrations of 2-nonanone when compared to air. Similarly, to P. expansum, B. cinerea was not killed at 303 pl-liter’l , and it grew again after being removed to air. Unlike the response of P. axpansum, the decline in growth rate with increasing 2-nonanone concentration was not linear. However, the 29 relationship seemed to be linear when treatment concentration above 100 pl-liter". The growth of B. cinerea was reduced approximately 50% when treated with 240 pl-liter‘. B. cinerea on apple slices in the air control started to grow after 58 to 82 h. The growth of B. cinerea on slices was delayed by 24 hr when treated with the concentration below 222 pl-liter". It did not grow when treated with a 2-nonanone concentration of 222 pl-liter"(Table 4). Effect of temperature on the fungal growth. In air, P. expansum grew more slowly at 5°C than at 23°C as shown in the table 5 and Figure 4. The fungal growth rate at room temperature (0.25 mm-h") was approximately 4 times greater than that at 5°C (0.06 mm-h"). At low temperature, the fungus was more susceptible to 2-nonanone. At a 2-nonanone concentration greater than 50 pl-liter“, no fungi grew. P. expansum grew 50% more slowly than in air when the 2—nonanone concentration was at 26 pl-liter". The following equation presents the linear relationship between the 2-nonanone concentration and growth rate Y = 0.0608 - 0.00116X when X is the concentration of 2-nonanone (pl-liter") and Y is Growth rate of P.expansum (mm.h“). The coefficient of determination (r2) was 0.902. Standard error was less than 10%. The slices in the air control started to decay after 5 to 9 days. P. axpansum behaved similarly to colonies on PDA (Table 6). P. expansum lesions on slices developed at 26 pl-liter‘l 2-nonanone, however, no lesion development occurred when 3O 2-nonanone concentrations were greater than 50 pl-liter“. P. expansum was killed by 100 pl-liter’l (Table 6). At 5°C B. cinerea did not grow on either PDA agar or fruits at control or 2- nonanone treatment condition. Additionally, the V8 agar, which is the enriched media for B. cinerea, was not capable of inducing the fungal growth, therefore no growth data for B. cinerea was presented for the low temperature study. Skin damage. Physiological damage to the skin of apple slices was induced by 2-nonanone. 2-Nonanone concentrations of greater than 133 pl-liter" at 23°C and greater than 26 pl-liter'l at 5°C which were effective by retarded fungal growth, damaged the apple skin. The skin color of apple slices changed from red to yellow and brown less than 24 hr after treatment at room temperature. At 5°C, the slices started to change its color after only 5 hr of treatment with 2-nonanone at 50 Ill-liter". This skin damage resembled the symptoms of superficial scald in color, tissues affected and the presence of ’clear zones’ around the lenticels during the early stages of the disorder development. Discussion The lack of a humidity effect on fungal growth in air or when exposed to 2- nanone may be a result of a humid microclimate around the low RH dishes and slices. At 70%RH, because the inoculated apple slices and PDA were contained in the petri 31 dishes with the glass cover, the moisture inside the petri dishes was prevented from loss to the headspace of the chambers. 2-Nonanone was slightly more effective against P. expansum than B. cinerea. For comparison, at 23°C the concentration which reduced the growth rate of P. expansum by 50% was 156 pl-liter‘ and that of B. cinerea was 240 pl-liter". This means that the use level of 2-nonanone concentration to control the decay on apple slices should be based on the threshold level to inhibit growth of B. cinerea. Both fungi appeared to be more sensitive to 2-nonanone when growing on slices than on the culture media. Fungi on apple slices were completely inhibited while some growth took place on the media at 2~nonanone concentration between 224 to 303 pl-liter". This could be due to the sliced apple tissue preventing the absorption of more 2-nonanone than the PDA. Since a 2-nonanone concentration between 133 to 303 pl-liter'l did not kill the fungi. If a level which only temporarily inhibits fungi is used, the shelf life of the slices after removal from the treatment chamber or package needs to be determined. However, the concentration which is applied to the slices must be safe to consumers and operators. 2-Nonanone was categorized as a medium toxic compound with a LDSO of 400-4000 mg-kg‘l (Lewis, 1992). Other compounds such as hexanal and acetaldehyde were classified as highly toxic (LD50 below 400 mg-kg“). Based on this result, the 2-nonanone level which will be applied to retard the decay on slices needs to be within the safe range. However, the data were based on the experiment on rats, not on human consumption. Vaughn et a1. (1993) reported that the 2-nonanone concentration of 400 pl-liter’l inhibited all the growth of fungi (A. alremata, B. cinerea, 32 C. gloeosporioides) sprayed on raspberry and strawberry kept in sealed chamber (in dark) at 10°C. The threshold level of fungal inhibition at 10°C was studied on V-8 media. Those fungi were inhibited by 100 pl-liter‘. Although these data can not be compared directly to our experiment, since the sensitivity of the fungi to 2-nonanone on V-8 media and PDA may be different, the fact that these fungi were suppressed by a lower concentration than found at 23°C could be significant. P. expansum was more susceptible to 2-nonanone exposure at 5°C than that at 23°C. This should be explained by temperature effect since 2-nonanone might absorp more to the surface of apple slice and PDA at the low temperature. Additionally, the fungi, themselve, grow more slowly at the low temperature. This research has shown the possibility of use the naturally occurring compounds for extending the shelf—life of minimally processed fruits and vegetables. While it appears, 2-nonanone would be inappropriate for preserving apple slices due to skin damage, other antifungal compounds likely exist which are less toxic to the treated tissue. Some flavor constituents of apple fruit having antifungal properties include ketones, aldehydes, alcohols and acids. Aldehydes such as hexanal might provide the best way for modified atmosphere package of precut apple slices in that it is a strong antifungal vapor which does not affect fresh-like characteristics of slices but also enhances fruit flavor (Song, 1994; Song et al., unpublished). The threshold level of each volatile that completely inhibits decay should be determined and compared to safe level for the human exposure. The attempt to use natural volatile to control decay to fulfill the commercial demands needs further investigation. 33 Additionally, integrating the use of volatile compounds and gas exchange requirements for apple slices in a modified atmosphere package (MAP) system must be investigated more detail. System design needs include characteristics of apple slices (respiration rate, tissue sensitivity, etc.), packaging materials (permeability, adsorption, temperature sensitivity, etc.), and the release rate and temperature sensitivity of the compound delivery system. MAP involves matching the respiratory rate of apple slices to Oz and C02 permeability of the package. MAP should provide the proper atmosphere with low 02 and high CO; to influence the best quality of the slices in the package. The film permeability and respiration rate of slices as temperature-dependent should be considered. Lakakul (1994) suggested a mathematical model which presented the relationship between steady state 0; partial pressure and O; uptake and film permeability to O, of packages. This model predicted the effect of oxygen permeability, activation energy, temperature, film type, and film thickness on the 02 partial pressure. The method of delivery and vapor release inside the package need further attention. In addition, matching organic compound release from the package to release within the package to maintain the target headspace concentration in the MAP needs to achieved. 2-Nonanone is adsorbed and/or absorbed by many materials including glass, metal, non-polar polymers, etc. Vaughn et al.(1993) suggested the use of starch encapsulation of 2-nonanone for preserving raspberry and strawberry fruit. In this case 2-nonanone was combined with Miragel (Pregelatinized cornstarch, A.E. Staley, 34 Decatur, IL) for a ’fast release’ of the compound to the headspace of packages. A ’slow release’ formulation was achieved by adding some amount of glycerine to the Miragel. For any a release or delivery system for anti-fungal compound to be successfully employed, sorption characteristics of the compound need to be assessed. Other factors outside the scope of this study that result in a reduced shelf-life of the apple slices in the MAP must also be diminished to create a commercially viable product. Approaches to reduce browning in the fruit slices are being investigated. A Sea wwd-based edible coating (Stephens, 1994), and ascorbic acid (El-shimi, 1993) can reduce browning in the fruits. Importantly, these chemicals are reported to not adversely affect the original taste or flavor of the precut fruits. Foodbome human pathogen have been an important concern when one deals with the MAP for minimally processed fruits and vegetables. A great number of microorganisms on the products in the MAP have been found (Nguyen-the, 1994). No data on the apple slices are available at present. However, from our work, growth of other microorganisms (apparently bacteria) on the apple slices at the low temperature (5°C) were found. The proper handling and storage condition need to be further defined for the precut fruits. Slicing, peeling or other types of cutting, all impose substantial physical injury. Therefore, specific guidelines should be generated for slicing of fresh product (Will, 1989). For pie filling, cutting blades must be sharp, and rotating blades have been suggested (Price, 1993). Rinsing with or submerging of slices in chlorinated water may be helpful to reduce or remove nutrients and enzymes released after cutting to avoid extensive degradation and browning (Price, 1993). 35 REFERENCES Atlas, RM. 1993. Handbook of microbiological media. CRC Press, Boca Raton. p. 973. Buckingham, J. 1994. Dictionary of natural products. lst ed. Chapman & Hall, NY. Cook, R.L. 1992. The dynamic U.S. fresh produce industry an overview. p.3-13.In: A.A.Kader(ed.). Postharvest technology of horticultural crops. 2nd ed. Univ. of California Oakland,CA. El-shimi, N .M. 1993. Control of enzymatic browning in apple slices by using ascorbic acid under different conditions. Plant Foods Hum. Nutr. 43:71-76. Food and Drug Administration 21CFR ch.1(4-l-93 Edition). Gildemeister, E.,1913. The Volatile Oils. Wiley, N.Y. p.28. Hamilton-Kemp, T.R., C.T. McCracken, J.H. Loughrin, R.A. Andersen, and D.F.Hildebrand. 1992. Effects of some natural volatile compounds on the pathogenic fungi Alternaria alternata and Botrytis cinerea. J. Chem. Ecol. 18(7):]083-1091. Hoag, D. 1995. Fresh-cut fruit flowers in an array of ways. Produce Business. June p.35-45. How, RB. 1991. Marketing fresh fruits and vegetables. In: V. Norstrand (ed.) AVI book. Reinhold, NY. Kader, A.A. 1992. Postharvest biology and technology: an overview. p.15-20.In: A.A.Kader(ed.). Postharvest technology of horticultural crops. 2nd ed. Univ. of California Oakland,CA. Lewis, R.J.Sr. 1992. Sax’s dangerous properties of industrial material. Van Nostand Reinhold, N .Y. Lakakul, R. 1994. Modified-atmosphere packaging of apple slices: Modeling respiration and package oxygen partial pressure as function of temperature and film characteristics. Thesis. Michigan State Univ., Mich. Nguyen-the, C., and F. Carlin. 1994. The microbiology of minimally processed fresh fruits and vegetables. Crit. Rev. Food Sci. Nutr. 34(4):371-401. Perry, H.R., D. Green, and J.O. Maloney. 1984. Perry’s chemical engineering handbook. McGraw-Hill, N .Y. 36 Prasad, K. and G.J. Stadelbacher. 1973. Control the postharvest decay of fresh raspberries by acetaldehyde vapor. Plant Dis. Rep. 57:795-797. Prasad, K. and G.J. Stadelbacher. 1974. Effect of acetaldehyde vapor on postharvest decay and marketing quality of fresh strawberries. Phytopathol 64:948-951. Price, J .L. and JD. Floros. 1993. Quality decline in minimally processed fruits and vegetables. In: G. Charalambous (ed.). Food flavors,ingredients, and composition: proceedings the 7th international flavor conference, Pythagorion, Samos of Greece, 24—26 June 1992. Amsterdam, N .Y. Schlimme, D.V. 1995. Marketing lightly processed fruits and vegetables. HortScience 30(1):15-17. Singleton, P. 1987. Dictionary of microbiology and molecular biology. 2nd ed. Wiley, N .Y. Snowdon, A.L. 1990. Color atlas of post-harvest diseases and disorders of fruits and vegetables. CRC Press, Boca Raton. p.178-179,]88-l89. Song, J. 1994. Production and development of volatile aroma compounds of apple fruits at different times of maturity. Acta Hort. July, p.368. Song, 1., R. leepipattanawit, W. Deng, and R. Beaudry. 1996. Hexanal vapor inhibits fungal growth and decay development and enhances aroma biosynthesis in apple slices. (unpublished). Stephens, D. 1994. Edible coatings slice open new market. Fruit Grow. June, p.6—7. Vaughn, S.F., G.F. Spencer, and BS. Shasha. 1993. Volatile compounds from raspberry and strawberry fruit inhibit postharvest decay fungi. J. Food Sci. 58:793-796. Wills, R.B.H., W.B. McGlasson, D. Graham, T.H. Lee, and E.G. Hall. 1989. Postharvest: An Introduction to the physiology and handling of fruits and vegetables. Van Nostrand, Reinhold, N .Y. Wilson, C.L., J .D. Franklin, and BE. Otto. 1987. Fruit volatile inhibitory to Monilim'a Fructicola and Botrytis cinerea. Plant Dis. 71(4):316-319. 37 Table 1 Effect of 2-nonanone treatment on growth rate of P. expansum on PDA held for 7 days at 23°C. concentration Growth rate (pl-liter") (mm-h") 0 0.2804 0 0.2375 0 0.2194 133 0.1503 166 0.1440 207 0.0272 224 0.0696 290 0.0454 303 0 439 0 38 Table 2 Effect of 2-nonanone on Penicillium expansum growth on inoculated apple slices held for 7 days at 23°C. Concentration "Lesion BSkin Damage (pl°liter’" development 0 (control) + 133 166 207 224 290 - 303 - ++++ ##WWNNO " ’+’indicated lesion growth, and -’indicated no visible lesion growth. 3 level of the skin damage, 0 = no damage, 4 = the most severity (the color turned yellow-brown). 39 Table 3 Effect of 2-nonanone treatment on growth rate of B. cinerea on PDA held for 7 days at 23°C. concentration Growth rate (pl-liter") (mm-h") 0 0.4315 0 0.4180 0 0.41 l 1 1 16 0.4168 133 0.3330 161 0.3773 180 0.2947 214 0.2652 222 0.2938 303 0 439 0 39 Table 3 Effect of 2—nonanone treatment on growth rate of B. cinerea on PDA held for 7 days at 23°C. concentration Growth rate (pl-liter") (mm-h") 0 0.4315 0 0.4180 0 0.4111 116 0.4168 133 0.3330 161 0.3773 180 0.2947 214 0.2652 222 0.2938 303 0 439 0 40 Table 4 Effect of 2-nonanone on Botrytis cinerea growth on inoculated apple slices held for 7 days at 23°C. Concentration ALesion BSkin Damage (pl liter” development 0 (control) + 1 16 133 161 180 2 14 222 - 303 - 440 - +++++ #ADWWNNNO A ’+’indicated lesion growth, and -’indicated no visible lesion growth. 3 level of the skin damage, 0 = no damage, 4 = the most severity (the color turned yellow-brown). 41 Table 5 Effect of 2-nonanone treatment on growth rate of P. expansum and B. cinerea on PDA held for 30 days at 5°C. Growth rate concentration (mm.h“) (pl°liter") . P. expansum B. cmerea 0 0.0700 0 0 0.0492 0 26 0.0359 0 50 0 0 60 0 0 100 0 0 160 0 0 42 Table 6 Effect of 2-nonanone on decay of apple slices inoculated with P. expansum and B. cinerea and held for 30 days at 5°C. Conc AGrowth of Growth of BSkin Damage (pl-liter" P. expansum B. cinerea 0 (control) + - 26 + - 50 - - 60 _ _ 100 - - 160 - - 303 - - MNNNNHO " ’+’indicated lesion growth, and -’indicated no visible lesion growth. 3 level of the skin damage, 0 = no damage, 4 = the most severity (the color turned yellow-brown). 43 8.2.. E25 con—=58 EoE.8C.H .528 3:6 8:55» o:o§:0=-m.n 3.89:3 .893 835.». ton wEEEmmd .888 >6: .._<.N 8:... 3833232; 823m 8:89am .593 finch—.732; ._ Bani 44 1 1 1 r A 1 l J 160“ Y = -0.9957 4' 0.08083X - R-square = 0.999 .a N ‘1’ I GC-response (thousand) 8 r l m ' T . l ' l ‘ l a 0 400 800 1200 1 1600 2000 Concentration (pl.liter' ) Figure 2. Calibration curve for 2-nonanone (the concentration of 2-nonanone was estimated from Perry’s Chemical Engineering Handbook). 45 40 ll 11111 lIAAJrIAllr11111.111111rlr 353 + P.expansum at .90%RH . :_ 3 —O— B.cinerea at >90%RH E E 30% --v-- P.expansum at70%RH ' V :_ é : ---~®-~- B.cinerea At 70%RH , ' ; .52 25: :- ‘é’ ‘ C .22 2°? 3‘ '0 g : 2 ~ . 0 .: _~_ 0 1o: : 5f :_ 0d"WW"'IfiT'fi‘ILT‘TflT‘HHI‘H"Ii'h O 24 48 72 96 120 Time (hrs) Figure 3. Fungal growth on inoculated petri dishes in air as affected by RH. 46 ‘T I .C': .. E A Y = 0.2462 - 0.0007875X (230) : E a Y = 0.06081 - 0.001161X (so) - v i- p E - . 3 — _ w 0.2 q c . m a g - Q I A C 0- 1 : T5 0 1 i i .9 : 9 43 g 4: A _ e ‘ ‘ i o 0.0 T /I\ I I A l i n I 1 IA I r I i 1 Li 1 0 100 200 300 400 500 2-N0nanone concentration (pl.liter'1) Figure 4. Effect of 2-nonanone treatment on growth rate of Penicillium expansum at 5°C and 23°C (more than 90%RH). 47 .0 .0 .0 (JO -§ 01 ' w . At room temperature .0 N 1 Growth rate of B.cinerea (mm.h'1) .0 .—l l ' l . l 1 l- 1 " 0 100 200 300 _ 400 500 Concentration (pl.liter ) .0 0 Figure 5. Effect of 2-nonanone treatment on growth rate of Botyrtis cinerea at 23C. CHAPTER 3 METHOD DEVELOPMENT 48 49 A.Bioassay. A number of volatile exposure chambers have been utilized in various studies, each with its own shortcomings. Generally, 2 types of apparati aere constructed; closed and open systems (although there are some in-between closed and open systems). For closed systems, concentrations were calculated from a mount of compound vaporized in a chamber of known volume. A bioassay system developed by Hamiton and Kemp (1992) consisted of a 9-cm-petri dish in which a 5-cm-glass petri dish with agar block and l—cm-diameter glass sample dish was placed. Fungi were inoculated on the agar dish. The liquid compound or crushed leaf which released the volatile was added into the l-cm-diameter dish and vaporized to the headspace of the system. A 250-pl sample of headspace vapor was withdrawn through the septum with a gas tight syringe and injected to the gas chromatograph (Varian 3700). A similar system was employed by Vaughn et al.(1993). A 275-ml airtight glass jar with aluminum foil cap liners was constructed as a treatment chamber. An exact amount of compound was added to the filter paper disk (whatman No.1) and then placed in the test jar. The total headspace was 250 ml, and the vapor concentration was measured by GC analysis. Prasad and Stadelbacher (1973) and Prasad and Stadelbacher ( 1974) treated fruits with acetaldehyde in the fumigation chamber, a 8-liter desiccator with a gas tight injection port at the top. An accurate amount of liquid acetaldehyde was injected to the test chamber. Acetaldehyde vapor in the headspace of the treatment jar was maintained with a magnetic stirrer. Vapor concentration was determined in percentage of atm by volume (V/V). The standard curve of percentage of acetaldehyde in vapor phase versus detector response was 50 constructed by GC analysis. Temperature and humidity were also regulated. Those bioassay systems were set up as the similar methodology. They were closed systems with known headspace volume. Thereby, the headspace concentration can be estimated. The benefit of close system is a convenience in operating and controlling. Our experiment involved with the living tissue, therefore, the open or flow- through system was developed. The volatile vapor was delivered through the treatment chamber by mixing with sterilized air. The humidifier was placed in the chamber and moisture content was controlled. This system provided a similar model to the real product-package system in that the oxygen, carbon dioxide, and other gases or vapors in the package can exchange with those in the atmosphere to achieve steady state. The idea was to maintain the steady state of the volatile vaporization and other gases ; 02 and C02 at the headspace. However, some difficulties in operating this system were found. Although, the retention time to reach the desired concentration (equilibrium) in the test chamber was longer than 24 hr (24-36 hr), the estimated time was only 6 hr at the flow rate of 120 ml/min for a 9.3-liter chamber (see appendix 2). The reasons might be the properties of 2-nonanone, itself, and the inappropriate apparatus. 2-nonanone will adsorb to glass, stainless steel, and non-polar polymers. Therefore, the glass chamber, the petri dishes, teflon tubing and connection, PDA media, apple slices, water, etc. placed inside the system needed additional time to equilibrate with the gas phase. This resulted in the different vapor concentration at the inlet and outlet ports. 2-nonanone with high boiling point (194°C) causes the difficulty in controlling the vaporization. In this experiment, a water bath was employed to heat the 2-nonanone generator to elevate the vapor pressure. Although 51 higher temperature induced higher concentration of 2-nonanone at the headspace, it caused 2-nonanone and water to condense in the system. A similar flow-through system was set up for determination of the effect of hexanal, trans-Z-hexenal, and trans-Z-nonenal on md germination of soybean, Glycine max (Gardner, 1990). Most of its design was similar to our system except that the humidifier was located outside the test chamber. In our experiment, in order to eliminate the water vapor contamination in the line connection, the humidifier was placed inside the test chamber. The compounds were delivered as gas vaporized in air by flow through water jar (humidifier) in the test chamber. Thereby, this system is acceptable to compounds which are water insoluble. Gardner also found some problems in operating the system. The hexanal was auto-oxidized during a 3-day treatment. In our hexanal experiment, with a 2-day treatment, the oxidation did not occur (Song et al., unpublished). Gardner added the antioxidant, 0.05% 2,6-di-tert-butyl-p-cresol (BHT), lmM B—carotene, and 0.012% ethylenediaminetetraacetic acid (EDTA), were added. Gardner controlled vapor concentration by controlling by the air flow. In our experiment, flow rate was controlled by adjusting the caliper of capillaries to achieve target concentrations. Reducing the size of the test chamber may minimize the time to reach the equilibrium of the gas phase. The glass chamber with flat shape and thin wall is suggested such as a Pyrex growth tube which was employed to test strawberry varietal resistance to B. cinerea (Irvine, 1959). The growth tube consisted of a horizontal pyrex glass tube with 0.5 inch bore, 15 inches long, and bent up 60° angle at the ends. The PDA media was contained in the tube, and the fungus was inoculated from 52 the ends on the surface of PDA. This growth tube may be modified for use in the volatile experiment. The compound vapor will be delivered through the media. Injection ports at both ends and in the middle might be added. Therefore, the headspace concentration can be determined. However, this system may be tested only on culture media not apple slices. B.Gas analysis. Quantification of 2-nonanone concentration in the treatment chambers was determined by a gas chromatographic analysis. Standard concentrations were prepared by two methods. First, the saturated headspace method was from calculating the equilibrium vapor pressure for 2-nonanone in the headspace of closed (50 ml) vials containing pure (99%) compound. The concentration was calculated from Perry ’3 Chemical Engineering Handbook. The relationship between the logarithm of vapor pressure and the reciprocal of temperature should be linear in accordance with Clapeyron-Clausius equation (Appendix 2). For our work, a calibration curve of GC response versus 2-nonanone concentration calculated from the equilibrium vapor pressure was constructed. Liquid 2-nonanone was added to 3 small vials insulated by styrene containers and incubated at three different temperature to achieve equilibrium between the liquid and vapor phases. The linear relationship between GC-response and 2-nonanone concentration was found (Fig.1). The unsaturated headspace method for standard generation was by evaporation of a known quantity of 2-nonanone into a 4.36 liter jar with a gas tight septum. The GC-response from the two methods did not significantly differ if analysis was within 24 hr of 53 preparing the standard. Thereafter, the response of the second standard decreased gradually with time (Table 1). However, the GC response from the first standard method appeared very constant. The first method for the standard concentration was selected for our experiment. The injection technique was optimized so as to obtain the most accurate data (Table 2, Fig. 2, and Fig. 3). Needle types (stainless steel, and glass capillary), size of the needle and number of times the plunger was pumped altered GC response. As the number of times the plunger was pumped increased, GC response increased. It was therefore evident that 2-Nonanone was absorbed and/or adsorbed inside the needle. In addition, the desorption time in the GC inlet also affected the GC-response ( data not shown). Consequently, the injection technique was standardized. The syringe was pumped ten times before withdrawing the gas sample. The needle, Hamilton No.1705, was used through out the experiment. The desorption time was 3 sec. 2-nonanone concentration was calculated using a standard for each analysis. A newly developed syringe, SPME (solid-phase microextraction) is suggested as an alternative volatile sampling systeme. SPME was developed by Pawliszyn and co—workers (Arthur and Pawliszyn, 1990 ; Arthur et al., 1992 ; Potter and Pawliszyn 1992). The needle consists of a fused silica fiber coated with an adsorbent. Use of SPME may offer more accurate results with GC analysis. 54 C. Biological technique. Preliminary test 1. The purpose of this test was to develop the technique used in the research and to study the range of 2-nonanone concentrations which decreased the growth of Penicilliwn expansum at room temperature. Most of the techniques were similar to those described in materials and methods. Besides, the spore inoculation was used mycelium transferring instead of spore suspension. In this experiment, the fungal spore were transferred from the donor petri dishes to the sample plates. Fungi inoculated on the PDA plates with glass cover were treated with 2-nonanone. This method did not allow the vapor to directly contact with fungi. 2-nonanone was continuously delivered to the treatment chambers. The headspace concentration was monitored and controlled at the desired level. The relative humidity in the test chamber was approximately 70% (the same as the relative humidity of air). The results showed that fungal growth rate under 250 pl-liter’l of 2-nonanone was not significant different from that in control condition. Fungi were entirely inhibited at 750 pl-liter‘. In addition, growth rate was reduced by 2-nonanone treatment at 400 to 750 pl'liter‘1 (Table 3). This was the primary evidence exhibiting the potential of 2-nonanone in fungal suppression. However, the preliminary result showed the effective concentrations of 2-nonanone (greater than 750 pl'liter') to completely control fungal growth which were much higher than those of the final result ( higher than 303 pl-liter"). The reason may be that the fungi were not directly exposed to the vapor. Furthermore, the inoculation technique needed to be standardized because of the 55 variable amount of the fungal spores. The use of liquid culture was recommended. The concentration of spore suspension was determined. Preliminary test 2. The purpose was to determinate the proper concentration and volume of inoculum (fungal liquid culture). The spore suspensions of Botrytis cinerea at several concentrations (10", 10’, and 10° cells/ml) were inoculated on the PDA and treated with 2-nonanone (not direct contact with compound). The inoculation volume varied from 5 to 10 pl. The technique was similar to that which was defined in materials and methods. The results showed no significant variation of colony diameters of fungi inoculated by different concentration and volume of fungal suspension (Table 4). Hence the level of 105 cells/ml was selected as a suitable concentration. After fungi were inoculated on the surface of PDA or apple slices, fungal suspension needed some time to be dry before placed in the test chambers. The volume of 5 pl'liter’l was preferred due to less time requirement. Preliminary test 3. The purpose was to determine the effect of indirect 2-nonanone exposure on the fungal growth. 5 pl of liquid culture of B. cinerea was inoculated on the agar and apple slices which were contained in petri dishes with covers. The covers were used to minimize moisture loss, but may have prevented fungi from being directly exposed to the desiccator atmosphere. Under these conditions, 2-nonanone was 56 effective in reducing fungal growth (Fig.4) with concentration of 517 pl-liter" completely inhibiting the fungal growth on both PDA agar and apple slices. At lower concentrations (270, 338, and 438 pl-liter"), the growth rates (0.26, 0.27, and 0.11 mm°h", respectively) were reduced relative to air controls which grew at 0.35 mm-h". The fungus on the apple slices was completely controlled by 2-nonanone at 517 pl-liter'1 but it still grew at the lower concentrations. When compared to fungal growth on petri dish without glass cover (directly contacted), these data showed higher concentration of 2-nonanone that can inhibit fungal growth. 57 Table 1 Comparison of the accuracy between saturated headspace method (1) and unsaturated headspace method (2) in generation of the standard calibration curve. Time after F-factor (ratio of concentration prepared the (pl/l) and GC-response) Standard “1"” Method 1 Method 2 24 hours 0.00870 0.008660 48 hours 0.00877 0.010005 % difference 0.39 14.3 58 Table 2 Comparison of GC-response between using stainless steel needle and glass-column needle (PB-l). APB-l Stainless steel Test 1 Test 2 Test 1 Test 2 GC-response 208128 212480 58214 57549 Std dev. 12749 13798 4306 2539 % std error 6.13 6.49 7.40 4.42 A needle made of capillary column. 59 Table 3 Preliminary test of the effect of 2-nonanone on Penicillium expansum growth rate at 23°C. 2-nonanone concentration Growth rate (pl-liter") (mm/h) 0 (control) 0.290 400 0. 1 13 575 0.027 750 0 60 Table 4 Comparison of the fungal colony diameter on agar inoculated with different concentrations and volumes of spore suspension (6 days after inoculation). Spore concentration (cells/ ml) 2-Nonanone concentration V01 104 105 100 (pl-liter”) (pl) 0 (control) 5 42 44 45 10 44 190 5 38 40 40 260 5 36 35 35 517 5 0 0 0 61 l r l 1 l 1 1 160; Y = 09957 + 0.08083X _ A ‘ R-square = 0.999 'U C. m . ‘6’ 120— o _ 5 L- 3 _ I- r: 804 ._ 0 . L D. (D a; - 0 40— — (D . 0 f I I ' I . I ' I I O 400 800 1200 1600 2000 Concentration (pl.liter'1) Figure 1. Calibration curve for 2-nonanone (the concentration of 2-nonanone was estimated from Perry’s Chemical Engineering Handbook). 62 200“11“11111‘11113111111111111114‘.1.1... 180— . " _ 1604 _ 140$ ; 120; _ 100! ; 80: "_ so! ; 40$ ; 20,: ggeple Test _ GC-response (thousands) O TifIlIIIrrfITITITITIITTTIITIITTfi'TIIIIII‘F 0 10 20 30 40 50 60 70 80 90 100 Injection Volume (pl/l) Figure 2. Variation in GC-response when using different injection volumes. 63 280 Lxrelrrrirr...1,,,,1,,,111,111 260; 2405 220~ 2005 1803 160? 140— 120i 1009 GC-Response (x 512 ) Metal needle 80 7 Test 60 I I I I I I I I I I I I . I I II I I I I I I I I I I I I 0 5 10 15 20 25 30 N0. of pumps (times) Figure 3. Variation in GC-response in response to the number of times the plunger was pumped. p 01 L. r. .0 ‘1’ ' <—‘—< .0 .0 N (a) l GrOVvth Rate of B.cinerea (mm. h'1) .0 - f I ‘r I I I I I I I I I I I I I I I I I 0 100 200 300 400 500 600 2-N0nanone concentration (pl.|iter' ) .0 o T Figure 4. Effect of 2-nonanone treatment on growth rate of Botrytis cinerea at 23C when fungi were not directly in contact with 2-nonan0ne vapor (fungi were covered by loose fitting petri lid). CHAPTER 4 CONCLUSIONS 65 66 Modified atmosphere packaged fruits and vegetables have become increasingly interest in the market. MAP technology involving use of natural volatile compounds to retard microbial decay is an emerging approach which is expected to achieve longer product shelf life or storage life. The goal is to maintain the quality and physiology of the enclosed product. This research showed the effect of 2-nonanone, a volatile compound from nature, on the inhibition of decay on apple slices. However physical damage was found on the apple skin. The possibility of use the naturally occurring volatiles has been proven, even other appropriate compounds that are less toxic to plants and mammals are required to investigate. The effect of the 2-nonanone was examined with emphasis on the temperature dependence. This should show the temperature effect on both fungal growth and sorption coefficient of the 2-nonanone on the apple cuticle. Role of 2-nonanone which influences the inhibition of fungal growth has not been explained. However mechanism of six-carbon aldehyde and six-carbon alcohol which control the fungal decay and insects may be defined. These volatile compounds are produced by green leaves through the lipoxygenease/hydoperoxide lyase pathway (Hatanaka et al., 1987). Increasing amount of these compounds after leaves were damaged has been presented as plant defense against microorganism and insects. (Deng et al.,l993). Therefore, mechanism of 2-nonanone which is also produced by plants might be investigated as natural defense of fruits. Future research needs to be studied further to understand the 2-nonanone function in inhibition of fungal growth. 67 A new methodology to study the action of organic vapors on sliced apples was developed. A set up apparatus, flow-through vapor exposure system can generate and deliver continuously the 2-nonanone vapor to contact surface of apple slices. Microbiological study was then conducted to evaluate the effect of different concentration of 2-nonanone in air on the fungal growth. However, the success of MAP depends on many factors including internal and external factors. The internal factor is good initial product or fruit quality. External factors include any techniques to retard decay on fruits, controlling the storage condition and proper package, and hygiene during processing. APPENDICES 68 APPENDIX 1 Potato Dextrose Agar (PDA) This medium is available as a premixed powder from Difco Laboratories. Composition per liter: Bacto dextrose 20 gram Bacto agar 15 gram Potatoes, infusion from 200 gram Preparation of medium : Suspend 39 grams in 1 liter distilled or deionized water and boil to dissolve completely. Sterilize at l2l-124°C for 15 minutes. 69 V-8 Agar Composition per liter: V-8 canned vegetable juice 200 milliter Cacao3 4.0 gram Agar 20 gram Preparation of medium : Add components to distilled or deionized water and make the volume to 1 liter. Mix thoroughly. Alter the reaction to pH 7.3 i 0.2 at 25 °C. Heat or boil to mix well. Autoclave for 15 minutes at 15 psi pressure or 121°C (Atlas, 1993). 70 APPENDIX 2 1.Calculation of partial pressure of 2-nonanone. From Perry’s Chemical Engineering Handbook (Perry et al., 1984), the relationship of 2-nonanone vapor pressure and the temperature indicated by following equation (Fig. 1). 1 3 L =7.40 -2. 20 — 10 0gp 6 5 (TX ) (1) Where, p is the partial pressure of 2-nonanone and T is temperature in Kelvin. From equation 1, the concentration of 2-nonanone at the headspace in equilibrium with liquid in standard Vial can be calculated. 2.Calcu1ation of the fungal growth rate. The fungal growth rates were estimated from the slopes of the linear relationships of change the colony diameter by time.(see Fig. 2,3 and 4) 71 3.Calculation of the retention time to reach the equilibrium of the treatment chamber Volume of test chamber (V) = 9300 ml Flow rate (F) = 120 ml/min T = time C = Saturated concentration V T = -—-9 (In ———C ) (2) F C-O.99C T = 9300 ml C 120 ml/min ( 0.10C') T=6h 72 10: l 1 l I l I 1 I 1 l 1 Log P = 7.406 - 2.520 (1fo 103) N .a O l I I J I lLlll O 2-N0nanone pressure (KPa) 8 3 r r l l I l 10.1 I I I I I 2.2 2.4 2.6 1rr (1/K*10'3) Figure 1. Vapor pressure of 2-nonanone as a function of temperature. r I . . 2.8 3.0 3.2 73 l A E A I A 2 A I . I ‘ ' ‘ ’ 3C) P. a Colon .3 _ 8 b O A . A y diamotar( M O --a- control (on - 024 W") -e- m; 14 may“ (on - 0.:0 ma“) ' 0 ' l ' l ' ' I ' I 24 ‘40 72 0'0 120 144 Time (hrs) P. sum 23C Commas: (mat) -e- mica-0 am") —e- 20132414014? panorama“) o I I ' l I V I ' I ' I e ' 0 24 49 72 99 120 144 199 192 Time (hrs 50 I I l I I I l ' -e- mum-o m") 40- -e- 2003,44 m’ (on - 0.04 m“) P. expansum (230) Colony diameter (mm) .5 O l Figure 2. Growth of Penicillium exp 24 4a 72 . 00 '120 'l'ima(hrs) l ' c 144 168 various concentrations at 23°C. I ‘ j— 168 192 5° 1 l l A l . I . I A 1 A . -e- comm-022m“) 9‘5- ‘0- -e- 10030014016 Ion-0mm") _ 81' ' ’ 3 30.. . §§ “ 1 C gu 20- > 1 (fig 10. D r“ I A I . I I I I I ' 0 24 49 72 99 120 144 168 Time (hrs) 6c . 9 1 I . I . I . J A l ,, 50- -e- mu (ca-02,2 m“) , g ‘ -e- 224 3 am‘, Ion-0.07 m") a; 40- . .. SE a“ - Io ' - ' 3" 20- - ' .5 . - 8 10‘ M' c A “A - b I ' i ' I ' 9 ' l 9 ' O 24 48 72 99 120 144 169 Time (hrs) ansum in air and exposed by 2-nonanone at 74 I 1 . L . L I r . r . r I r . r A 1 . , A 44 1 A r . 1 - r - r ! ' '9' wmwfi’ m“) .1 ’ ‘ -e- Wren-0.41m") I 001 -e- 11091.net (68.9.42Mh . ,0. _._ 133312 I. .1 ‘63.”, In) _ 1 I 4 I 4 i .1 D 4°"I . 40.. L A A if B. cinerea (230) Colony diameter (mm.) M O A A B. cinema (230) Colony diameter (mm.) A vvv'. 3 L A f‘ v ' v A _-.- " Y—Ofi—VTTVTfiTV ' IA' 7AAfi‘I—Afi1 * IA fit - I . I fr ' I r r I 0 24 49‘ 72 99 120 144 199 192. 219 240 O 24 49 72 99 120 144 199 192 Time (hrs) Time (hrs) 7c L L A l J l A l L l A I . J l a l A l . l 4 1+! A l . l A L4 1 L . -e- moron-0.45m") -e- won-afresh") .1 Qw" -e- 101:24m"cca-0.sam") 00- +100300m Ion-020m 1 - § 0 I i” D B. cinerea (230) Colony diameter (mm) A D ‘l'l'l‘l'yevlv ' I A ‘ I I I I . I 40—7'TA fin‘A‘JI—A' j +1? 12+ I I 1 f O 24 ' 49 72 99 120 144 199 9 24 49 72 99 120 144 199 192 219 24 Time (hrs) TWO (hrs) -. DO A 1 A 1 L I , 1 . 1 , J 4 . A r 7: I 1 I L . I I l . l . r A l . -e- wise-211m") .1 . . -e- mum-0.45m") t ".‘50- +214g14m (GR-0.26am b 03‘90- +m:31m"m-ogom") .. -g . . o E sol L g ‘0‘ P a: 1 i a 4 . 10 g 40.1 f 2 3°... - 8.5 4 , g . . g” 00~ . >0 20" .- >. ‘ D o 10‘ ._ - 3 10- b : v“‘0‘ A' AA4_0A_'A AI'A/o T 0 l 0‘ {if '— ' I P I v j v r 0 24 4a 72 00 120 144 100 102 0 24 48 72 96 120 144 160 Time (hrs) “"10 (hrs) O Figure 3. Growth of Bonytis cinerea in air and exposed by 2-nonan0ne at (various concentrations at 23°C. 75 50 1 l 1 l 1 l 1 J 1 i 1 l 1 l —9— control (GR = 0.1070 mm.h") 1 —o— 26 1 3.7 leiter' (GR = 0.033 mm.r1' ) OD -5 O O l I At 50 Colony diameter (mm.) N O o “1 1‘? ‘Te—r‘ 1“ ‘1 1“? 1 1 1 1 1 o 120 240 360 480 600 720 840 Time (hrs) Figure 4. Growth of Penicillium expansum in air and exposed by 2—nonanone at an average of 26 pl'litel" at 5°C BIBLIOGRAPHY 76 BIBLIOGRAPHY Andersen, R.A., T.R. Hamilton, D.F. Hildebrand, C.T. McCracken, Jr., R.W. Collins, and P. D. Fleming. 1994. Structure-antifungal activity relationships among volatile C5 and C9 aliphatic aldehydes, ketones, and alcohols. J. Agric. Food Chem. 42:1563-1568. Arthur, C.L., and J. Pawliszyn. 1990. Solid phase microextraction with the desorption using fused silica optical fibers. Anal. Chem. 62:2145-2148. Arthur, C.D.,D.W. Potter, K.D. Buchholz, S. Motlagh, and J. Powliszyn. 1992. Solid phase microextraction for the direct analysis of water. In Theory and Practice. p.656-66l. Atlas, R.M. 1993. Handbook of microbiological media. CRC Press, Boca Raton. p. 973. Bailey, LB. 1930. Hortus; a concise dictionary of gardening, gemeral horticulture and cultivated plants in North American. Macmillan , N .Y. Beaumont, F., H.F. Kauffman, J.G.R. de Monchy, H.J. Sluiter, and K. Vries. 1985. A volumetric aerobiological survey of conidial fungi in the north-east of the Netherlands. 2. Comparison of aerobiological data and skin tests with mould extracts in an asthmatic population. Allergy. 40: 181-186. Barrett, D.M. 1989. Effects of controlled atmosphere storage on browning and softening reaction in ’delicious’ apples. Thesis. Cornell Univ. Beever, R.E., E.P. Laracy, and H.A. Pak. 1989. Stains of Bonyris cinerea resistant to dicarboximide and benzimidazole fungicides in New Zealand vineyards. Plant pathol. 38:427-437. Brecht, J .K. 1995 . Physiology of lightly processed fruits and vegetables. HortScience. 30(1):18—22. _ Buckingham, J. 1994. Dictionary of natural products. 1st ed. Chapman & Hall, NY. Burns, J. K. 1995. Lightly processed fruits and vegetables: Introduction to the colloquium. HortScience. 30(1):14-17. Chambers, K.R. 1990. Benzyl alcohol as an inhibitor of the development of Botrytis cinerea in vitro and in packed grapes during storage. Am. J. Enol. Vitic. 41(4):265—268. 77 Cook, R.L. 1992. The dynamic U.S. fresh produce industry an overview. p.3-13.In: A.A.Kader(ed.). Postharvest technology of horticultural crops. 2nd ed. Univ. of California Oakland,CA. Couey, H.M., and J.M. Wells. 1970. Low-oxygen or high carbon dioxide atmospheres to control postharvest decay of strawberries. Phytopathol. 60:47- 49. Couey, H.M., M.N. Follslad, and M. Uota. 1966. Low-oxygen or high carbon dioxide atmospheres for control of postharvest decay of fresh strawberries. Phytopathol. 56: 1339- 1341. Deng, W., T.R. Hamiton-Kemp, M.T. Nielsen, R.A. Andersen, G.B. Collins, and D. F. Hildebrand. 1993. Effects of six-carbon aldehydes and alcohol on bacterial proliferation. J. Agric. Food Chem. 41:506-510. Doehlert, D.C., D.T. Wicldow, and H.W. Gardner. 1993. Evidence implicating the lipoxygenase pathway in providing resistance to soybeans against Aspergillus flavos. Phytopathol. 83(12): 1473-1477. Eckert, J .W., and J .M. Ogawa. 1988. The chemical control of postharvest diseases: Deciduous fruits, berries, vegetables and roots/tuberscrops. Annu. Rev. Phytopathol. 26:433-469. Elliot, RP, and H.D. Michener. 1965. Factor affecting the growth of psychrophilic micro-organisms in foods. Tech. Bull. 1320, US. Dept. of Agriculture, Washington, DC. El-shimi, N.M. 1993. Control of enzymatic browning in apple slices by using ascorbic acid under different conditions. Plant Foods Hum. Nutr. 43:71-76. Fallik, E., and S. Grinberg. 1992. Hinokitiol: a natural substance that controls postharvest diseases in eggplants and pepper fruits. Post. Bio. Tech. 2:137- 144. Food and Drug Administration 21CFR ch. 1(4-1-93 Edition). Gardner, R.W., D.L. Dombos, Jr., and A.E. Desjardins. 1990. Hexanal, trans-2- hexenal, and trans-Z-nonenal inhibit soybean, Glycine max, seed germination. J.Agric. Food. Chem. 38:1316-1320. 78 Gildemeister, E.,1913. The Volatile Oils. Wiley, N.Y. p.28. Hamilton-Kemp, T.R., C.T. McCracken, J .H. Loughrin, R.A. Andersen, and D.F.Hildebrand. 1992. Effects of some natural volatile compounds on the pathogenic fungi Altemaria altemata and Botrytis cinerea. J. Chem. Ecol. 18(7):1083-1091. Hanson, E.J.,J.L. Beggs, and R.M. Beaudry. 1993. Applying calcium chloride postharvest to improve highbush blueberry firmness. Hortscience. 28(10): 1033-1034. Hatanaka, A., T. Kajimara, and J. Sekiya. 1987. Biosysthetic pathway for C6- aldehyde formation from linolenic acid in green leaves. Chem. Phys. Lipids. 44:341—361. Hayes, ER. 1963. Acetaldehyde, p.77-95. In: A. Standen (ed) Encyclopedia Chem. Tech. Kirk-Othmer. Hildebrand, DP. 1989. Lipoxygenase. Physiol. Plant. 75:1-5. Hoag, D. 1995. Fresh-cut fruit flowers in an array of ways. Produce Business. June p.35-45. How, RB. 1991. Marketing fresh fruits and vegetables. In: V. Norstrand (ed.) AVI book. Reinhold, NY. Huxsoll, C.C., and HR. Bolin. 1989. Processing and distribution alternatives for minimally processed fruit and vegetables. Food technol. 43:124. Irvine T.B. 1959. A study of laboratory methods to determine strawberry varietal resistance to grey mold (Botryris cinerea). Thesis. Michigan State Univ., Mich. Kader, A.A. 1992. Postharvest biology and technology: an overview. p.15-20.In: A.A.Kader(ed.). Postharvest technology of horticultural crops. 2nd ed. Univ. of California Oakland,CA. Kiss,I. 1984. Testing methods in food microbiology. In: Developments in Food Science Elsevier Science Publishing Co., Inc., N.Y. Lewis, R.J.Sr. 1992. Sax’s dangerous properties of industrial material. Van Nostand Reinhold, N .Y. 79 Lakakul, R. 1994. Modified-atmosphere packaging of apple slices: Modeling respiration and package oxygen partial pressure as function of temperature and film characteristics. Thesis. Michigan State Univ., Mich. Nguyen-the, C., and F. Carlin. 1994. The microbiology of minimally processed fresh fruits and vegetables. Crit. Rev. Food Sci. Nutr. 34(4):371-401. Nicoli, M.C., M. Anese, and C. Severini. 1993. Combined effects in preventing enzymatic browning reactions in minimally processed fruit. J. Food Sci. 17:221-229. Perry, H.R., D. Green, and J .0. Maloney. 1984. Perry’s chemical engineering handbook. McGraw-Hill, N .Y. Potter, D.W., and J. Powliszyn. 1992. Detection of the substituted benzene in water at the pg/mL level using silid-phase microextraction and Gas Chromatograph- Ion Trap Mass Spectrometry. J. Chromatogr. 625, 247—255. Prasad, K. and G.J. Stadelbacher. 1973. Control the postharvest decay of fresh raspberries by acetaldehyde vapor. Plant Dis. Rep. 57:795-797. Prasad, K. and G.J. Stadelbacher. 1974. Effect of acetaldehyde vapor on postharvest decay and marketing quality of fresh strawberries. Phytopathol 64:948-951. Price, J .L. and JD. Floros. 1993. Quality decline in minimally processed fruits and vegetables. In: G. Charalambous (ed.). Food flavors,ingredients, and composition: proceedings the 7th international flavor conference, Pythagorion, Samos of Greece, 24-26 June 1992. Amsterdam, N.Y. Schlimme, D.V. 1995. Marketing lightly processed fruits and vegetables. HortScience 30(1):15-17. Shimoni, M., E. Putievsky, U. Ravid, and R. Reuveni. 1993. Antifungal activity of volatile fractions of essential oils from four aromatic wild plants in Israel. J. Chem. Ecol. l9(6):1129-1133. Sholberg, P.L. Agriculture and Agri-Food Canada, Reseach Center, Summerland, British columbia, Canada. Sholberg, P.L., and A.P. Gaunce. 1995. Fumigation of fruit with acetic acid to prevent postharvest decay. HortScience 30(6): 1271-1275. Singleton, P. 1987. Dictionary of microbiology and molecular biology. 2nd ed. Wiley, N .Y. 80 Snowdon, A.L. 1990. Color atlas of post-harvest diseases and disorders of fruits and vegetables. CRC Press, Boca Raton. p.178-179,188-189. Sommer, N .F. 1985 . Role of controlled environments in suppression of postharvest diseases. Can. J. Plant Pathol. 7:331-339. Song, J. 1994. Production and development of volatile aroma compounds of apple fruits at different times of maturity. Acta Hort. July, p.368. Song, J ., R. leepipattanawit, W. Deng, and R. Beaudry. 1996. Hexanal vapor inhibits fungal growth and decay development and enhances aroma biosynthesis in apple slices. (unpublished). Spotts, R.A., and LA. Cervantes. 1986. Populations, Pathogenicity, and benomyl resistance of Botrytis spp., Penicillium spp. , and Mucor pirifromis in packinghouses. Plant Dis. 70: 106-108. Stadelbecher, G.J., and K. Prasad. 1994. Postharvest decay control of apple by acetaldehyde vapor. J. Amer. Soc. Hort. Sci. 99(4):364-368. Stephens, D. 1994. Edible coatings slice open new market. Fruit Grow. June, p.6—7. Tronsmo, A., and J. RAA. 1977. Life cycle of the dry eye rot pathogen Botrytis cinerea Pres. on apple. Phytopath. Z. 89:203-207. Urbasch, I. 1984. Production of C6-wound gases by plants and the effect on some phytopathogenic fungi. Z. Naturforsch. 39:1003-1007. Vaughn, S.F., G.F. Spencer, and BS. Shasha. 1993. Volatile compounds from raspberry and strawberry fruit inhibit postharvest decay fungi. J. Food Sci. 58:793-796. Wills, R.B.H., W.B. McGlasson, D. Graham, T.H. Lee, and E.G. Hall. 1989. Postharvest: An Introduction to the physiology and handling of fruits and vegetables. Van Nostrand, Reinhold, N.Y. Wilson, C.L., J .D. Franklin, and BE. Otto. 1987. Fruit volatile inhibitory to Monilinia Frucricola and Bonytis cinerea. Plant Dis. 71(4):316-319. “I11111111111111?