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The inhibition of yeast spoilage of blueberries during modified atmosphere packaging storage 1988

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c -I THE INHIBITION OF YEAST SPOILAGE OF BLUEBERRIES DURING MODIFIED ATMOSPHERE PACKAGING STORAGE By NGOC BICH DAY B . S c , The U n i v e r s i t y of B r i t i s h Co lumbia , 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE THE FACULTY OF GRADUATE STUDIES Department of Food Sc ience We accep t t h i s t h e s i s as conforming to the r e q u i r e d s t andard THE UNIVERSITY OF A p r i l © c o p y r i g h t Ngoc BRITISH COLUMBIA 1988 B l c h Day, 1988 3 9 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) ABSTRACT Mo d i f i e d atmosphere packaging storage combines an atmosphere of higher carbon d i o x i d e and lower oxygen l e v e l s than a i r , with c h i l l i n g temperatures to extend s h e l f - l i f e of f r e s h f r u i t s . In three m o d i f i e d atmosphere packaging storage t r i a l s , b l u e b e r r i e s were packaged i n f i l m bags with d i f f e r e n t gas p e r m e a b i l i t i e s , and s t o r e d at about 4<>C. Storage of b l u e b e r r i e s i n packages of a f i l m with intermediate gas p e r m e a b i l i t y produced an a e r o b i c atmosphere and a r e l a t i v e l y low carbon d i o x i d e l e v e l , r e s u l t i n g i n r a p i d growth of yeast and molds on b l u e b e r r i e s . Packaging b l u e b e r r i e s i n a f i l m with very low gas p e r m e a b i l i t y c r e a t e d a high carbon d i o x i d e almost anaerobic atmosphere, which s u c c e s s f u l l y i n h i b i t e d yeast and mold growth on b l u e b e r r i e s f o r up to e i g h t weeks. The p o s s i b i l i t y of yeast i n h i b i t i o n by a n t i f u n g a l compounds accumulated i n b l u e b e r r i e s s t o r e d under mo d i f i e d atmosphere packaging c o n d i t i o n s was i n v e s t i g a t e d by using the d i s k d i f f u s i o n assay. The r e s u l t s of these assays showed the absence of a n t i f u n g a l a c t i v i t y a g a i n s t two Rhodotorula s p e c i e s , a Zygosaccharomyces s p e c i e s , a Cryptococcus s p e c i e s , a Debaryomyces s p e c i e s , and i n d i c a t e d t h a t the i n h i b i t i o n of yeast growth was due to low temperature, high carbon d i o x i d e l e v e l and a naerobic — i i i — c o n d i t i o n s . The e f f e c t s of temperature and atmosphere composition were i n v e s t i g a t e d by using n a t u r a l f l o r a of b l u e b e r r y j u i c e and two yeast i s o l a t e s grown i n s t e r i l i z e d j u i c e . At 21°C, yeast growth was slow i n the presence of carbon d i o x i d e and absence of oxygen. At low temperature, yeast growth was slow i n the presence of oxygen, but was i n h i b i t e d i n the anaerobic, high carbon dioxide environment. I t i s proposed t h a t the m i c r o - a e r o b i c environment of modified atmosphere packaging storage might have allowed slow d e s a t u r a t i o n of yeast membrane f a t t y a c i d s which enabled yeasts to maintain membrane f l u i d i t y and f u n c t i o n at low .temperature. Furthermore, yeast growth during storage of modified atmosphere packaged b l u e b e r r i e s may be a f f e c t e d by low temperature and high carbon d i o x i d e c o n d i t i o n s . - i v - TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES V i LIST OF FIGURES v i i ACKNOWLEDGEMENTS ix Chapter 1: INTRODUCTION AND LITERATURE REVIEW 1.0 I n t r o d u c t i o n 2 1.1 L i t e r a t u r e review 3 1.1.1 Important b l u e b e r r y d i s e a s e s and a p p l i c a t i o n of f u n g i c i d e s 3 1.1.2 E f f e c t s of MAP storage on mold growth 4 1.1.3 Yeast metabolism i n r e l a t i o n to MAP storage c o n d i t i o n s 6 1.1.3.1 Anaerobic growth of yeasts 6 1.1.3.2 In f l u e n c e of low temperature on yeast growth...9 1.1.4 Background on ph y t o a l e x i n s 12 1.1.4.1 S t r u c t u r e and d i s t r i b u t i o n 13 1.1.4.2 B i o s y n t h e s i s of phy t o a l e x i n s 17 1.1.4.3 S t r u c t u r e - a c t i v i t y r e l a t i o n s h i p 19 1.1.4.4 Modes of a c t i o n 19 1.1.5 Methodology i n yeast s u s c e p t i b i l i t y t e s t i n g with a n t i f u n g a l compounds 21 1.1.5.1 Broth d i l u t i o n method.... 22 1.1.5.2 Agar d i l u t i o n method 25 1.1.5.3 Disk d i f f u s i o n method 26 1.2 Summary of l i t e r a t u r e review and o b j e c t i v e s of the study 27 Chapter 2: EXPERIMENTATION, RESULTS AND DISCUSSION: MODIFIED ATMOSPHERE PACKAGING STORAGE OF BLUEBERRIES 2.1 A n a l y s i s of b l u e b e r r i e s i n MAP storage 30 2.1.1 M a t e r i a l s and methods 30 2.1.2 R e s u l t s and d i s c u s s i o n 35 2.1.2.1 Headspace atmosphere of packages with low gas p e r m e a b i l i t y 35 2.1.2.2 Headspace atmosphere of packages with intermediate gas p e r m e a b i l i t y 39 2.1.3 pH of b l u e b e r r i e s i n MAP storage 43 2.1.4 So l u b l e s o l i d contents of b l u e b e r r y f r u i t 49 2.1.5 Growth of microorganisms on b l u e b e r r i e s d u r i n g MAP storage 51 2.1.5.1 Anaerobic p l a t e counts 51 2.1.5.2 Aerob i c p l a t e counts of b l u e b e r r i e s 54 2.1.5.3 Yeast and mold counts 58 2.2 I d e n t i f i c a t i o n of yeasts i s o l a t e d from b l u e b e r r y . . . . 62 -v- 2.3 Conclusions 64 Chapter 3: EXPERIMENTATION, RESULTS AND DISCUSSION: DETECTION OF ANTIFUNGAL ACTIVITY IN BLUEBERRIES STORED UNDER A MODIFIED ATMOSPHERE. EFFECTS OF TEMPERATURE AND CARBON DIOXIDE ON YEAST GROWTH 3.0 Purpose of experiments 67 3.1 Disk d i f f u s i o n assays 67 3.1.1 M a t e r i a l s and methods 67 3.1.2 R e s u l t s and d i s c u s s i o n 70 3.2 E f f e c t s of carbon d i o x i d e and low temperature on yeast growth 76 3.2.1 M a t e r i a l s and methods 76 3.2.1.1 Studies u s i n g c u l t u r e s of yeast i s o l a t e s 76 3.2.1.2 Studies u s i n g n a t u r a l yeast and mold f l o r a of blueberr i e s 80 3.2.2 R e s u l t s and d i s c u s s i o n 80 3.2.2.1 Studies with n a t u r a l f l o r a 80 3.2.2.2 Studies with yeast i s o l a t e s 84 3.3 Yeast growth i n b l u e b e r r i e s d u r i n g MAP storage 93 3.4 Conclusions 97 REFERENCES 99 APPENDIX: I d e n t i f i c a t i o n of yeast i s o l a t e s 108 - v i - LIST OF TABLES Table 1-1 Examples of antimicrobial compounds isolated from plant tissues 15 Table 2-1 Properties of packaging films 31 Table 2-2 Headspace gas composition of samples packed in the high barrier f i l m 36 Table 2-3 Headspace gas content of samples packed in the intermediate barrier f i l m 42 Table 2-4 pH of blueberries and exudates in MAP storage 46 Table 2-5 Soluble s o l i d contents of blueberry f r u i t in MAP storage 50 Table 3-1 Fatty acid composition of yeast membranes ... 91 Table A - l Colonial morphology of yeast isolates I l l Table A-2 Sexual reproduction of yeast isolates 118 Table A-3 U t i l i z a t i o n of carbon compounds by yeast isolates 122 Table A-4 Fermentation of sugars by two isolates 125 - v i i - F i g u r e 1-1 F i g u r e 1-2 F i g u r e 1-3 F i g u r e 1-4 F i g u r e 1-5 F i g u r e 2-1 Fi g u r e 2-2 Fi g u r e 2-3 Fi g u r e 2-4 Fi g u r e 2-5 Fi g u r e 2-6 Fi g u r e 2-7 Fi g u r e 2-8 Fi g u r e 2-9 F i g u r e 3-1 F i g u r e 3-2 LIST OF FIGURES Fermentation of glucose by yeasts 7 R e l a t i o n s h i p between g l y c o l y s i s , t r i c a r b o x y l i c a c i d c y c l e and e l e c t r o n t r a n s p o r t system....10 S t r u c t u r e s of s e v e r a l p h y t o a l e x i n s 16 R e l a t i o n s h i p between r e s p i r a t i o n and s y n t h e s i s of p h y t o a l e x i n s 18 MIC of 5 - f l u o r o c y t o s i n e a t d i f f e r e n t inoculum s i z e s f o r three yeast s t r a i n s 23 A model of headspace composition i n the high b a r r i e r f i l m packages 40 Gas exchange between f r u i t t i s s u e s , headspace of package and environment 44 Anaerobic p l a t e counts of Bluecrop b l u e b e r r i e s i n two MAP c o n d i t i o n s 53 Aerob i c p l a t e counts of Bluecrop b l u e b e r r i e s i n three storage c o n d i t i o n s (1986) 55 Ae r o b i c p l a t e counts of J e r s e y b l u e b e r r i e s i n three storage c o n d i t i o n s (1986) 56 Aerob i c p l a t e counts of Bluecrop b l u e b e r r i e s i n two storage c o n d i t i o n s (1987) 57 Yeast and mold counts of Bluecrop b l u e b e r r i e s i n t hree storage c o n d i t i o n s (1986) 59 Yeast and mold counts of J e r s e y b l u e b e r r i e s i n ,three storage c o n d i t i o n s (1986) 60 Yeast and mold counts of Bluecrop b l u e b e r r i e s i n two storage c o n d i t i o n s (1987) 63 General procedures of b l u e b e r r y e x t r a c t i o n and d i s k d i f f u s i o n assay 71 Disk d i f f u s i o n assay using a Rhodotorula s p e c i e s i n 21°C-air c o n d i t i o n 73 - v i i i - F i g u r e 3-3 Disk d i f f u s i o n assay using a Rhodotorula s p e c i e s i n 4°C-air c o n d i t i o n 74 F i g u r e 3-4 Disk d i f f u s i o n assay using a Rhodotorula s p e c i e s i n 4°C-carbon d i o x i d e i n c u b a t i o n . . . . 75 F i g u r e 3-5 F l u s h i n g of c u l t u r e f l a s k s with a gas mixture 78 F i g u r e 3-S Yeast and mold p o p u l a t i o n i n b l u e b e r r y j u i c e a t 21<>C 81 F i g u r e 3-7 Yeast and mold p o p u l a t i o n i n b l u e b e r r y j u i c e a t 4<>c 83 F i g u r e 3-8 Growth of a Zygosaccharomyces s p e c i e s i n b l u e b e r r y j u i c e 85 F i g u r e 3-9 Growth of a Debaryomyces s p e c i e s i n b l u e b e r r y j u i c e 86 Fi g u r e 3-10 A model of phase t r a n s i t i o n of f a t t y acids..89 F i g u r e A - l V e g e t a t i v e c e l l s of yeast i s o l a t e A 112 F i g u r e A-2 V e g e t a t i v e c e l l s of yeast i s o l a t e B 113 Fig u r e A-3 V e g e t a t i v e c e l l s of yeast i s o l a t e C 114 F i g u r e A-4 V e g e t a t i v e c e l l s of yeast i s o l a t e D 115 Fi g u r e A-5 V e g e t a t i v e c e l l s of yeast i s o l a t e 116 Fi g u r e A-6 Standard curve of a Rhodotorula s p e c i e s ( i s o l a t e A) 126 F i g u r e A-7 Standard curve of a Zygosaccharomyces s p e c i e s ( i s o l a t e B) 127 F i g u r e A-8 Standard curve of a Rhodotorula s p e c i e s ( i s o l a t e C) 128 F i g u r e A-9 Standard curve of a Cryptococcus s p e c i e s ( i s o l a t e D) 129 F i g u r e A-10 Standard curve of a Debaryomyces s p e c i e s ( i s o l a t e E) 130 - i x - ACKNOWLEDGEMENTS I am g r a t e f u l to Drs. W.D. Powrie, S. Nakai and P. Townsley who commented on ideas throughout the study and c a r e f u l l y reviewed the manuscript. Dr. C.H. Wu provided guidance i n the MAP storage t r i a l s and gas chromatography. Dr. B.J. Skura s u p e r v i s e d t h e s i s r e s e a r c h with patience and gave much-needed encouragement. V. Skura and S. Yee a s s i s t e d when t e c h n i c a l problems were encountered. D. Yan and W. Kam helped with photography. F i g u r e s were d r a f t e d by Stephen who a l s o gave much understanding and i n s p i r a t i o n . -1- CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW -2 - 1.0 INTRODUCTION The s h e l f - l i f e of fresh blueberries in the d i s t r i b u t i o n system is limited primarily by fungal spoilage (Ballinger and Kushman, 1970; Ceponis and C a p p e l l i n i , 1978). Postharvest deterioration not only causes economic loss to producers but also influences the a v a i l a b i l i t y and cost of blueberries to consumers. The use of Modified Atmosphere Packaging (MAP) storage to extend the s h e l f - l i f e of fresh f r u i t s is currently being studied in the Department of Food Science, University of B r i t i s h Columbia. MAP implies the packaging of foods in f i l m bags with selective permeabilities, and blanketing the product with an atmosphere of gas having higher carbon dioxide and lower oxygen levels compared with those in a i r . MAP storage combines modified atmosphere with c h i l l i n g temperature to retard decay and delay senescence. Growth of molds is inhibited in MAP storage due to their i n a b i l i t y to grow in low oxygen atmosphere and s e n s i t i v i t y to high carbon dioxide. Under anaerobic atmosphere, many yeast species can generate energy via fermentation of sugars. However, yeast spoilage of blueberries has been found to be absent in a low oxygen-high carbon dioxide environment. The mechanism of yeast i n h i b i t i o n in blueberries under MAP storage - 3 - conditions demanded attention. This study approached the problem in three stages: 1. The growth trend of yeasts and molds on blueberries with d i f f e r e n t gas storage conditions was followed as an indication of their i n h i b i t i o n under MAP storage. 2. The possible synthesis of antifungal compound(s) by blueberry f r u i t in response to temperature and atmospheric stresses of MAP storage was investigated. 3. The effects of low temperature and anaerobic atmospheres on yeasts isolated from blueberries were studied. 1.1 LITERATURE REVIEW 1.1.1 Important blueberry diseases and application of fungicides Spoilage l e v e l of blueberries varies from year to year, with decay incidence possibly being as high as 18% (Ceponis e_t a l . , 1973). Anthracnose, gray mold rot and black rot are the most common types of spoilage a f f e c t i n g blueberries (Cappellini e_t al.., 1972; Ceponis et a l . , 1973 ) . Anthracnose is a defect with black or dark brown spots covering tissue. Pink spore masses of Co l l e t o t r icum gloeospor ioides (the imperfect stage of Glomerella cingulata) may appear on the spots. - 4 - Gray mold rot is evidenced by grayish-brown spots covered with mycelia of B o t r i t i s cinerea. Black rot of blueberries is due to Altern a r i a tenuis. The decay areas on the blueberries are brown, soft and watery. Application of chemicals to reduce postharvest decay of blueberries has been suggested. Ballinger (1983) reported reduction of anthracnose decay by dipping blueberries in 100 ppm solution of captafol. However, this treatment l e f t v i s i b l e white residues on the berries. Anthracnose, gray mold rot and black rot were decreased by dipping blueberries in a solution of 5000 ppm of 2-aminobutane and 100 ppm sodium hypochlorite, but the natural bloom of the berries was removed by sodium hypochlorite (Ceponis and Cap p e l l i n i , 1978). When application of fungicides is considered, the possible t o x i c i t y of their residues to mammals must be extensively studied. 1.1.2 Effects of MAP storage on mold growth The presence of carbon dioxide and low oxygen le v e l in the atmosphere together with low temperature of MAP storage generally retards mold growth. El-Kazzas e_t a l . (1983) reported that under 21% oxygen and 15% carbon dioxide, fewer strawberries were infected by B. cinerea than those under a i r . Diameter of A. tenuis -5- and B. cinerea colonies decreased with decreasing oxygen levels to 1% (Follstad, 1966; Adair, 1971). Colony diameter on s o l i d media is not a very accurate measurement of growth, since molds often produce a e r i a l mycelia in addition to diffuse surface growth. Wells and Uota (1970) used mass of mycelia grown in l i q u i d medium as a growth index. These authors found that at 4% oxygen, mycelial mass of A. tenuis was 31%, of Fusarium roseum was 38%, of B. cinerea was 45%, and of Cladosporium herbarum was 50% of the mass in a i r . Carbon dioxide greatly inhibited mycelial growth even in the presence of 21% oxygen: growth of B. cinerea and C. herbarum was completely abolished at a 45% carbon dioxide l e v e l , growth of A. tenuis and Rhizopus stoloni fer were about 20% compared to growth in a i r . At 1% oxygen, spore germination of B. cinerea was inhibited by 16% carbon dioxide, while germination of C. herbarum and R. s t o l o n i f e r were inhibited by 32% carbon dioxide. Svircev et al_. ( 1984 ) noted that carbon dioxide caused d i s t o r t i o n of fungi germ tubes, and interfered with the normal germination process. - 6 - 1.1.3 Yeast metabolism in r e l a t i o n to MAP storage conditions 1.1.3.1 Anaerobic growth of yeasts In contrast to molds which are obligate aerobes, many yeasts can grow under anaerobic conditions. Fermentation of sugars supplies energy and metabolic precursors for yeast growth in anaerobic environments. Fermentation is an oxidation-reduction process where molecular oxygen is replaced by organic compounds as terminal electron acceptors. When fermentative a b i l i t y e x i s t s , glucose is always fermented; other sugars may also be fermented by some yeast species. The biochemical pathway for the fermentation of glucose can be divided into three major parts (Figure 1-1). The f i r s t part is a series of reactions leading to production of glyceraldehyde-3-phosphate. In the second part, pyruvate and adenosine triphosphate (ATP) molecules carrying the high-energy phosphate bond are produced. In the thi r d part, ethanol and carbon dioxide are released. The series of reactions leading to pyruvate formation is called glycolys i s . Ethanol and carbon dioxide are the most common end products of glucose fermentation, although other products also appear in minor proportions. In one g l y c o l y t i c step, the oxidation of glyceraldehyde-3-phosphate to 1,3- FRUCTOSE-1,6-P ^ADP 1,3-DIPHOSPHOGLYCERATE If cz 3-PHOSPHOGLYCERATE u 2-PHOSPHOGLYCERATE N PHOSPHOENOLPYRUVATE K PYRUVATE ACETALDEHYDE + CO2 1̂ ETHANOL Fi g u r e 1-1: Fermentation of glucose by yeasts 1979). - 8 - diphosphoglycerate, an oxidized coenzyme nicotinamide adenine dinucleotide (NAD) acts as an acceptor to the hydrogen atom removed from glyceraldehyde-3-phosphate and becomes reduced (NADH). The c e l l has only a limited supply of NAD, so NAD must be regenerated for g l y c o l y s i s to proceed. The l a s t step in fermentation, reduction of acetaldehyde to ethanol, f u l f i l l s t his objective: NADH donates a hydrogen atom to acetaldehyde leading to formation of ethanol and NAD. Regeneration of NAD can be carried out by an alternative route, namely the reduction of dihydroxyacetone phosphate to glycerol-3-phosphate which is then hydrolyzed to g l y c e r o l . When the growth medium is very a l k a l i n e , another type of fermentation occurs to produce ethanol, acetic acid, gly c e r o l and carbon dioxide. The biochemical explanation of t h i s fermentation route is that in alkaline media, an aldehyde dehydrogenase with an alkaline pH optimum, converts acetaldehyde to acetic acid. This enzyme requires NAD, so that the c e l l s must u t i l i z e the reduction of dihydroxyacetone phosphate to glycerol-3-phosphate to re-oxidize NADH. The role of this pathway is to bring medium pH within the range favourable for growth (Sols e_t a l . , 1971). When oxygen is available, glucose is oxidized completely in res p i r a t i o n process. The early step in - 9 - glucose oxidation follows g l y c o l y s i s , but pyruvate is oxidized to carbon dioxide, through a series of reactions known as the t r i c a r b o x y l i c acid cycle. NADH molecules produced during both g l y c o l y s i s and the t r i c a r b o x y l i c acid cycle can be reoxidized by the electron-transport system, with oxygen as the terminal electron acceptor. The electron-transport system is coupled with ATP synthesis so that 3 ATP molecules are produced per NADH, and a t o t a l of 38 ATP molecules are produced by aerobic u t i l i z a t i o n of glucose. The relationship of gl y c o l y s i s , t r i c a r b o x y l i c acid cycle and electron-transport system is i l l u s t r a t e d in Figure 1-2. A comparison of ATP y i e l d from aerobic r e s p i r a t i o n to ATP y i e l d from fermentation shows that aerobic u t i l i z a t i o n is much more favourable energetically than fermentation. The pathways involved in aerobic u t i l i z a t i o n of glucose are similar in aerobic molds, yeasts and bacteria. Molds generally lack the a b i l i t y to ferment glucose, and f a i l to grow under anaerobic conditions. 1.1.3.2 Influence of low temperature on yeast growth Temperature can exert a profound e f f e c t on growth and surv i v a l of yeasts. Most yeasts can grow slowly at or near 0<>C. The optimum temperature, at which growth rate is maximum, occurs in the range of 20°C to 3QOC for most -10- Figure 1-2: Relationship between g l y c o l y s i s , t r i c a r b o x y l i c acid cycle and electron-transport system. -11- yeasts, except the psychrophilic species. Maximum temperature for growth of most yeasts, with a few exceptions, is in the 30°C to 40°C range (Stokes, 1971). The biochemical processes affected by temperature are complex and numerous and include enzyme a c t i v i t y , substrate transport, and protein synthesis. One factor which determines the upper temperature l i m i t is thermal resistance of enzymes. A Cryptococcus species f a i l e d to grow at 30°C unless a-ketoglutarate, c i t r a t e or i s o c i t r a t e were added to the growth medium, which indicated that several enzymes of the t r i c a r b o x y l i c acid cycle were inactivated at this temperature (Hagen and Rose, 1962). A psychrophilic Candida n i v a l i s suffered severe membrane damage at 25°C, with leakage of soluble phosphates, amino acids and small peptides (Nash and S i n c l a i r , 1968). These results suggested that enzyme inact i v a t i o n and membrane damage set the upper temperature l i m i t for yeast growth. Baxter and Gibbons (1962) reported that a psychrophilic Cand ida species could oxidize glucose at 0°C, whereas another Candida species was inactive at t h i s temperature. At 10OC and 15°C, psychrophilic Candida gelida exhibited more rapid fermentation rates than those for Saccharomyces cerevisiae . Amino acid uptake by Candida u t i l i s ceased -12- below 4°C (Quetsch and Danforth, 1964), but uptake remained operative in Rhodotorula g l u t i n i s at -4°C (Clinton, 1968). These data indicate that enzyme a c t i v i t y and substrate transport are cr u c i a l factors governing the minimum growth temperature of yeasts. Preliminary MAP storage experiments with fresh f r u i t s showed that both mold rots and yeast spoilage were absent in a low oxygen-high carbon dioxide atmosphere. The i n h i b i t i o n of mold rots was not surprising, but i n h i b i t i o n of yeast spoilage was unexpected. When a population of natural f l o r a present on berry surfaces is subjected to c h i l l i n g temperature and anaerobic atmosphere, some yeast species w i l l be unable to grow, but some species which can grow at low temperature and possess fermentative a b i l i t y w i l l continue to p r o l i f e r a t e . In a survey of yeasts of strawberry, Cand ida sake, Kloeckera apiculata, Torulops is candida, and Torulopsis fragaria were found to have both the a b i l i t y to ferment sugars and grow at 50C (Buhagiar and Barnett, 1971). Fermentation of sugars by yeasts associated with blueberries at low temperature were investigated in t h i s study. 1.1.4 Background on phytoalexins The mechanism of yeast i n h i b i t i o n in MAP storage of f r u i t s demanded attention. A possible explanation is the -13- accumulation of antifungal compounds in f r u i t s in response to a c h i l l i n g temperature and an anaerobic environment. 1.1.4.1 Structure and d i s t r i b u t i o n Muller and BSrger (1940) proposed the concept of plant resistance. These authors used a potato c u l t i v a r which was resistant to one species of Phytophthora infestans, but susceptible to another species. The viru l e n t species rapidly colonized tuber s l i c e s but growth of the avirulent species was r e s t r i c t e d to dead tuber c e l l s . The v i r u l e n t species, however, was unable to infect tissues previously inoculated with spores of the avirulent species. Muller and BSrger (1940) concluded that protection was caused by production of an antifungal compound by potato c e l l s in response to the i n i t i a l inoculation. They ca l l e d t h i s substance a phytoalexin. Phytoalexins are defined as "antimicrobial compounds that are synthesized by and accumulated in plants which have been exposed to microorganisms" (Mansfield and Bailey, 1982). Phytoalexin accumulation may be triggered not only by exposure to microorganisms but also by a number of stresses such as cut injury, u l t r a v i o l e t l i g h t , temperature adversity, and ethylene (Haard and Cody, 1978; Cheema and Haard, 1978; Currier and Kuc, 1975). The term stress metabolite has been coined to include compounds -14- formed in response to injury, physiological stress and microbial i n f e c t i o n (Haard and Cody, 1978). In contrast to the term phytoalexin, stress metabolite does not imply a functional role of these compounds. Table 1-1 l i s t s several antimicrobial compounds known to accumulate in plants in response to both fungal and non-fungal i l l i c i t o r s . Their structures are presented in Figure 1-3. A stimulus which i l l i c i t s production of a metabolite type does not necessarily promote accumulation of other metabolites in a tissue. In sweet potato roots, ethylene stimulated accumulation of phenolic compounds, terpenes accumulated in response to c h i l l i n g storage, cut injury and UV radiation promoted coumarin formation. Accumulation of stress metabolites also depends on the physiological condition of tissues: mercuric acetate r e a d i l y caused formation of r i s h i t i n in potato tubers after cold storage, but was i n e f f e c t i v e on freshly harvested tubers (Cheema and Haard, 1978). In general, the formation of stress metabolites in response to a wide array of stimuli may be part of a general repair mechanism operating in damaged tissues (Kuc and Shain, 1977). -15- Table 1-1: Examples of antimicrobial compounds isolated from plant tissues (adapted from Haard and Cody, 1978). Compounds Plant sources Fungal I l l i c i t o r s Non-fungal I l l i c i t o r s P i s a t i n Garden pea Moni1ia fruct i c o l a UV radiation, DNA-intercalators Phaseollin Green bean Coll e t o t r icum 1indemuthiamum Heavy metal ions, t r a n s c r i p t i o n a l i n h i b i t o r s . R i s h i t i n I r i s h potato Phytophthora infestans AgNOa 6-methoxy- me 11 e i n Carrot Ceratocystis f imbr iata C h i l l i n g , HgClz Ipomeamarone Sweet potato Ceratocystis f imbr iata C h i l l i n g , SDS, HgCl 2 -16- Figure 1-3: Structures of several phytoalexins (Kuc and Shain, 1977). (a) P i s a t i n , (b) phaseollin, (c) r i s h i t i n , (d) 6-methoxy-mellein, and (e) Ipomeamarone. -17- 1.1.4.2 Biosynthesis of phytoalexins The interactions between primary and secondary metabolism in phytoalexin synthesis, and the role of key intermediates such as phenylalanine, acetyl-Coenzyme A (CoA), malonyl-CoA, and mevalonic acid are outlined in Figure 1-4. Development of an understanding of metabolic controls of phytoalexin synthesis requires studies on the properties and a c t i v i t i e s of enzymes involved. Enzymes studied have included those catalyzing synthesis of early precursors, as well as those d i r e c t l y responsible for formation of phytoalexins. Recent studies indicated that phytoalexin formation may involve a c t i v a t i o n of the biosynthetic pathways by de. novo enzyme synthesis (Manfield, 1983). Stoessl (1982) proposed two possible mechanisms which lead to diversion of normal metabolic processes to phytoalexin synthesis: (1) the imposition or removal of metabolic blocks on the normal metabolic route, or (2) increased synthesis of general biosynthetic precursors. Both mechanisms may be operating in d i f f e r e n t plants for synthesis of d i f f e r e n t compounds. -18- SUGAR£ GLYCOLYSIS PENTOSE PHOSPHATE CYCLE PHOSPHOENOLPYRUVATE + ERYTHROSE-4-P ^PHENYLALANINE CINNAMIC ACID ^COUMARINS COUMAROYL-COA PYRUVATE ACETYL-CO MEVALONIC ACID- TRICARBOXYLIC ACID CYCLE { FLAVONOIDS ISOFLAVONOIDS ACETYLENES MONOTERPENES ^DITERPENES ^ SESQUITERPENES Fi g u r e 1-4: R e l a t i o n s h i p between r e s p i r a t i o n and s y n t h e s i s of p h y t o a l e x i n s (adapted from M a n s f i e l d , 1983). -19- 1.1.4.3 St r u c t u r e - a c t i v i t y relationship L i t t l e can be stated d e f i n i t e l y concerning the rela t i o n s h i p between molecular structure and a c t i v i t y of phytoalexins. A common feature is the hydrophobic nature of phytoalexins, which probably enhances effective penetration of fungal membranes (Smith, 1982). A hydrophobic side chain is essential for antifungal a c t i v i t i e s of wighteone (Ingram e_t a_l., 1977), and kievitone (Smith, 1978). In the investigation of s t r u c t u r e - a c t i v i t y patterns of vignafuran analogues, Carter et al,. (1978) demonstrated that presence of one phenolic hydroxyl in the molecule was v i t a l for a c t i v i t y , since f u l l y methylated analogues were inactive. The t o x i c i t y of some isoflavonoid phytoalexins was postulated to be dependent on s t e r i c and compositional requirements: the two aromatic rings must be almost perpendicular to one another and small oxygen-containing substituents must be present (Perrin and Cruickshank, 1969). However, these results were disputed by subsequent research (VanEtten, 1976). The relationship between structure and t o x i c i t y of antifungal compounds requires clar i f i c a t i o n . 1.1.4.4 Modes of action Among many studies of phytoalexins, there have been few -20- attempts to determine the modes of action. The precise mode of action of any phytoalexin remains to be defined. The available information suggests two possible modes of a c t i v i t y : phytoalexins are multi-site toxicants, and damage of membrane systems is instrumental in their a c t i v i t y . M u l t i - s i t e a c t i v i t y of phytoalexins offers advantages to plants. S i t e - s p e c i f i c compounds are more e a s i l y countered by fungi as small mutations may be s u f f i c i e n t to induce a change at the s i t e of action and confer resistance (Smith, 1982). Phytoalexins could cause such damage as disorganization of c e l l u l a r components in fungi after a few minutes' treatment (Harris and Dennis, 1977). Selective i n h i b i t i o n of one particular metabolic process seems unlikely, since c e l l u l a r damage would not be apparent in a short time after treatment. Evidence of t o x i c o l o g i c a l effects of phytoalexins indicate that membrane dysfunction plays an important role in a c t i v i t y . Harris and Dennis (1977) reported that treatment of three Phytophthora species with terpenoids resulted in swelling of the c e l l cytoplasmic granulation, bursting of the c e l l membrane and loss of c e l l u l a r contents. These c y t o l o g i c a l changes were comparable to changes observed after treatment of fungal c e l l s with the membranolytic agent Triton X-100. Membrane damage is -21- r e f l e c t e d in leakage of e l e c t r o l y t e s and metabolites, which inevitably leads to loss of mycelial dry weight (VanEtten and Bateman, 1971). Membrane damage adversely affected substrate intake: glucose uptake by germinating spores of Stemphylium botryosum was inhibited after phytoalexin treatment (Higgins, 1978). For many phytoalexins, there may not be only one s i t e of action but many targets. A compound which induces membrane damage may also a f f e c t other reactions, r e s u l t i n g in gross f u n g i s t a t i c or fungicidal e f f e c t s . 1.1.5 Methodology in yeast s u s c e p t i b i l i t y testing with antifungal compounds When investigating the i n h i b i t i o n of yeast growth by phytoalexin(s) which may accumulate in blueberries during MAP storage, an assay which can provide meaningful information is required. Since the phytoalexin concept evolved from plant resistance to mold infections, studies on phytoalexin t o x i c i t y have been carried out almost exclusively with molds. These assays included mycelial growth on agar surfaces (Skipp and Bailey, 1977), growth of mycelia in l i q u i d media (VanEtten and Bateman, 1971), growth of germ tubes (Harris and Dennis, 1977; Higgins, 1978). Methodology for testing s u s c e p t i b i l i t y of yeasts is based mainly on c l i n i c a l tests with antifungal drugs used -22- to treat human infections. Three procedures, broth d i l u t i o n , agar d i l u t i o n and disk d i f f u s i o n have been recommended for yeast s u s c e p t i b i l i t y t esting (Shadomy and Espinel-Ingroff, 1980). At present, there is no standardized method of testing the s u s c e p t i b i l i t y of yeasts to antifungal agents. 1.1.5.1 Broth d i l u t i o n method In broth d i l u t i o n method, antifungal compounds are incorporated into a series of tubes containing a l i q u i d medium to achieve a range of concentrations. After inoculation and incubation, the tubes are examined for the minimum i n h i b i t o r y concentration (MIC). MIC is defined as the lowest concentration of drug which i n h i b i t s v i s i b l e t u r b i d i t y . This v i s u a l end-point has the advantage of si m p l i c i t y , however, i t enta i l s some serious problems. Visual end point determination requires a subjective judgement, and var i a t i o n between observers is an obvious problem. In addition, attempts to distinguish "almost no v i s i b l e growth" to "complete absence of v i s i b l e growth" can be d i f f i c u l t and erroneous. Another drawback is dependence of MIC on inoculum s i z e : MIC for some drugs increased with heavy inocula (Galgiani and Stevens, 1976 and 1978). The inoculum-dependence of v i s u a l end point method is i l l u s t r a t e d in Figure 1-5. The basis of t h i s -23- Inoculum (cfu/ml) Figure 1-5: MIC of 5-fluorocytosine at d i f f e r e n t inoculum sizes for three yeast strains (Galgiani and Stevens, 1976). Curves 1: Candida albicans, 2: Candida t r o p i c a l i s , 3: Torulops is glabrata. - 2 4 - problem is the use of vis u a l t u r b i d i t y as an index of growth. At the time set for reading the end point, tests with large s t a r t i n g inocula w i l l have more tubes in the d i l u t i o n series reaching v i s i b l e t u r b i d i t y than tests using the same drug concentrations but sta r t i n g with smaller inocula. This is because fewer doublings are required for a large inoculum to become turbid. At time of test reading, a small inoculum would have fewer turbid tubes, and the f i r s t clear tube would occur at a lower drug concentration than in the tubes with a large inoculum. Therefore, a high drug concentration i s necessary to keep tubes with a large inoculum from reaching v i s u a l t u r b i d i t y within a fixed time of end point reading. Galgiani and Stevens (1976) replaced v i s u a l t u r b i d i t y by spectrophotometric measurements to determine growth i n h i b i t i o n in a broth d i l u t i o n method. They calculated the i n h i b i t o r y concentration (IC) as the lowest drug concentration that met the c r i t e r i o n : % T > % Tcontrol + n(100 - % Tcontrol) where control = drug-free tube % T = percent transmission n = a selected f r a c t i o n less than 1 This formula defines a fr a c t i o n of i n h i b i t i o n (set by -25- n) as a function of t u r b i d i t y in drug-free controls. This end point determination is independent of inoculum size, i t eliminates s u b j e c t i v i t y , and variation among observers. 1.1.5.2 Agar d i l u t i o n method Broth d i l u t i o n tests can be adapted for use with agar media. In this method, a yeast isolate is inoculated into a series of agar plates containing d i f f e r e n t d i l u t i o n s of an antifungal drug. Inoculated drug-free plates serve as controls. MIC is defined as the lowest concentration of drug preventing macroscopic formation of colonies. Dependence of MIC on inoculum size is also a problem in this method (Stevens, 1984). MIC increases with heavy inocula. Tests with large st a r t i n g inocula have more plates showing v i s i b l e colonies than tests with small inocula, at time of reading r e s u l t s . This is because a colony s t a r t i n g with a small number of c e l l s w i l l not become v i s i b l e at the same time as a colony s t a r t i n g with a large c e l l number. A solution to this problem would be to define MIC as the drug concentration which reduces the number of colony forming units on test plates to a preset f r a c t i o n of the number of colony forming units on drug- free control plate. The application of t h i s c a l c u l a t i o n has not been reported. - 2 6 - 1.1.5.3 Disk d i f f u s i o n method In t h i s method, a high inoculum of a yeast isolate is spread on surface of agar plates to form a lawn of dense growth. Antifungal compounds are impregnated into assay disks, which are then placed on the agar surface. Growth i n h i b i t i o n by an antifungal compound is shown by a clear zone around the corresponding disk. The diameter of i n h i b i t i o n zone is influenced by d i f f u s i o n and concentration of antifungal compounds and s e n s i t i v i t y of yeast i s o l a t e s . The disk d i f f u s i o n method has the advantage of r a p i d i t y , the ease of performance, and economy. Several disks containing d i f f e r e n t concentrations of a compound or drug-free control could be tested on one plate. Dependence of test results on inoculum size is l a r g e l y eliminated, because a high s t a r t i n g inoculum is required to achieve a lawn of dense growth. C l i n i c a l use of the disk d i f f u s i o n method for yeast s u s c e p t i b i l i t y testing has become popular. Commercially- prepared disks of antifungal drugs are now available in Europe (Kostiala and Kostiala, 1984). - 2 7 - 1 . 2 SUMMARY OF L I T E R A T U R E R E V I E W A N D O B J E C T I V E S OF THE STUDY 1 . The s h e l f - l i f e o f f r e s h b l u e b e r r i e s i s l i m i t e d p r i m a r i l y b y m o l d d e c a y . C . g l o e o s p o r i o i d e s , B . c i n e r e a , a n d A . t e n u i s a r e common s p o i l a g e o r g a n i s m s o f b l u e b e r r i e s . MAP s t o r a g e may be u s e d t o i n h i b i t m o l d d e c a y a n d e x t e n d t h e s h e l f - l i f e o f b l u e b e r r i e s . I n p r e v i o u s MAP s t o r a g e e x p e r i m e n t s , y e a s t s p o i l a g e o f b l u e b e r r i e s was a l s o a b s e n t , a l t h o u g h many y e a s t s p e c i e s c a n g r o w a t l o w t e m p e r a t u r e a n d a l s o h a v e t h e a b i l i t y t o f e r m e n t s u g a r s i n a n a e r o b i c c o n d i t i o n . The g r o w t h t r e n d o f y e a s t s , m o l d s a n d b a c t e r i a , t h e h e a d s p a c e a t m o s p h e r e o f p a c k a g e s o f m o d i f i e d a t m o s p h e r e p a c k a g e d b l u e b e r r i e s , a n d b e r r y p H , s o l u b l e s o l i d s c o n t e n t w i l l be f o l l o w e d d u r i n g MAP s t o r a g e . 2 . Y e a s t i n h i b i t i o n may be a t t r i b u t e d t o t h e p r o d u c t i o n o f a n t i f u n g a l c o m p o u n d ( s ) b y b l u e b e r r y f r u i t i n r e s p o n s e t o l o w t e m p e r a t u r e a n d a n a e r o b i c e n v i r o n m e n t . A v a r i e t y o f s t r e s s m e t a b o l i t e s a r e k n o w n t o a c c u m u l a t e i n p l a n t s e x p o s e d t o s t r e s s e s s u c h a s c h i l l i n g a n d c u t i n j u r y , UV r a d i a t i o n , a n d f u n g a l i n f e c t i o n . Some s t r e s s m e t a b o l i t e s e x h i b i t f u n g i c i d a l o r f u n g i s t a t i c a c t i v i t i e s t o p r o t e c t p l a n t s a g a i n s t m i c r o b i a l i n v a s i o n . E x t r a c t s o f - 2 8 - b l u e b e r r i e s h e l d u n d e r MAP s t o r a g e w i l l be t e s t e d f o r a n t i f u n g a l a c t i v i t y . 3. D i f f i c u l t i e s i n v o l v e d i n i n . v i t r o a s s a y a r i s e b e c a u s e t h e r e a r e no s t a n d a r d i z e d p r o c e d u r e s f o r t e s t i n g y e a s t s u s c e p t i b i l i t y , a n d t h e s t r u c t u r e o f a n y a n t i f u n g a l c o m p o u n d , i f p r e s e n t , i s u n k n o w n . The d i s k d i f f u s i o n m e t h o d w i l l be u s e d b e c a u s e i t i s r a p i d , a n d r e s u l t s o f t h i s m e t h o d a r e r e l a t i v e l y i n d e p e n d e n t o f s t a r t i n g i n o c u l u m s i z e . I n a d d i t i o n , t h i s m e t h o d i s more e a s i l y a d a p t e d f o r u s e w i t h m o d i f i e d a t m o s p h e r e s t o m i m i c MAP s t o r a g e t h a n t h e b r o t h d i l u t i o n m e t h o d , a n d i t i s l e s s t i m e a n d l a b o u r c o n s u m i n g t h a n t h e a g a r d i l u t i o n m e t h o d . CHAPTER 2 EXPERIMENTATION, RESULTS AND DISCUSSION MODIFIED ATMOSPHERE PACKAGING STORAGE OF BLUEBERRIES. -30- 2.1 ANALYSES OF BLUEBERRIES IN MAP STORAGE In i n i t i a l studies, yeast and mold, and t o t a l plate counts were followed with time during storage of blueberries, to establish the microbial growth trends. F i n a l l y , headspace composition, f r u i t pH, and soluble s o l i d contents were determined in addition to enumeration of microorganisms on blueberries. 2.1.1 MATERIALS' AND METHODS In 1986, two blueberry v a r i e t i e s Bluecrop (mid-season) and Jersey (late season), were used in separate storage t r i a l s . Blueberries were purchased from the B.C. Blueberry Co-op (Richmond, B.C.). In each t r i a l , a l l berries were pooled to form one population before packaging. Approximately 50 g of berries were packaged in each 20 cm x 12 cm bag. Two storage conditions of low-oxygen and intermediate-oxygen were established using packaging films whose transmission properties are shown in Table 2-1. A l l MA packages were heat sealed; in the control (air) condition, berries were stored in unsealed p l a s t i c bags of the type frequently used for f r u i t and vegetable packaging at homes and r e t a i l stores. After packaging, the bags were stored at 4°C. Two samples from each MAP storage condition and control -31- Table 2-1: Properties of the packaging films. Film Transmiss ion rates 1 M.V.T . R.2 Nitrogen Oxygen Carbon dioxide High Barr ier 0.015 0.05 0 .3 0. 3 Intermediate Barr ier 22 83 420 0. 6 1 expressed as cm3 / 100in 2 / 24 hours / atm 2 Moisture Vapor Transmission Rate, g/100 i n 2 / 24 hours at 95% ..Relative Humidity. -32- were used to determine the t o t a l aerobic plate count, anaerobic plate count, and yeast and mold counts every week for six weeks and every two weeks for six more weeks. Twenty-five grams of berries were stomached in 225 ml of (0.1%) peptone water with a Colworth Stomacher; 1 ml- samples of appropriate d i l u t i o n s were f i l t e r e d through the Hydrophobic Grid Membrane F i l t e r (HGMF), which was housed in the Isogrid F i l t r a t i o n units (QA Laboratories, Toronto, Ont), The HGMF is a polysulfone membrane f i l t e r (0.45 Mm), on which is printed a grid of hydrophobic material. The hydrophobic grid prevents spreading of mold colonies. After f i l t r a t i o n , the f i l t e r s were placed on appropriate media for incubation. Enumeration media included: Plate Count agar (PCA) with 0.005% Congo Red dye for aerobic or anaerobic plate counts, PCA supplemented with 0.01% chloramphenicol and 0.01% chlo r t e t r a c y c l i n e HC1 for yeast and mold counts, PCA supplemented with 0.01% chloramphenicol, 0.01% chlortetracycline HC1 and 0.5% sodium pyruvate for yeast and mold counts. The l a t t e r medium was used because pyruvate might have a b e n e f i c i a l e f f e c t on yeast and mold enumeration (Koburger, 1986). The two media for yeast and mold counts contained 0.01% Trypan Blue so that colonies appeared blue against the black and white background of f i l t e r s . PCA was purchased from Difco Co., Detroit, MI, while a l l a n t i b i o t i c s and - 3 3 - dyes were obtained from Sigma Chemicals, St. Louis, MO. Plates for the determination of anaerobic plate counts were incubated in an anaerobic jar (Oxoid, Nepean, Ont), and anaerobic environment was achieved using the Gas- Generating k i t and anaerobic catalyst (Oxoid, Nepean, Ont). A l l plates were incubated at 21°C for 4 days, except plates incubated anaerobically (7 days, 21°C). Most probable numbers (MPN) were obtained by the formula (Sharpe and Michaud, 1975): MPN = 1600 x ln [1600 / (1600 - X)] where X = t o t a l number of squares containing colonies in a f i l t e r which has a maximum of 1600 squares. Geometric means of transformed (log base 10) MPN from antibiotic-supplemented PCA and antibiotic-pyruvate- supplemented PCA were compared by paired t - t e s t . In 1987, one MAP storage t r i a l was set up. Bluecrop blueberries were stored in two conditions: intermediate- oxygen and low-oxygen content; a i r storage was omitted. The packaging films, procedure and storage temperature were i d e n t i c a l to those used in the previous t r i a l s . Aerobic and anaerobic plate counts, yeast and mold counts, f r u i t pH, soluble s o l i d content, and headspace gas -34- analysis were carried out at two-week intervals for 12 weeks. Four samples from each storage condition were used for microbial counts. The media and plating procedure were i d e n t i c a l to those used in the previous t r i a l s , except for the elimination of antibiotic-pyruvate-supplemented medium. For anaerobic plate counts, the Gas-Generating k i t was replaced by a i r evacuation followed by flushing of the anaerobic jars with a gas mixture (10% carbon dioxide, 10% hydrogen, 80% nitrogen). Composition of headspace atmosphere was obtained from four samples of each storage condition. The analyses were carri e d out using a gas chromatograph (Shimadzu GC-9A) equipped with a thermal conductivity detector, and a Carbosieve S-II column (Supelco, Oakville, Ont), with helium c a r r i e r gas (30 ml/min). The temperature was programmed to hold for 7 minutes at 35°C, then increased to 225°C at 32°C/min. Peak areas' were integrated by a Shimadzu C-R 3A Chromatopac integrator. Four bags from each storage condition were sampled, and internal pH of 10 berries from each bag were measured using a surface electrode (Fisher S c i e n t i f i c , Vancouver, B.C.). When berry exudates were present, their pH was also measured. Four bags from each storage condition were sampled for -35- s o l u b l e s o l i d s . B e r r i e s were homogenized with a Waring blender ( h a l f speed, 1 min), and c e n t r i f u g e d (4,080 x g, 15 min, 20<>C) to c l a r i f y j u i c e . Soluble s o l i d contents were determined with a refTactometer (Bausch and Lomb, Rochester, NY) operated a t 20°C. 2.1.2 RESULTS AND DISCUSSION 2.1.2.1 Headspace atmosphere of packages with low gas p e r m e a b i l i t y Changes i n headspace atmosphere of l o w - p e r m e a b i l i t y packages are presented i n Table 2-2. Since the packaging f i l m had a very low t r a n s m i s s i o n r a t e s f o r oxygen, n i t r o g e n and carbon d i o x i d e , d i f f u s i o n of these gases a c r o s s the f i l m can be con s i d e r e d n e g l i g i b l e r e l a t i v e to b e r r y r e s p i r a t i o n r a t e s , and changes i n the headspace composition are the r e s u l t of f r u i t r e s p i r a t i o n . During the process of energy u t i l i z a t i o n , carbohydrates provide r e a d i l y a v a i l a b l e energy and are p r e f e r r e d s t a r t i n g m a t e r i a l s . Glucose c a t a b o l i s m begins with g l y c o l y s i s , a sequence of r e a c t i o n s which r e s u l t s i n the co n v e r s i o n of one glucose molecule to two molecules of pyruvate ( S e c t i o n 1.1.3.1). The o v e r a l l process of g l y c o l y s i s o c c u r r i n g i n p l a n t t i s s u e s i s s i m i l a r to g l y c o l y t i c breakdown of glucose by yeasts and other organisms. -36- Table 2-2: Headspace gas composition of blueberry packages. The high barrier f i l m was used. 1 Time Headspace composition (%) (weeks) Oxygen Nitrogen Carbon dioxide 0 21.28 (0.17) 78.72 (0.17) 0 2 0.82 (0.19) 52.87 (5.32) 46.30 (5.49) 4 0.65 (0.18) 42.80 (3.69) 56.55 (3.76) 6 0.41 (0.10) 38.45 (4.11) 61.14 (4.62) 8 0.63 (0.16) 34.58 (2.14) 64.79 (1.87) 10 0.42 (0.11) 32.68 (1.37) 66.93 (1.39) 12 0.48 (0.14) 30.56 (1.32) 68.96 (1.55) 1 Data represent the means of four r e p l i c a t e s and the sample standard deviations are shown in brackets. -37- The fate of pyruvate depends primarily on the a v a i l a b i l i t y of oxygen. In the presence of oxygen as an electron acceptor, pyruvate is channelled in the d i r e c t i o n of aerobic r e s p i r a t i o n . The distinguishing c h a r a c t e r i s t i c s of aerobic r e s p i r a t i o n are: (1) oxygen is the ultimate electron acceptor, (2) the complete oxidation of pyruvate to carbon dioxide and water, and (3) the e f f i c i e n t conservation of free energy as ATP. Oxygen serves as the terminal electron acceptor to provide for the continuous reoxidation of reduced coenzyme molecules. These coenzyme molecules (in their oxidized form) carry out the stepwise oxidation of intermediates derived from pyruvate. The reoxidation of these reduced coenzymes by transfer of electrons to oxygen is accompanied by the generation of ATP. The o v e r a l l equation for aerobic r e s p i r a t i o n can be written as: C e H j z O e + 6 0 2 + 38 ADP + 38P± = 6 C0= + 6 H z0 + 38 ATP The low permeability of the packaging f i l m for oxygen r e s t r i c t e d i t s d i f f u s i o n into the storage bags from the atmospheric a i r , so that oxygen consumed by aerobic r e s p i r a t i o n could not be replenished. Oxygen l e v e l became l i m i t i n g after two weeks of storage, and thus berries must have switched to anaerobic r e s p i r a t i o n . Ethanol is the -38- most common end-product of anaerobic r e s p i r a t i o n in plants (Devlin and Witham, 1983). Pyruvate i s decarboxylated to acetaldehyde which acts as an acceptor molecule in the re- oxidation of NADH to regenerate NAD. The ov e r a l l equation for anaerobic r e s p i r a t i o n of glucose by plants is written: C 6 H i a 0 6 + 2 ADP + 2 P ± = 2 C 2H=OH + 2 C0=> + 2 H 3 0 + 2 A T P During anaerobic r e s p i r a t i o n of plants, l a c t i c acid could also appear as end-product. This mechanism for anaerobic reoxidation of NADH involves reduction of the carbonyl group of pyruvate to form l a c t a t e . L a c t i c acid formation may protect plants from ethanol poisoning (Zemlianukhin and Ivanov, 1978): C 6 H i 2 0 6 + 2 ADP + 2 P i = 2 C a H s 0 3 + 2 H z 0 + 2 ATP The continuous increase of carbon dioxide content in storage packages over time (Table 2 - 2 ) indicated that ethanol formation might be the predominant mode of anaerobic r e s p i r a t i o n by berries, since l a c t i c acid production involves no net gain of carbon dioxide. The changes in oxygen and carbon dioxide contents in packages are expected, because these gases p a r t i c i p a t e in berry metabolism. The change in nitrogen content was - 3 9 - u n e x p e c t e d s i n c e b e r r i e s c a n n o t m e t a b o l i z e t h i s i n e r t g a s . The m o d e l p r e s e n t e d i n F i g u r e 2-1 h e l p s t o e x p l a i n t h e o b s e r v e d c h a n g e i n n i t r o g e n . F i g u r e 2-1 ( a ) s h o w s a h y p o t h e t i c a l a t m o s p h e r e o f a c l o s e d s y s t e m w h e r e t h e r e i s no g a s e x c h a n g e w i t h t h e e n v i r o n m e n t . When c a r b o n d i o x i d e i s i n t r o d u c e d i n t o t h e s y s t e m ( F i g u r e 2-1 b ) , n i t r o g e n c o n t e n t e x p r e s s e d a s p e r c e n t o f t o t a l v o l u m e i s d e c r e a s e d a l t h o u g h i t s v o l u m e was n o t c h a n g e d . I n F i g u r e 2-1 ( c ) , more c a r b o n d i o x i d e i s a d d e d a n d f u r t h e r d e p r e s s i o n o f n i t r o g e n p e r c e n t i s o b s e r v e d . I n b o t h F i g u r e 2-1 ( b ) a n d ( c ) , o x y g e n v o l u m e was s m a l l r e l a t i v e t o t h e o t h e r g a s e s s o i t s d e p r e s s i o n c a u s e d b y c a r b o n d i o x i d e was n o t d r a m a t i c . The c h a n g e s i n h e a d s p a c e c o m p o s i t i o n o f p a c k a g e s w i t h l o w g a s p e r m e a b i l i t y w e r e s i m i l a r t o t h e s i t u a t i o n d e m o n s t r a t e d i n F i g u r e 2-1. C a r b o n d i o x i d e p r o d u c e d b y b e r r y r e s p i r a t i o n was r e t a i n e d i n t h e s t o r a g e b a g s , w h i c h l e d t o a n i n c r e a s e i n t o t a l h e a d s p a c e v o l u m e . The i n c r e a s e o f c a r b o n d i o x i d e q u a n t i t y c a u s e d a n a p p a r e n t d e c r e a s e i n p e r c e n t n i t r o g e n i n t h e h e a d s p a c e , e v e n t h o u g h t h e a c t u a l q u a n t i t y o f n i t r o g e n r e m a i n e d u n c h a n g e d w i t h t i me. 2.1.2.2 H e a d s p a c e a t m o s p h e r e o f p a c k a g e s w i t h i n t e r m e d i a t e g a s p e r m e a b i l i t y I n t h e s e p a c k a g e s , d i f f u s i o n o f p e r m a n e n t g a s e s a c r o s s t h e p a c k a g i n g f i l m m u s t be c o n s i d e r e d i n r e l a t i o n t o -40- 80 ml N 2 (80%) 80 ml (72%) 20 ml 0= (20%) 1 ml 0 2 (0.9%) 30 ml C0 2 (27%) 80 ml N 2 (66%) 1 ml 0 2 (0.8%) 4 0 ml C0 2 (33%) (a) (b) (c) Figure 2-1: A model of headspace composition in the high barrier f i l m packages. (a) a closed system with s t a r t i n g headspace composition; (b) carbon dioxide is added, and oxygen is taken out; (c) more carbon dioxide is added with no change to other gases. - 4 1 - changes i n the headspace atmosphere. Ae r o b i c r e s p i r a t i o n of b e r r i e s caused the decrease of oxygen content and an i n c r e a s e of carbon d i o x i d e during storage (Table 2-3). The decrease i n oxygen l e v e l r e f l e c t e d the s h o r t f a l l between oxygen consumption by b e r r i e s and inward oxygen d i f f u s i o n through the packaging f i l m . If b e r r y r e s p i r a t i o n was c o n s i d e r e d alone, then one mole of carbon d i o x i d e would be produced per mole of oxygen consumed. However, at low storage temperature, s o l u b i l i t y of carbon d i o x i d e i n aqueous s o l u t i o n s w i t h i n the b e r r y t i s s u e s i n c r e a s e d and a p o r t i o n of carbon d i o x i d e produced by r e s p i r a t i o n would be d i s s o l v e d i n t i s s u e s , while a p a r t would be l i b e r a t e d i n t o the package headspace. The i n c r e a s e of carbon d i o x i d e q u a n t i t y i n the headspace c r e a t e d a g r a d i e n t of higher carbon d i o x i d e c o n c e n t r a t i o n i n s i d e the packages than i n a i r . Carbon d i o x i d e i n s i d e the bags then had a tendency to d i f f u s e outward a c c o r d i n g to t h i s g r a d i e n t . The r a t e of i t s d i f f u s i o n was i n f l u e n c e d by the p e r m e a b i l i t y of the packaging f i l m . T h e r e f o r e , the q u a n t i t y of carbon d i o x i d e observed i n the package headspace was the net d i f f e r e n c e between t o t a l carbon d i o x i d e produced by r e s p i r a t i o n and the sum of d i s s o l v e d carbon d i o x i d e i n f r u i t t i s s u e and the q u a n t i t y l o s t by d i f f u s i o n through the packaging f i l m . T o t a l pressure of permanent gases i n f r u i t t i s s u e s , -42- Table 2-3: Headspace gas content of blueberry packages. The intermediate b a r r i e r f i l m was used * . Time Headspace composition (%) (weeks) Oxygen Nitrogen Carbon dioxide 0 21.28 (0.17) 78.72 (0.17) 0 2 18.04 (2.23) 80.92 (2.33) 1.04 (0.23) 4 12.52 (3.03) 84.91 (1.97) 2.57 (0.77) 6 9 .48 (3.06) 86.94 (3.29) 3.54 (0.64) 8 7 .77 (1.53) 89.16 (1.12) 3.07 (0.44) 10 7.38 (1.12) 88.33 (2.80) 4 .29 (1.60) 12 6.32 (1.10) 89.42 (2.60) 4.26 (1.53) 1 Data represent the means of four rep l i ca te s and the sample standard dev iat ion are shown in brackets . -43- which is the sum of p a r t i a l pressures of oxygen, nitrogen and carbon dioxide, must be equal to the t o t a l pressure of gases in the headspace (Figure 2-2). As shown in Table 2-3, the change (decrease) in oxygen content was larger than the change (increase) of carbon dioxide in the headspace, due to outward d i f f u s i o n of carbon dioxide and i t s increased s o l u b i l i t y in berry tissue. This difference created an imbalance in t o t a l pressure of gases between f r u i t tissue and headspace. Nitrogen would be exchanged from i n t e r c e l l u l a r spaces of the f r u i t tissue, and possibly dissolved nitrogen, to gaseous nitrogen in the headspace, in order to balance t o t a l pressure between f r u i t tissue and headspace. This exchange resulted in an increase of nitrogen content in headspace. 2.1.3 pH of blueberries in MAP storage Growth of bacteria is profoundly influenced by pH: there usually is a maximum and minimum l i m i t for growth and an optimum range for each species. Yeasts and molds are generally less sensitive to low pH than bacteria (Koburger and Marth, 1984). pH also a f f e c t s the a c t i v i t y of antimicrobial compounds. This may be an e f f e c t on s t a b i l i t y or binding to target s i t e s (Stevens, 1984). -44- F r u i t P(tota l ) = P ( O a ) + P ( N a ) + P ( C 0 2 ) Headspace F i l m P(tota l ) = P (0 2 ) + P(N2) + P (C0 2 ) A i r Gas exchange regulated by f i l m permeabi l i ty . Figure 2-2: Gas exchange between f r u i t t i s sues , headspace of package and environment. -45- In a low oxygen environment (packages of low gas permeability), pH of the berries remained stable over 12 weeks (Table 2-4). Anaerobic r e s p i r a t i o n of berries led to production of ethanol, carbon dioxide, and possibly a small amount of l a c t i c acid. A part of the carbon dioxide produced was released into the package headspace, and a small part dissolved in berry tissues as carbonic acid. The amount of protons produced by d i s s o c i a t i o n of l a c t i c acid and carbonic acid would be small since these are weak acids. Weak di s s o c i a t i o n of these acids could not cause a pH decrease in berry tissue, because berry juice had an a b i l i t y to r e s i s t pH changes. This buffer capacity is attributed to organic s a l t s , and acid phosphates because of their a b i l i t y to bind protons. Berries stored in an intermediate oxygen environment (packages of intermediate gas permeability) also exhibited a stable pH. A portion of carbon dioxide produced by aerobic r e s p i r a t i o n of berries existed in tissue as carbonic acid, which weakly dissociated to release protons. The buffering agents in berry tissue were able to maintain a stable pH. Berry exudate was observed after four weeks in intermediate oxygen storage, and no exudate was present in the low oxygen packages. Exudate is probably a resu l t of -46- Table 2-4: pH of blueberries and exudates during MAP storage . Time (weeks) pH (cut berr i e s ) 1 pH (exudate) 2 Low 0 2 Intermediate 0 2 Intermediate 0 2 0 3 .49 (0. 38 ) 3.49 (0.38) no exudate 2 3 .69 (0. 49 ) 3.61 (0.53) no exudate 4 3 .68 (0. 41) 3.55 (0.47) no exudate 6 3 .68 (0. 47) 3.54 (0.36) 3.24 (0.20) 8 3 .50 (0. 40) 3.43 (0.35) 3.05 (0.14) 10 3 .54 (0. 53) 3.49 (0.21) 2.97 (0.17) 12 3 .49 (0. 48) 3.47 (0.22) 2.99 (0.15) x Data represent the means of pH taken from 40 berries, and the sample standard deviations are shown in brackets. 3 no exudate was present in low oxygen samples throughout storage time. Data represent the means of exudates in four bags and the sample standard deviations are shown in brackets. - 4 7 - juice leakage from berries due to f r u i t senescence and autolysis as well as microbial penetration. In intact berries and other a e r i a l organs of plants, the c u t i c l e constitutes the f i r s t barrier against invasion of fungi. Plant c u t i c l e consists of the insoluble polymer cutin, embedded in a mixture of waxy materials. Cutin is a polyester composed of C i 6 and C s e hydroxy and epoxy fatty acids (Kolattukudy, 1985). Since cutin i s the main barrier in the c u t i c l e , i t was proposed that penetrating fungi secrete a cutin degrading enzyme, cutinase. Cutinase isolated from B.cinerea and other fungi can hydrolyze cutin into oligomers and monomers (Soliday and Kolattukudy, 1976; Dickman et §JU, 1982). After breaking the f i r s t barrier with cutinase, fungi encounter the c e l l wall. Therefore, successful penetration requires both cutinase and c e l l wall degrading enzymes such as c e l l u l a s e and pectinase. P e c t i n o l y t i c a c t i v i t y has been found in many yeast genera: Kluyveromyces, Cand ida, Torulopsis, and Saccharomyces (Luh and Phaff, 1951). Pectin hydrolysis by species of Debaryomyces, Candida, and Rhodotorula which leads to softening of cucumbers and olives has been reported (Bell and E t c h e l l s , 1956; Vaughn et a l . , 1969). The hydrolytic a c t i v i t y of cutinase, pectinase and c e l l u l a s e on cutin and c e l l wall might contribute to - 4 8 - leakage of juice from berries. In contrast to intact berries where microbial growth is largel y confined to berry surface with limited nutrient supply, the pool of exudate provided a r i c h growth medium. U t i l i z a t i o n of exudate sugars by microorganisms would proceed i n i t i a l l y v i a aerobic oxidation pathway, u n t i l rapid r e s p i r a t i o n led to depletion of oxygen, which has a low s o l u b i l i t y in non-aerated solutions. When oxygen tension is low, yeasts and bacteria which have the a b i l i t y to ferment sugars would u t i l i z e this pathway to generate energy and metabolic precursors. Fermentation of exudate sugars by microorganisms produced ethanol, carbon dioxide and other compounds such as glyc e r o l , acetic acid and l a c t i c a c i d . Many yeasts could also switch to fermentative metabolism when a large concentration of sugars i s available in the medium, even under aerobic conditions (Sols e_t al.., 1971). This impairment of respiratory c a p a b i l i t y is known as the "Crabtree e f f e c t " ; i t s mechanism is not well understood. The "Crabtree e f f e c t " might operate in some yeast species, and contribute to fermentation of exudate sugars. If the buffering s a l t s of exudate were exhausted to balance protons dissociated from carbonic acid produced by berry metabolism, then further d i s s o c i a t i o n of acid products of microbial fermentation would cause a decrease of exudate -49- pH. 2.1.4 S o l u b l e s o l i d contents of b l u e b e r r y f r u i t Table 2-5 shows r e f r a c t i v e index v a l u e s expressed as s o l u b l e s o l i d contents of b l u e b e r r i e s d u r i n g MAP s t o r a g e . S o l u b l e s o l i d contents of b l u e b e r r i e s remained at the same l e v e l throughout storage i n packages with low oxygen or intermediate oxygen atmosphere. El-Kazzaz e_t a l . (1983) a l s o r e p o r t e d no s i g n i f i c a n t d i f f e r e n c e i n s o l u b l e s o l i d contents among s t r a w b e r r i e s i n s e v e r a l c o n t r o l l e d - atmosphere treatments. During b e r r y metabolism, carbohydrates are not always broken down completely f o r s y n t h e s i s of e n e r g y - r i c h ATP molecules, i n s t e a d they can serve as precursors to i n t e r m e d i a t e s i n the s y n t h e s i s of amino a c i d s , l i p i d s , and pigments ( D e v l i n and Witham, 1983). The compounds d e r i v e d from o x i d a t i o n of carbohydrates supply the b u i l d i n g b l o c k s f o r other compounds. Solu b l e s o l i d contents of b e r r i e s remained at the same l e v e l d u r i n g storage most l i k e l y as a r e s u l t of the dynamic i n t e r a c t i o n between anabolism and c a t a b o l i s m i n t i s s u e s . During i n f e c t i o n , microorganisms produced enzymes which hydrolyzed the components of b e r r y c e l l w a l l s . Evidence f o r t h i s process was the presence of b e r r y exudates i n the intermediate-oxygen packages (see Table 2-4). H y d r o l y s i s of c e l l w a l l p o l y s a c c h a r i d e s c o u l d r e s u l t i n r e l e a s e of -50- Table 2-5: Soluble s o l i d contents of blueberry f r u i t in MAP storage. Time Soluble solids (as % sucrose) 1 (weeks) Low 02 Intermediate 0 2 0 12.3 (0.4) 12.3 (0.4) 2 12.0 (0.2) 12.0 (0.3) 4 11.6 (0.3) 11.8 (0.2) 6 11.8 (0.3) 11.4 (0.2) 8 11.8 (0.2) 11.2 (0.3) 10 12.0 (0.4) 11. 4 (0.1) 12 12.0 (0.4) 11.6 (0.2) 1 Data represent the means of four replicates and the sample standard deviations are shown in brackets. -51- sugars which might cause an increase in soluble s o l i d contents of blueberries. The soluble s o l i d contents of berries taken from the intermediate-oxygen packages did not increase, probably because sugars of berry c e l l wall were u t i l i z e d by microorganisms. 2.1.5 Growth of microorganisms on blueberry during MAP storage 2.1.5.1 Anaerobic plate counts Anaerobic plate counts represented the most probable number of bacteria capable of growing under anaerobic conditions. In the two storage t r i a l s of 1986, anaerobic plate counts showed large fluctuations due to inconsistent performance of the Gas-Generating k i t (Oxoid, Nepean, Ont), which produced hydrogen and carbon dioxide. In the presence of palladium catalyst, hydrogen released by the Gas-Generating k i t reacts with oxygen in the anaerobic jar to form water; th i s reaction depletes oxygen and creates an anaerobic atmosphere. This reaction which depends on the rate of hydrogen release and a c t i v i t y of catalyst could sometimes proceed slowly, and res u l t in delay of oxygen depletion. When anaerobic atmosphere was not promptly established in the anaerobic j a r s , growth of aerobic organisms probably occurred on the Hydrophobic -52- G r i d Membrane f i l t e r , c a u sing e r r a t i c a l l y high MPN v a l u e s . T h i s problem was overcome i n the 1987 storage t r i a l by the use of a i r evacuation and subsequent f l u s h i n g of i n c u b a t i n g j a r s with a gas mixture (10% carbon d i o x i d e , 10% hydrogen, 80% n i t r o g e n ) . F i g u r e 2-3 shows anaerobic p l a t e counts f o r b l u e b e r r i e s s t o r e d i n low and intermediate oxygen environments. The i n c r e a s i n g growth trend i n intermediate oxygen s t o r a g e , combined with the a b i l i t y to grow i n anaerobic i n c u b a t i o n i d e n t i f i e d these b a c t e r i a as f a c u l t a t i v e aerobes. F a c u l t a t i v e aerobes are able to o b t a i n energy by e i t h e r a e r o b i c r e s p i r a t i o n or fermentation, and do not r e q u i r e oxygen f o r b i o s y n t h e s i s (Brock, 1979). These organisms grow b e t t e r with oxygen because a e r o b i c r e s p i r a t i o n i s f a r more e f f i c i e n t than fermentation i n energy p r o d u c t i o n . In low oxygen s t o r a g e , growth i n c r e a s e d u r i n g the f i r s t two weeks was f o l l o w e d by a p l a t e a u where counts remained at the same l e v e l f o r 10 weeks. T h i s s h i f t corresponded to the increase i n carbon d i o x i d e l e v e l of packages. The i n h i b i t o r y e f f e c t s of carbon d i o x i d e on a n a e r o b i c a l l y grown B a c i l l u s cereus and Streptococcus cremoris have been r e p o r t e d by Enfors and Molin (1980). At present there are two proposed mechanisms of carbon d i o x i d e i n h i b i t i o n to b a c t e r i a : (1) Carbon d i o x i d e i n h i b i t s enzymatic r e a c t i o n s such as c a r b o x y l a t i o n , d e c a r b o x y l a t i o n which are c r i t i c a l -53- Time (weeks) F i g u r e 2-3: Anaerobic p l a t e counts of Bluecrop b l u e b e r r i e s i n two MAP storage c o n d i t i o n s (1987). Curves 1: inte r m e d i a t e oxygen, 2: low oxygen s t o r a g e . P o i n t s r e p r e s e n t means of four samples. Geometric means of two storage c o n d i t i o n s were s i g n i f i c a n t l y d i f f e r e n t o n l y d u r i n g 4-12 weeks by Student's t - t e s t . -54- f o r growth (King and Nagel, 1975), and (2) Carbon d i o x i d e a f f e c t s spore germination (Enfors and M o l i n , 1978). Both mechanisms may operate i n d i f f e r e n t organisms. 2.1.5.2 Aerob i c p l a t e counts of b l u e b e r r i e s A e r o b i c p l a t e counts represented p o p u l a t i o n s of o b l i g a t e and f a c u l t a t i v e a e r o b i c b a c t e r i a on b l u e b e r r i e s . Because of t h e i r slow growth and poor c o m p e t i t i v e a b i l i t y , y e a s t s and molds can o n l y develop i n media which i s unfavorable f o r b a c t e r i a . A e r o b i c p l a t e counts of samples from a i r and intermediate oxygen storage i n c r e a s e d up to 8 weeks of s t o r a g e , followed by a p l a t e a u between 8-12 weeks (Fig u r e s 2-4, 2-5, 2-6). The growth increase i n d i c a t e d s p o i l a g e of the b e r r i e s , and r a p i d growth of microorganisms. The p l a t e a u showed exhaustion of n u t r i e n t s , and c o m p e t i t i o n by l a r g e numbers of b a c t e r i a . A e r o b i c b a c t e r i a i n the intermediate oxygen atmosphere reached the p l a t e a u at a lower p o p u l a t i o n than those i n a i r . T h i s i s probably a r e s u l t of oxygen l i m i t a t i o n i n the l a s t s i x weeks of i n t e r m e d i a t e oxygen storage (Table 2-3). A e r o b i c p l a t e counts of samples from low oxygen atmosphere showed no i n c r e a s e over time. Under the v e r y low oxygen atmosphere, o b l i g a t e aerobes c o u l d not develop because they r e q u i r e oxygen f o r energy g e n e r a t i o n and -55- T i m e (weeks) F i g u r e 2-4: Ae r o b i c p l a t e counts of Bluecrop b l u e b e r r i e s i n three storage c o n d i t i o n s (1986). Curves 1: a i r , 2: intermediate oxygen, 3: low oxygen atmosphere. P o i n t s represented means of 2 samples. Geometric means of a i r and intermediate oxygen samples were s i g n i f i c a n t l y d i f f e r e n t o n l y a t 10-12 weeks by Student's t - t e s t (a = 0.05). Means of int e r m e d i a t e and a i r samples were s i g n i f i c a n t l y d i f f e r e n t from means of low oxygen samples a t 3-12 weeks by Student's t - t e s t (a = 0.05). -56- 3 10 4— 1 1 • • 1 - — " r- 0 2 4 6 8 10 12 Time (weeks) Figure 2-5: Aerobic plate counts of Jersey blueberries in three storage conditions (1986). Curves 1: a i r , 2: intermediate oxygen, 3: low oxygen atmosphere. Points represented means of 2 samples. Geometric means of a i r and intermediate oxygen samples were s i g n i f i c a n t l y d i f f e r e n t only at 8-12 weeks by Student's t-test (a = 0.05). Means of intermediate and a i r samples were s i g n i f i c a n t l y d i f f e r e n t from means of low oxygen samples at 3-12 weeks by Student's t - t e s t (a = 0.05). -57- 1 0 4 J 1 • . . , , r - 0 2 4 6 8 10 12 Time (weeks) F i g u r e 2-6: Ae r o b i c p l a t e counts of Bluecrop b l u e b e r r i e s i n two storage c o n d i t i o n s (1987). Curves 1: intermediate oxygen, 2: low oxygen atmosphere. P o i n t s represented means of 4 samples. Means of intermediate oxygen samples were s i g n i f i c a n t l y d i f f e r e n t from means of low oxygen samples a t 4-12 weeks by Student's t - t e s t (a = 0.05). -58- b i o s y n t h e s i s of c e l l u l a r components. F a c u l t a t i v e a e r o b i c b a c t e r i a were i n h i b i t e d by the high carbon d i o x i d e l e v e l of t h i s storage c o n d i t i o n . 2.1.5.3 Yeast and mold counts The use of p y r u v a t e - a n t i b i o t i c - s u p p l e m e n t e d medium f o r yeast and mold counts was d i s c o n t i n u e d a f t e r 8 weeks of the 1986 t r i a l s , because no s i g n i f i c a n t b e n e f i t over the standard a n t i b i o t i c - s u p p l e m e n t e d medium was found by p a i r e d t - t e s t at the 95% confidence l e v e l . Yeast and mold counts of b l u e b e r r i e s from the intermediate oxygen packages reached a p l a t e a u at lower counts than counts of samples i n a i r ( F i g u r e s 2-7 and 2- 8), due to the s e n s i t i v i t y of molds to carbon d i o x i d e and low oxygen atmosphere. S v i r c e v e_t al_. (1984) r e p o r t e d that a t 7.5% carbon d i o x i d e (17.5% oxygen) germ tubes of B. c i n e r e a were c o n s i d e r a b l y d i s t o r t e d and s h o r t e r than germ tubes developed i n a i r . At 4% oxygen, m y c e l i a l growth of B. c i n e r e a was o n l y 45%, and t h a t of A. t e n u i s was 31% compared with growth i n a i r (Wells and Uota, 1970). Under carbon d i o x i d e and the low oxygen atmosphere of intermediate oxygen s t o r a g e , m y c e l i a l growth and s p o r u l a t i o n of molds would be l e s s than growth and s p o r u l a t i o n i n a i r . T h i s d i f f e r e n c e e x p l a i n e d the smaller -59- 103-! , , , , , r - 0 2 4 6 8 10 12 Time (weeks) F i g u r e 2-7: Yeast and mold counts of Bluecrop b l u e b e r r i e s i n three storage c o n d i t i o n s (198G). Curves 1: a i r , 2: intermediate oxygen, 3: low oxygen atmosphere. P o i n t s r e p r e s e n t means of two samples. Geometric means of a i r and intermediate oxygen samples are not d i f f e r e n t by Student's t - t e s t (a = 0.05). Means of a i r and intermediate oxygen samples were d i f f e r e n t from means of low oxygen samples d u r i n g 2-12 weeks by Student's t - t e s t (a = 0.05) . -60- V Time (weeks) F i g u r e 2-8: Yeast and mold counts of J e r s e y b l u e b e r r i e s i n three storage c o n d i t i o n s (1986). Curves 1: a i r , 2: intermediate oxygen, 3: low oxygen atmosphere. P o i n t s r e p r e s e n t means of two samples. Geometric means of a i r and intermediate oxygen samples are s i g n i f i c a n t l y d i f f e r e n t between 4-12 weeks by Student's t - t e s t (a = 0.05). Means of a i r and intermediate oxygen samples were d i f f e r e n t from means of low oxygen samples d u r i n g 1- 12 weeks by Student's t - t e s t (a = 0.05). -61- yeast and mold counts from b l u e b e r r i e s s t o r e d i n the i n t e r m e d i a t e oxygen environment than from b l u e b e r r i e s s t o r e d i n a i r . However, there was no d i f f e r e n c e between these counts i n a t r i a l with Bluecrop (mid-season) b e r r i e s ( F i g u r e 2-7). In the t r i a l where d i f f e r e n c e s of yeast and mold counts e x i s t e d between a i r and intermediate oxygen s t o r a g e , J e r s e y ( l a t e season) v a r i e t y was used. The d i s c r e p a n c y between two t r i a l s may be due to d i f f e r e n t mold s p e c i e s making up the f l o r a present on the s u r f a c e of b l u e b e r r i e s . C. g l o e o s p o r i o i d e s , B. c i n e r e a , and A. t e n u i s were the predominant molds i s o l a t e d from b l u e b e r r i e s grown i n New J e r s e y , U.S. A ( C a p p e l l i n i e_t a l . , 1972). Those authors r e p o r t e d an i n t e r e s t i n g r e l a t i o n s h i p between these organisms: i n f e c t i o n s by these molds occurred a t almost equal f r e q u e n c i e s i n b e r r i e s picked e a r l y i n the season, but d u r i n g l a t e season the o c c u r r i n g frequency of C. g l o e o s p o r i o i d e s was much higher than B. c i n e r e a and A. t e n u i s . A d i r e c t comparison between r e s u l t s obtained from b l u e b e r r i e s grown i n New J e r s e y and l o c a l b l u e b e r r i e s i s i n a p p r o p r i a t e ; n e v e r t h e l e s s , a s i m i l a r s h i f t of predominant s p e c i e s might a l t e r r e s p e c t i v e s p e c i e s numbers and c o n t r i b u t e to d i f f e r e n t growth trends of f u n g i observed for Bluecrop and J e r s e y b l u e b e r r i e s . -62- Yeast and mold counts from low oxygen b l u e b e r r y samples were low compared to counts from b l u e b e r r i e s s t o r e d i n intermediate oxygen environment (Figure 2-9). M y c e l i a l growth and s p o r u l a t i o n of molds were i n h i b i t e d by high carbon d i o x i d e and low oxygen environment ( S v i r c e v e_t a l . , 1984; Wells and Uota, 1970; F o l l s t a d , 1966). In a l l three storage t r i a l s , b l u e b e r r i e s i n the low oxygen atmosphere remained f r e e of mold i n f e c t i o n throughout 12 weeks. Yeast growth was expected s i n c e some yeast s p e c i e s have the a b i l i t y to ferment sugars under anaerobic c o n d i t i o n . However, no in c r e a s e was observed i n yeast and mold counts up to 6-8 weeks of st o r a g e . T h i s o b s e r v a t i o n suggested the presence of an i n h i b i t o r y mechanism. Since b e r r y pH remained s t a b l e d u r i n g s t o r a g e , growth i n h i b i t i o n by pH i s not a p p l i c a b l e . A s m a l l i n c r e a s e of yeast growth was observed a f t e r 8 weeks. T h i s i n c r e a s e showed a re c o v e r y process, and must be co n s i d e r e d i n r e l a t i o n to the mechanism of yeast i n h i b i t i o n i n low oxygen s t o r a g e . 2.2 IDENTIFICATION OF YEASTS ISOLATED FROM BLUEBERRY F i v e yeast s p e c i e s were i s o l a t e d from l o c a l b l u e b e r r i e s . I d e n t i f i c a t i o n to the genus l e v e l was c a r r i e d out us i n g macroscopic and m i c r o s c o p i c morphology, a b i l i t y to ferment sugars, a b i l i t y to u t i l i z e v a r i o u s compounds as s o l e source of carbon, the mode of v e g e t a t i v e -63- 103-l i • - 1 1 r 0 2 4 6 8 10 12 Time (weeks) F i g u r e 2-9: Yeast and mold counts of Bluecrop b l u e b e r r i e s i n two storage c o n d i t i o n s (1987). Curves 1: intermediate oxygen, 2: low oxygen atmosphere. P o i n t s r e p r e s e n t means of 4 samples. Geometric means of intermediate oxygen samples were d i f f e r e n t from means of low oxygen samples d u r i n g 2-12 weeks by Student's t - t e s t (a = 0.05). -64- and sexual reproduction, production of ammonia from urea, production of e x t r a c e l l u l a r amyloid compounds, and growth at 37°C (Van der Walt and Yarrow, 1984). The procedures and results of these tests are presented in the Appendix. I d e n t i f i c a t i o n was based on genus c h a r a c t e r i s t i c s reported in the l i t e r a t u r e (Kregger Van R i j , 1984; Barnett et al.., 1983). The yeast species were i d e n t i f i e d as two Rhodotorula species, a Zygosaccharomyces species, a Cryptococcus species, and a Debaryomyces species. 2.3 CONCLUSIONS 1. The packaging f i l m with intermediate gas permeability allowed some outward d i f f u s i o n of carbon dioxide produced by berry r e s p i r a t i o n . As a r e s u l t , the atmosphere within the storage packages remained aerobic with a r e l a t i v e l y low carbon dioxide l e v e l . Yeast, mold and b a c t e r i a l growth were vigorous in the early part of storage, and were somewhat inhibited by oxygen l i m i t a t i o n after 6 weeks of storage. This MAP storage condition could not suppress spoilage of berries, e s p e c i a l l y mold rot. 2. Packaging blueberries in a f i l m with very low gas permeability created a high carbon dioxide and almost - 6 5 - anaerobic atmosphere. Oxygen was r a p i d l y consumed by a e r o b i c r e s p i r a t i o n of b e r r i e s w i t h i n two weeks of s t o r a g e . In an almost anaerobic atmosphere, b l u e b e r r i e s began anaerobic r e s p i r a t i o n to generate energy with e t h a n o l , carbon d i o x i d e , and p o s s i b l y l a c t i c a c i d as end-products. Anaerobic r e s p i r a t i o n r a i s e d the carbon d i o x i d e l e v e l to about 70% of the headspace atmosphere at the end of the storage t r i a l . T h i s packaging f i l m and storage temperature s u c c e s s f u l l y i n h i b i t e d b l u e b e r r y s p o i l a g e . Molds, o b l i g a t e and f a c u l t a t i v e a e r o b i c b a c t e r i a could not grow i n an almost anaerobic atmosphere with a high carbon d i o x i d e content. Yeast growth was a l s o r e t a r d e d , even though some s p e c i e s can generate energy v i a fermentation. I n h i b i t i o n by low pH i s not a p p l i c a b l e because berry pH remained s t a b l e and w i t h i n the growth range throughout s t o r a g e . The mechanism of yeast i n h i b i t i o n i n MAP storage m e r i t s f u r t h e r r e s e a r c h . 3 . F i v e yeast s p e c i e s i s o l a t e d from b l u e b e r r y were i d e n t i f i e d as two Rhodotorula s p e c i e s , a Zyqosaccharomyces s p e c i e s , a Cryptococcus s p e c i e s , and a Debaryomyces spec i e s . - 6 6 - CHAPTER 3: EXPERIMENTATION, RESULTS AND DISCUSSION: DETECTION OF ANTIFUNGAL ACTIVITY IN BLUEBERRIES STORED UNDER A MODIFIED ATMOSPHERE. EFFECTS OF TEMPERATURE AND CARBON DIOXIDE ON YEAST GROWTH. - 6 7 - 3.0 PURPOSE OF EXPERIMENTS Th i s r e s e a r c h phase c l a r i f i e d the p o s s i b l e presence of p h y t o a l e x i n s i n b l u e b e r r i e s , and the e f f e c t s of low temperature, high carbon d i o x i d e on yeast growth. The d i s k d i f f u s i o n method was used to d e t e c t presence of p h y t o a l e x i n s i n b l u e b e r r i e s h e l d i n MAP s t o r a g e . Yeast growth i n b l u e b e r r y j u i c e under d i f f e r e n t temperatures and atmosphere compositions was s t u d i e d as a model of yeast behaviour during MAP s t o r a g e . 3.1 DISK DIFFUSION ASSAYS 3.1.1 MATERIALS AND METHODS B l u e b e r r i e s from low-oxygen packages were used a f t e r two, three and ten weeks of MAP s t o r a g e . A l l b e r r i e s from a storage package (approximately 50 g) were homogenized i n a Waring blender ( h a l f speed, 1 min), and c e n t r i f u g e d (4,080 x g, 15 min, 4°C) to separate j u i c e and pulp. J u i c e and pulp were f r e e z e - d r i e d and e x t r a c t e d with a s e r i e s of s o l v e n t s : hexanes, e t h a n o l , and water. Water e x t r a c t i o n was not necessary f o r the pulp p o r t i o n , because water-soluble compounds would be present i n b e r r y j u i c e . In each e x t r a c t i o n , 2 ml of a s o l v e n t was added to f r e e z e - d r i e d r esidue ( j u i c e or pulp) and s t i r r e d with a glass rod for f i v e minutes. Three s u c c e s s i v e e x t r a c t i o n s y i e l d e d -68- 6 ml of each solvent ex trac t . When extract ions with a solvent were completed, berry residues were dr ied under flowing nitrogen at room temperature before addi t ion of another so lvent . Freeze-dr ied residue of blueberry ju ice (from a 50 g of berr ies ) was a lso reconst i tuted with 6 ml of water, and subsequently used in the assays as unfractionated j u i c e . Berr ies from r e p l i c a t e storage packages were extracted separate ly . Each solvent extract from berr ies of each package was tested independently and also as a pooled sample. The pooled sample was made up by pooling extracts from berr ies from two or four rep l icated packages, and the f i n a l volume was reduced under flowing nitrogen to the volume of a s ingle sample. If phytoalexin concentrations in extracts from an i n d i v i d u a l package were below l eve l s necessary for yeast i n h i b i t i o n , then pooling of extracts from r e p l i c a t e packages increased their concentrat ions, so that yeast i n h i b i t i o n could be detected. Blueberries which had not undergone MAP storage were treated by the same procedure, and the i r extracts served as controls in the assays. The yeast s t r a i n s i so lated from blueberries were test organisms. Yeast cul tures were grown in a 250 ml- Erlenmeyer f lask containing 100 ml of Trypt icase Soy broth (Difco , D e t r o i t , MI). The cul ture flasks were incubated - 6 9 - o v e r n i g h t a t 21°C, i n a shaking incubator (80 rpm, New Brunswick S c i e n t i f i c Co., E d i s o n , NJ). The overnight c u l t u r e s were then c o l l e c t e d by c e n t r i f u g a t i o n (7,710 x g, 15 min, 21°C), and resuspended i n s t e r i l e 0.1% peptone water. The yeast i n o c u l a were a d j u s t e d to c o n t a i n approximately 10 s colony-forming u n i t s (cfu)/ml using standard curves (see Appendix). The t e s t medium was prepared by a c i d i f y i n g Yeast Morphology agar (Shadomy and E s p i n e l - I n g r o f f , 1980) to pH 3.5 with c i t r i c a c i d to achieve the average pH of b l u e b e r r i e s . C i t r i c a c i d was r e p o r t e d to be the major o r g a n i c a c i d i n b l u e b e r r i e s (Meyer, 1968; Markakis et a l . , 1963). A s t e r i l e c o t t o n swab dipped i n a yeast inoculum was used to i n o c u l a t e agar p l a t e s . Swabbing of the agar s u r f a c e was repeated three times, with a 60-degree r o t a t i o n of the t e s t p l a t e each time, to ensure an even d i s t r i b u t i o n of inoculum. A l l s o l v e n t e x t r a c t s and u n f r a c t i o n a t e d j u i c e were f i l t e r s t e r i l i z e d . P e c t i n a s e (Sigma, S t . L o u i s , MO) was added to u n f r a c t i o n a t e d j u i c e and the water e x t r a c t at 2% (w/v) f i n a l c o n c e n t r a t i o n (10 min, room temperature) to f a c i l i t a t e f i l t e r s t e r i l i z a t i o n . F i l t e r s made of p o l y v i n y l i d e n e d i f l u o r i d e polymer (0.45 p\m, M i l l i p o r e C o r p o r a t i o n , Bedford, MA) were used. High absorbency paper d i s k s (6.4 mm diameter, S c h l e i c h e r & S c h n e l l , Keene, NH) - 7 0 - were dipped i n e x t r a c t s , and so l v e n t s were allowed to evaporate before the d i s k s were placed on the i n o c u l a t e d agar s u r f a c e . The procedures of berry e x t r a c t i o n and d i s k d i f f u s i o n assay are o u t l i n e d i n Figure 3-1. Each t e s t p l a t e contained d i s k s of pulp e x t r a c t s (hexanes, e t h a n o l ) , j u i c e e x t r a c t s (hexanes, e t h a n o l , water), and u n f r a c t i o n a t e d j u i c e . E x t r a c t s of b l u e b e r r i e s which had not undergone MAP storage were i n c l u d e d on the c o n t r o l p l a t e s ; d i s k s c o n t a i n i n g s t e r i l e hexanes and etha n o l were used as blanks. The p l a t e s were incubated under four c o n d i t i o n s : 21°C-air; 4°C-air; 21°C-carbon d i o x i d e ; 4°C-carbon d i o x i d e . The l a s t two c o n d i t i o n s were e s t a b l i s h e d by packaging p l a t e s i n the h i g h - b a r r i e r f i l m used i n MAP storage (see Table 2-1) with a i r evacuation and carbon d i o x i d e b a c k f l u s h . pH of the agar medium d i d not decrease upon exposure to the carbon d i o x i d e atmosphere. 3.1.2 RESULTS AND DISCUSSION The in. v i t r o assays were designed to approximate i n v i v o c o n d i t i o n s of b l u e b e r r i e s held i n MAP s t o r a g e . The agar medium was a d j u s t e d to the average pH of b l u e b e r r i e s (see Table 2 - 4 ) . A high carbon d i o x i d e , low temperature i n c u b a t i o n was a l s o i n c l u d e d . Since the s t r u c t u r e s and c h a r a c t e r i s t i c s of p o t e n t i a l phytoalexins i n b l u e b e r r i e s -71- BLUEBERRIES (2, 3, 10 WEEKS MAP STORAGE) HOMOGENIZATION I CENTRIFUGATION (4,080 x g, 15 MIN) JUICE FREEZE DRYING RECONSTITUTION WATER EXTRACTIONS HEXANES ETHANOL WATER DISCS PULP FREEZE DRYING EXTRACTIONS HEXANES ETHANOL DISCS SOLVENT EVAPORATION PLATES WITH YEAST LAWNS INCUBATION 21°C - AIR 40C - AIR 21°C - C0 2 4°C - CO* Fi g u r e 3-1: General procedures of b l u e b e r r y e x t r a c t i o n and dis k d i f f u s i o n assay. -72- were unknown, the use of d i f f e r e n t solvents for their extraction and concentration was necessary. A l l solvents were evaporated before deposition of disks on agar, to eliminate growth i n h i b i t i o n by solvents. Remaining solutes would be somewhat soluble in the agar medium since they existed in the aqueous environment of berry tissues. Growth of yeast species varied under d i f f e r e n t temperatures and atmosphere compositions. No i n h i b i t i o n was observed with any yeast species and any extract tested. Good growth of a l l isolates occurred in 21°C-air and 4°C-air incubations to produce confluent lawns on agar plates (Figures 3-2, 3-3). Two Rhodotorula species and a Cryptococcus species f a i l e d to grow under carbon dioxide incubation (Figure 3-4), probably because they could only u t i l i z e sugars a e r o b i c a l l y . Since these assays were carried out before i d e n t i f i c a t i o n of yeast i s o l a t e s , their i n a b i l i t y to ferment sugars was unknown. Only the Zygosaccharomyces s t r a i n and the Debaryomyces s t r a i n grew during 21°C-carbon dioxide incubation, because these isolates could ferment sugars under anaerobic conditions. Growth of a l l yeast isolates was inhibited in the 4°C- carbon dioxide incubation. The results of disk d i f f u s i o n assays showed an absence of antifungal a c t i v i t y in blueberries as tested against - 7 3 - Figure 3-2: Disk d i f f u s i o n assay using a Rhodotorula species in 210C-air incubation. The agar surface was covered with yeast growth. An i n h i b i t i o n zone was absent around a l l disks containing berry extracts. -74- Figure 3-3: Disk d i f f u s i o n assay using a Rhodotorula species in 4<>C-air incubation. The agar surface was covered with yeast growth. An i n h i b i t i o n zone was absent around a l l disks containing berry extracts . - 7 5 - F l g u r e 3 - 4 : D i s k d i f f u s i o n a s s a y u s i n g a R h o d o t o r u l a s p e c i e s i n 4 ° C - c a r b o n d i o x i d e i n c u b a t i o n . L a w n o f g r o w t h w a s a b s e n t o n t h e a g a r s u r f a c e . -76- the f i v e yeast s t r a i n s , and i n d i c a t e d t h a t i n h i b i t i o n of yeast growth was due to low temperature, high carbon d i o x i d e l e v e l and anaerobic c o n d i t i o n s . 3.2 EFFECTS OF CARBON DIOXIDE AND LOW TEMPERATURE ON YEAST GROWTH The d i s k d i f f u s i o n assays i n d i c a t e d i n h i b i t i o n of yeast growth under carbon d i o x i d e and low temperature. T h i s i n h i b i t o r y e f f e c t was s t u d i e d using n a t u r a l f l o r a of b l u e b e r r y j u i c e and yeast i s o l a t e s grown i n s t e r i l i z e d j u i c e a t d i f f e r e n t temperatures and atmospheres. 3.2.1 MATERIALS AND METHODS 3.2.1.1 Studie s using c u l t u r e s of yeast i s o l a t e s Two yeast i s o l a t e s Zygosaccharomyces sp. and Debaryomyces sp. were s e l e c t e d f o r t h i s study because they can ferment sugars when oxygen i s absent. Each yeast were grown i n a 250 ml-Erlenmeyer f l a s k c o n t a i n i n g 100 ml of T r y p t i c a s e Soy broth ( D i f c o , D e t r o i t , MI). The c u l t u r e f l a s k s were incubated (21°C, 48 hours) i n a shaking incubator (80 rpm, New Brunswick S c i e n t i f i c Co., Edison, NJ). C e l l s were harvested by c e n t r i f u g a t i o n (7,710 x g, 15 min, 21°C), and washed by suspending i n 0.1% peptone water. D i l u t i o n s were prepared to o b t a i n i n o c u l a of approximately 1 0 e cfu/ml. The t e s t medium was s t e r i l i z e d b l u e b e r r y j u i c e . -77- Homogenized b l u e b e r r i e s were c e n t r i f u g e d (4080 x g, 15 min, 21°C) f o r j u i c e c o l l e c t i o n . P e c t i n a s e (Sigma Chemicals, S t . L o u i s , MO) was added to j u i c e a t the 2% (w/v) f i n a l c o n c e n t r a t i o n to f a c i l i t a t e s t e r i l i z i n g f i l t r a t i o n . P e c t i n a s e treatment was c a r r i e d out a t room temperature, f o r 15 minutes with o c c a s i o n a l s t i r r i n g . J u i c e was f u r t h e r c l a r i f i e d by a s e r i e s of f i l t r a t i o n s u s i n g Whatman No.4, No.2, and No.5 f i l t e r s , s u c c e s s i v e l y . T h i s f i l t r a t i o n s e r i e s enhanced the speed of subsequent f i l t e r s t e r i l i z a t i o n (0.45 Hm, mixed e s t e r s of c e l l u l o s e , M i l l i p o r e C o r p o r a t i o n , Bedford, MA). F i f t y m i l l i l i t e r - a l i q u o t s of s t e r i l i z e d j u i c e were a s e p t i c a l l y dispensed to s t e r i l i z e d Square-Pak f l a s k s (100 ml, American S t e r i l i z e r Co., E r i e , PA), which were covered with g a s - t i g h t rubber caps. The headspace of each f l a s k (ca 50 ml) was f l u s h e d with a gas mixture (25% carbon d i o x i d e 75% n i t r o g e n , or 100% carbon d i o x i d e ) f o r 15 minutes at the r a t e of 50 ml/min. The gas mixtures were s t e r i l i z e d by hydrophobic gas f i l t e r s (0.3 Um, Gelman Sciences Inc., Ann Arbor, MI) before e n t e r i n g i n t o c u l t u r e f l a s k s . T h i s procedure i s i l l u s t r a t e d i n F i g u r e 3-5. The c o n t r o l f l a s k s were not f l u s h e d to maintain a i r i n the headspace. F l a s k s were incubated a t e i t h e r 4<>C or 21°C to achieve the f o l l o w i n g c o n d i t i o n s : - 7 8 - 2[ 2C 3 T 3»T 4 4 C 21C F i g u r e 3-5: F l u s h i n g of c u l t u r e f l a s k s with a gas mixture. Numbers 1: gas tubes, 2: s t e r i l i z i n g f i l t e r s , 3: a i r - t i g h t caps, 4: s t e r i l i z e d needles, 5: b l u e b e r r y j u i c e . Arrow heads p o i n t to d i r e c t i o n of gas flow. - 7 9 - 210C: a i r 4°C: a i r 25% carbon d i o x i d e 25% carbon d i o x i d e 100% carbon d i o x i d e 100% carbon d i o x i d e For the 21°C c o n d i t i o n , f l a s k s were incubated i n a o r b i t a l shaking incubator (80 rpm, New Brunswick S c i e n t i f i c Co., Edis o n , NJ), and f o r the 4<>C i n c u b a t i o n , an o r b i t a l shaking bath (80 rpm, Lab Lin e Instrument Inc., Melrose Park, IL) was used. A f t e r a sh o r t i n c u b a t i o n p e r i o d (30 min) to e s t a b l i s h d e s i r e d j u i c e temperatures, 0.5 ml of yeast inoculum was i n j e c t e d i n t o each f l a s k u s ing s t e r i l e needles and s y r i n g e s (Becton and Dickson, Rutherford, NJ). A f t e r i n o c u l a t i o n , d u p l i c a t e samples were taken from each f l a s k (time 0). Sampling was c a r r i e d out every 24 hours f o r three days. A l l samples were taken a t a point w e l l below the j u i c e s u r f a c e . J u i c e samples were plated u sing the Hydrophobic G r i d Membrane f i l t e r (QA L a b o r a t o r i e s , Toronto, Ont.) and P l a t e Count agar ( D i f c o , D e t r o i t , MI) which contained 0.01% Trypan Blue dye (Sigma Chemicals, St. L o u i s , MO). Use of a n t i b i o t i c s i n t h i s medium to suppress b a c t e r i a l growth was not necessary when working with pure yeast c u l t u r e s . U n i n o c u l a t e d j u i c e i n "dummy" f l a s k s (100% carbon d i o x i d e , 21°C or 4°C) d i d not show any change i n pH du r i n g - 8 0 - incubat i o n . 3.2.1.2 Studie s u s i n g n a t u r a l yeast and mold f l o r a of bl u e b e r r i e s J u i c e was prepared by the procedure d e s c r i b e d i n the preceding s e c t i o n , except for a few minor changes. Since j u i c e s t e r i l i t y was not d e s i r e d , p e c t i n a s e and f i l t e r s t e r i l i z a t i o n were omitted, and onl y Whatman No.4 f i l t e r was used f o r j u i c e c l a r i f i c a t i o n . The sampling and p l a t i n g procedures were i d e n t i c a l to those used i n s t u d i e s with pure c u l t u r e s . However, the p l a t i n g medium trypan blue-PCA was supplemented with 0.01% chloramphenicol (Sigma Chemicals, St. L o u i s , MO) and 0.01% c h l o r t e t r a c y c l i n e HC1 (Sigma Chemicals, St. L o u i s , MO) to suppress b a c t e r i a l growth on the HGMF. 3.2.2 RESULTS AND DISCUSSION 3.2.2.1 Studie s with n a t u r a l f l o r a In a mixed p o p u l a t i o n of yeast and mold f l o r a , d i f f e r e n t temperatures l i k e l y i n h i b i t growth of some s p e c i e s , t h e r e f o r e , the growth data from 4°C and 21°C should be t r e a t e d s e p a r a t e l y . At 21°C (Figure 3-6), mold growth was i n h i b i t e d under carbon d i o x i d e atmosphere, so t h a t counts of samples i n 25% and 100% carbon d i o x i d e i n c u b a t i o n s r e p r e s e n t e d yeast growth o n l y . Under ana e r o b i c c o n d i t i o n , yeasts could ferment sugars to supply -81- 10 3~l • r 0 1 2 3 Time (days) F i g u r e 3-6: Yeast and mold p o p u l a t i o n i n b l u e b e r r y j u i c e a t 21oc. Curves 1: a i r , 2: 25% C0 =, 3: 100% CO =. P o i n t s are means of two counts. Counts of samples i n a i r were s i g n i f i c a n t l y d i f f e r e n t from counts of 25% C0=> or 100% C 0 2 samples by Student's t - t e s t (a = 0.05). Counts of samples i n 25% C O z were s i g n i f i c a n t l y d i f f e r e n t from counts of samples i n 100% C 0 3 by Student's t - t e s t (a = 0.05). -82- energy and metabolic p r e c u r s o r s . An increase of carbon d i o x i d e l e v e l to 100% a p p a r e n t l y caused a decrease i n yeast growth, compared to growth i n 25% carbon d i o x i d e . These r e s u l t s i n d i c a t e d that some yeast s t r a i n s i n the b l u e b e r r y m i c r o f l o r a were s e n s i t i v e but some s t r a i n s were r e s i s t a n t to a high carbon d i o x i d e atmosphere, s i n c e growth of f l o r a was not completely i n h i b i t e d i n the 21°C- 100% carbon d i o x i d e c o n d i t i o n . S e v e r a l r e p o r t s have been p u b l i s h e d on the e f f e c t of carbon d i o x i d e on yeast growth, but the data were obtained under c o n d i t i o n s such as high carbon d i o x i d e pressures (Kunkee and Ough, 1966; Pe r i g o e_t a l . , 1964), high sugar contents i n media (Witter et a l . , 1958), and l i m i t i n g - s u b s t r a t e c o n c e n t r a t i o n s (Chen and Gutmans, 1976), so t h a t no comparison with t h i s study c o u l d be made. At 4°C and under 25% or 100% carbon d i o x i d e atmosphere, yeast and mold counts showed no in c r e a s e from i n i t i a l counts (Figure 3-7), which confirmed e a r l i e r r e s u l t s of low yeast and mold counts dur i n g low- oxygen storage of b l u e b e r r i e s . R e s u l t s of t h i s study (4°C) i n d i c a t e d t h a t the high carbon d i o x i d e l e v e l at 12 weeks of low-oxygen storage (68.96%) may not have g r e a t e r e f f e c t on yeast and mold f l o r a than i t s e f f e c t a t two weeks (46.30%, see Table 2-2). I n h i b i t i o n of yeast and mold f l o r a i n the 4<>c-25% CO=> and 4<>C-100% CO=» -83- 103-l • • - 0 1 2 3 Time (days) F i g u r e 3-7: Yeast and mold p o p u l a t i o n i n b l u e b e r r y j u i c e a t 4<>c. Curves 1: a i r , 2: 25% CO^, 3: 100% C0 2. P o i n t s r e p r e s e n t means of two counts. Counts of the sample i n a i r were s i g n i f i c a n t l y d i f f e r e n t from counts of samples i n 25% C 0 = or 100% CO= by Student's t - t e s t (a = 0.05). -84- environments was probably the combined e f f e c t s of low temperature, high carbon d i o x i d e and anaerobic atmosphere. 3.2.2.2 Studie s with yeast i s o l a t e s Growth of Zygosaccharomyces sp. and Debaryomyces sp. under d i f f e r e n t temperature and atmosphere combinations are presented i n F i g u r e s 3-8 and 3-9. At 21<>C, a growth l a g occurred i n the 25% and 100% carbon d i o x i d e environments, as compared to growth i n a i r . In the absence of oxygen, yeasts can d e r i v e l e s s energy v i a ferm e n t a t i o n than by a e r o b i c u t i l i z a t i o n of sugars. Since carbon d i o x i d e i s a product of d e c a r b o x y l a t i o n r e a c t i o n s , high carbon d i o x i d e c o n c e n t r a t i o n s w i l l a f f e c t r e a c t i o n e q u i l i b r i a , and the r a t e s of enzymatic d e c a r b o x y l a t i o n . When t h i s occurred, the d e c a r b o x y l a t i o n r e a c t i o n s such as those involved i n the t r i c a r b o x y l i c a c i d c y c l e would become r a t e - l i m i t i n g steps i n c e l l u l a r metabolism, i f other enzymes were o p e r a t i n g at normal r a t e s , and the e f f e c t of high carbon d i o x i d e would be slow growth (King and Nagel, 1975; Kritzman e_t a l . , 1977). Slow growth of Zygosaccharomyces sp. and Debaryomyces sp. under 25% or 100% carbon d i o x i d e atmosphere was probably the r e s u l t of both high carbon d i o x i d e and i n e f f i c i e n t energy g e n e r a t i o n i n absence of oxygen. These two e f f e c t s -85- 1 0 4_| , , r 0 1 2 3 Time (days) F i g u r e 3-8: Growth of a Zygosaccharomyces s p e c i e s i n b l u e b e r r y j u i c e . Curves 1: 21o c - a i r , 2: 210C-25% C 0 2 / 3: 21<>C-100% C0 a, 4: 4<>C-air, 5: 4<>c-25% C0 2, 6: 4°C-100% COa. P o i n t s are means of two counts. Sample means were compared by Duncan's M u l t i p l e Range t e s t (a = 0.05): counts of 21°C-air sample were s i g n i f i c a n t l y d i f f e r e n t from counts of other samples; counts of 21°C-25% C 0 2 sample, 21<>C-100% C 0 2 sample and 4°C-air sample were not d i f f e r e n t from one another, but they were s i g n i f i c a n t l y d i f f e r e n t from counts of 4<>c-25% CO=. and 4°C-100% C 0 2 samples a t 72 hours. -86- Time (days) F i g u r e 3-9: Growth of a Debaryomyces s p e c i e s i n b l u e b e r r y j u i c e . Curves 1: 21°C-air, 2: 21°C-25% C0=>, 3: 21<>c-100% C O a , 4: 4<>C-air, 5: 40C-25% C 0 2 / 6: 4<>C-100% C0=>. P o i n t s are means of two counts. Sample means were compared by Duncan's M u l t i p l e Range t e s t (a = 0.05): counts of 21<>C- a i r sample were s i g n i f i c a n t l y d i f f e r e n t from counts of other samples; counts of 21<>C-25% C 0 2 sample, 21°C-100% C O a sample and 4°C-air sample were not d i f f e r e n t from one another, but they were s i g n i f i c a n t l y d i f f e r e n t from counts of 4<>C-25% C0=> and 4<>C-100% C O z samples a t 72 hours. -87- c o u l d not be d i s t i n g u i s h e d under the c o n d i t i o n s of t h i s exper iment. Low temperature had a marked e f f e c t on yeast s p e c i e s : growth i n a i r a t 4 ° C was lower than a t 2 1 ° C / and growth was i n h i b i t e d under the 2 5 % carbon d i o x i d e atmosphere. S o l u b i l i t y of carbon d i o x i d e i n c r e a s e d a t low temperature and c o n t r i b u t e d to growth i n h i b i t i o n . However, the composition and f u n c t i o n a l i t y of yeast membranes might be the key f a c t o r a f f e c t e d by low temperatures and might e x p l a i n the i n h i b i t i o n of yeast growth i n MAP st o r a g e . Yeasts g e n e r a l l y have a high p r o p o r t i o n of unsaturated f a t t y a c i d s i n membrane l i p i d s when grown a t low temperatures (Kates and Baxter, 1962; Arthur and Watson, 1976). P h o s p h o l i p i d molecules, which have a g l y c e r o l - 3 - phosphate backbone a c y l a t e d with f a t t y a c i d s a t carbons 1 and 2, form the b i l a y e r s t r u c t u r e of b i o l o g i c a l membranes. Some membrane p h o s p h o l i p i d s may be s p e c i f i c a l l y a s s o c i a t e d with membrane p r o t e i n s and are e s s e n t i a l f o r t h e i r a c t i v i t i e s . The e n t i r e membrane s t r u c t u r e i s dynamic r a t h e r than s t a t i c , with most components capable of l a t e r a l d i f f u s i o n and r o t a t i o n a l motion about an a x i s p e r p e n d i c u l a r to the b i l a y e r plane. R o t a t i o n r a t e i n a l i p i d b i l a y e r i s a f u n c t i o n of both temperature and membrane composition. Depending on temperature, membranes - 8 8 - may pass from a g e l phase, i n which the f a t t y a c y l chains of p h o s p h o l i p i d s are h i g h l y ordered, to a l i q u i d phase in which the f a t t y a c y l chains are more mobile. T h i s phase t r a n s i t i o n process ( i l l u s t r a t e d i n F i g u r e 3-10a) i s accompanied by i n c r e a s e d r o t a t i o n a l motion about the carbon-carbon bonds of the hydrocarbon chains of p h o s p h o l i p i d s , which a l l o w s the hydrocarbon chains to assume random conformations. In g e n e r a l , unsaturated f a t t y a c y l chains undergo phase t r a n s i t i o n a t lower temperatures than s a t u r a t e d f a t t y a c i d s . T h i s i s because u n s a t u r a t i o n produces a "kink" i n the f a t t y a c y l chains which leads to more d i s o r d e r i n the b i l a y e r than s a t u r a t e d chains (Figure 3-10b). T h e r e f o r e , the p r o p o r t i o n of unsaturated f a t t y a c i d s w i l l a f f e c t the f l u i d i t y ( d e f i n e d as the i n v e r s e of v i s c o s i t y ) of membranes a t a given temperature. Arthur and Watson (1976) examined the membrane l i p i d c omposition of p s y c h r o p h i l i c (Leucospor i d ium f r i g idum, growth temperature l i m i t -2°C to 20°C), m e s o p h i l i c (Cand ida l i p o l y t i c a , temperature l i m i t 5° to 35°C), and t h e r m o p h i l i c ( T o r u l o p s i s bovina, temperature l i m i t 25° to 45°C) y e a s t s , and r e p o r t e d t h a t s a t u r a t e d f a t t y a c i d s composed 30-40% of membrane l i p i d of t h e r m o p h i l i c y e a s t s , whereas the p s y c h r o p h i l i c yeast contained approximately 90% unsaturated f a t t y a c i d s , 53% of which was l i n o l e n i c -89- F i g u r e 3-10: A model of phase t r a n s i t i o n of f a t t y a c i d s , (a) Changes i n p h o s p h o l i p i d b i l a y e r d u r i n g phase t r a n s i t i o n , (b) I n t r o d u c t i o n of a double bond i n t o a f a t t y a c y l c h a i n r e s u l t s i n an i n f l e x i b l e kink i n the p h o s p h o l i p i d t a i l , compared with a p h o s p h o l i p i d c o n t a i n i n g o n l y s a t u r a t e d f a t t y a c i d s (adapted from Jacobson and S a i e r , 1983). - 9 0 - a c i d ( T a b l e 3 - 1 ) . T h e s e a u t h o r s p r o p o s e d t h a t d e g r e e o f membrane l i p i d u n s a t u r a t i o n may be a n i m p o r t a n t f a c t o r w h i c h d e t e r m i n e s y e a s t a b i l i t y t o g r o w a t l o w t e m p e r a t u r e s . T h e h i g h c o n t e n t o f l i n o l e i c a c i d ( m i d p h a s e t r a n s i t i o n t e m p e r a t u r e - 5 ° C ) a n d l i n o l e n i c a c i d ( m i d p h a s e t r a n s i t i o n t e m p e r a t u r e - 1 1 ° C ) w o u l d p e r m i t y e a s t m e m b r a n e s t o be i n a s e m i - f l u i d s t a t e a n d r e m a i n f u n c t i o n a l a t l o w t e m p e r a t u r e s . W a t s o n (1978) c o r r e l a t e d t h e d e g r e e o f l i p i d u n s a t u r a t i o n o f L . f r i g i d u m a n d T . b o v i n a w i t h t h e i r g l u c o s e u p t a k e a t d i f f e r e n t t e m p e r a t u r e s . I n L . f r i g i d u m , g l u c o s e t r a n s p o r t was m o s t a c t i v e a t 2 ° C b u t n e g l i g i b l e a t 4 0 ° C ; g l u c o s e t r a n s p o r t o f T . b o v i n a was m o s t r a p i d a t 4 0 ° C b u t was m i n i m a l a t 2 ° C . He s u g g e s t e d t h a t a t l o w t e m p e r a t u r e s m e m b r a n e s o f T . b o v i n a w i t h a l a r g e a m o u n t o f s a t u r a t e d f a t t y a c i d s w e r e t o o r i g i d , a n d t h e h i g h d e g r e e o f u n s a t u r a t e d f a t t y a c i d s a l l o w e d m e m b r a n e s o f L . f r i g i d u m t o be f u n c t i o n a l . Y e a s t a d a p t a t i o n t o l o w t e m p e r a t u r e s v i a m o d i f i c a t i o n o f l i p i d c o m p o s i t i o n h a s b e e n r e p o r t e d ( B r o w n a n d R o s e , 1969; K a t e s a n d P a r a d i s , 1973; P u g h a n d K a t e s , 1975; A r t h u r a n d W a t s o n , 1976; F e r r a n t e §_t a l . , 1983). T h e l i n o l e i c a c i d c o n t e n t o f m e s o p h i l i c C . l i p o l y t i c a g r o w n a t 1 0 ° C d o u b l e d c o m p a r e d t o i t s c o n t e n t i n c e l l s g r o w n a t 250c ( K a t e s a n d B a x t e r , 1962) . K a t e s a n d P a r a d i s (1973) -91- Table 3-1: Fatty acid composition of yeast membranes (adapted from Arthur and Watson, 1976). Yeast Percentage of t o t a l f a t t y a c i d s 1 Saturated Unsaturated C l 2 ~ C l B C l 4 i 1 C1 ti« 1 C i a i 1 C l B l 2 ClBl3 L. frigidum 9 Trace Trace 11 27 53 C . l i p o l y t i c a 11 3 20 44 21 Trace T. bovina 28 3 42 . 23 ND ND ND: not detected 1 subscript numbers preceding colons denote number of carbons in f a t t y acid chains, numbers following colons denoted number of double bonds in f a t t y acids. -92- noted t h a t u n s a t u r a t i o n of f a t t y a c i d s i n ph o s p h o l i p i d s of C. l i p o l y t i c a was g r e a t e r a t 10°C than 25<>C. An inverse r e l a t i o n s h i p between contents of o l e i c and l i n o l e i c a c i d s was observed i n a l l p h o s p h o l i p i d s , which i n d i c a t e d d e s a t u r a t i o n of o l e i c a c i d to form l i n o l e i c a c i d . Yeast p h o s p h o l i p i d s are l o c a t e d almost e x c l u s i v e l y i n membranes. Changes i n degree of u n s a t u r a t i o n of f a t t y a c y l chains of ph o s p h o l i p i d s would a l t e r membrane f l u i d i t y and f u n c t i o n . A c t i v i t y of oleoyl-CoA desaturase, which c a t a l y z e s d e s a t u r a t i o n of CoA-ester of o l e i c a c i d , was higher i n membranes of C. l i p o l y t i c a grown a t 10°C than i n c e l l s grown a t 25°C; i n c r e a s e d enzyme a c t i v i t y was c o r r e l a t e d with a decrease i n o l e i c a c i d content and inc r e a s e i n l i n o l e i c a c i d content a t 10°C (Pugh and Kates, 1975; Fe r r a n t e et §_1., 1983). Increased a c t i v i t y of o l e y l - C o A desaturase was a l s o observed i n T o r u l o p s i s u t i l i s grown at suboptimum temperatures (Meyer and Bloch, 1963). The i n v e r s e r e l a t i o n s h i p between temperature and unsaturated f a t t y a c i d contents r e p r e s e n t s yeast e f f o r t s to maintain f l u i d i t y and f u n c t i o n of membranes a t a p a r t i c u l a r temperature. In the present study, growth of Zygosaccharomyces sp. and Debaryomyces sp. at 4<>C i n a i r was probably a r e s u l t of t h e i r a d a p t a t i o n to low temperature. Since low temperature a l s o caused a decrease -93- i n enzyme a c t i v i t y , growth of two yeast i s o l a t e s were slower i n 4<>C-air than i n 21<>C-air (Fi g u r e s 3-8 and 3-9). D e s a t u r a t i o n of f a t t y a c i d s r e q u i r e s presence of molecular oxygen ( B l o o m f i e l d and Bloch, 1960; Yaun and Bloch, 1961; Pugh and Kates, 1975). Two models of the e l e c t r o n t r a n s p o r t c h a i n i n v o l v e d i n the d e s a t u r a t i o n of f a t t y a c i d s have been summarized ( F u l c o , 1974). In both models, oxygen a c t s as the t e r m i n a l acceptor of the hydrogen atoms removed from f a t t y a c i d s d u r i n g d e s a t u r a t i o n . An anaerobic environment then i n h i b i t s d e s a t u r a t i o n of f a t t y a c i d s and prevents yeast a d a p t a t i o n to low temperatures. The i n a b i l i t y t o d e s a t u r a t e membrane f a t t y a c i d s may e x p l a i n i n h i b i t i o n of Zygosaccharomyces sp. and Debaryomyces sp. at 4°C, 25% or 100% carbon d i o x i d e , although the i n h i b i t o r y e f f e c t of carbon d i o x i d e c o u l d not be e n t i r e l y d i s c o u n t e d . 3.3 YEAST GROWTH IN BLUEBERRIES DURING MAP STORAGE When b l u e b e r r i e s were packaged i n a f i l m with low gas p e r m e a b i l i t y , i n h i b i t i o n of yeast growth l a s t e d s i x to e i g h t weeks which was followed by a growth i n c r e a s e (see F i g u r e s 2-7, 2-8, 2-9). In t h i s storage c o n d i t i o n , growth of yeast f l o r a was probably i n f l u e n c e d by three f a c t o r s : high carbon d i o x i d e , low temperature and low oxygen atmosphere. -94- Carbon d i o x i d e i s a product of c e l l u l a r d e c a r b o x y l a t i o n r e a c t i o n s . High carbon d i o x i d e c o n c e n t r a t i o n s i n MAP storage may a f f e c t r e a c t i o n e q u i l i b r i u m and r a t e s of enzymatic d e c a r b o x y l a t i o n . D e c a r b o x y l a t i o n r e a c t i o n s then would become r a t e - l i m i t i n g steps i n c e l l u l a r metabolism, i f other enzymes were o p e r a t i n g at normal r a t e s . High carbon d i o x i d e would r e s u l t i n slow growth (King and Nagel, 1975; Kritzman e t a l . , 1977). Low temperatures a f f e c t the f l u i d i t y and f u n c t i o n of c e l l membranes. Membrane-associated processes r e l y on a s e m i f l u i d environment f o r t h e i r o p e r a t i o n . Yeasts have a remarkable a b i l i t y to i n c r e a s e the p r o p o r t i o n of membrane unsaturated f a t t y a c i d s i n response to a decrease i n temperature. This ensures proper f l u i d i t y of membranes at low temperatures. Low temperatures may a l s o a f f e c t a c t i v i t y of enzymes i n v o l v e d i n c e l l u l a r metabolism, and cause decreased metabolic r a t e s and slow growth. Increased s o l u b i l i t y of carbon d i o x i d e at low temperature could c o n t r i b u t e to growth i n h i b i t i o n . Oxygen i s e s s e n t i a l f o r the s y n t h e s i s of e r g o s t e r o l which i s an important component of m i t o c h o n d r i a l membrane (Linnane e_t al.., 1972). In the absence of oxygen, m i t o c h o n d r i a l s t r u c t u r e s lack the f o l d e d inner membrane, and p r o t e i n s y n t h e s i z i n g a c t i v i t y . P i n t o and Nes (1983) showed t h a t 0.21% oxygen was s u f f i c i e n t f o r s y n t h e s i s of - 9 5 - e r g o s t e r o l , and promoted growth of Saccharomvces c e r e v i s i a e . During two to twelve weeks of MAP storage of b l u e b e r r i e s i n the present study, headspace of low-oxygen packages contained approximately 0.4% to 0.8% oxygen (see Table 2-2). T h e r e f o r e , a d e f i c i e n c y of e r g o s t e r o l i n yeast f l o r a was u n l i k e l y to occur during MAP s t o r a g e . The f u n g a l counts of b l u e b e r r i e s showed that yeast growth i n c r e a s e d a f t e r s i x to e i g h t weeks i n MAP storage (see F i g u r e s 2-7, 2-8, 2-9), which i n d i c a t e d a r e c o v e r y p r o c e s s . M i c r o b i a l a d a p t a t i o n to carbon d i o x i d e has not been s t u d i e d e x t e n s i v e l y , and r e c o v e r y u s u a l l y o n l y occurs when presence of carbon d i o x i d e i s removed ( I n t e r n a t i o n a l Committee f o r M i c r o b i a l S p e c i f i c a t i o n s f o r Foods, 1980), although Johnson and Ogrydziak (1984) have r e p o r t e d evidence of g e n e t i c a d a p t a t i o n to carbon d i o x i d e by Pseudomonas-1ike b a c t e r i a . Yeast recovery i n MAP storage of b l u e b e r r i e s was observed even though the carbon d i o x i d e contents of storage packages continued to i n c r e a s e . In view of t h i s r e c o v e r y process, yeast a d a p t a t i o n to low storage temperature v i a d e s a t u r a t i o n of f a t t y a c y l chains of membrane p h o s p h o l i p i d s may be the key f a c t o r a f f e c t i n g y e a st growth on b l u e b e r r i e s d u r i n g MAP s t o r a g e . Since oxygen i s r e q u i r e d f o r f a t t y a c i d d e s a t u r a t i o n , m i c r o - a e r o b i c c o n d i t i o n of MAP storage may i n h i b i t d e s a t u r a t i o n of f a t t y a c i d s , which i n t u r n , prevents yeast -96- a d a p t a t i o n to low temperatures. At 15°C, d e s a t u r a t i o n of f a t t y a c i d s occurred i n Candida u t i l i s , grown under an oxygen t e n s i o n of 1 mmHg, and i t s t o t a l f a t t y a c i d s c o n t a i n e d 34% l i n o l e i c a c i d and a sm a l l p r o p o r t i o n of l i n o l e n i c a c i d (Brown and Rose, 1969). During two to twelve weeks of MAP storage i n t h i s study, oxygen content of low-oxygen packages ranged approximately from 0.4% (760 mmHg x 0.4/100 = 3.04 mmHg) to 0.8% (6.28 mmHg). Thi s range of headspace oxygen composition was higher than 1 mmHg r e q u i r e d f o r s y n t h e s i s of l i n o l e i c and l i n o l e n i c a c i d s i n C. u t i l i s . Since the yeast f l o r a present on ber r y s u r f a c e s was made up of many s p e c i e s , a d i r e c t comparison with data from C. u t i l i s i s i n a p p r o p r i a t e . N e v e r t h e l e s s , s m a l l amounts of oxygen i n the MAP packages of b l u e b e r r i e s might have allowed a slow d e s a t u r a t i o n of f a t t y a c i d s which l e d to yeast r e c o v e r y and growth i n the l a t t e r p a r t of the storage t r i a l s (see F i g u r e s 2-7, 2-8, 2-9) . Since high carbon d i o x i d e , low temperature and low oxygen c o n d i t i o n s c o - e x i s t e d i n MAP st o r a g e , the e f f e c t s of these f a c t o r s on yeast growth c o u l d not be separated. T h e r e f o r e , when yeasts modified membranes to maintain proper f u n c t i o n s a t low storage temperature, t h e i r growth r a t e s may have been slow due to the e f f e c t s of carbon d i o x i d e and low temperature on metabolic r e a c t i o n s . These -97- phenomena require further study. 3.4 CONCLUSIONS 1. D i f fus ion disk assays were set up to detect ant i fungal phytoalexin accumulation in blueberries during MAP storage. The resu l t s showed an absence of ant i fungal compounds as tested against f ive yeast s t r a i n s , and indicated i n h i b i t i o n of yeast growth due to low temperature, high carbon dioxide l eve l and anaerobic cond i t i ons. 2. The ef fects of temperature and atmosphere were invest igated using natural f l o r a of blueberry juice and yeast i so la tes grown in s t e r i l i z e d j u i c e , under d i f f eren t temperatures and atmosphere compositions. At 2 1 ° C , yeast growth was slow in the presence of carbon dioxide and absence of oxygen. In a i r , slow growth accompanied exposure to low temperature. The responses of yeast and mold f l o r a as well as yeast cul tures in these experiments confirmed observations of low fungal populations on blueberries during MAP storage. 3. 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(ed s . ) , Ann Arbor Science P u b l i s h e r Inc., MI. pp.203-268. - 1 0 8 - APPENDIX: IDENTIFICATION OF YEAST ISOLATES. -109- Al.1 I s o l a t i o n of yeasts from b l u e b e r r y B l u e b e r r i e s purchased from the B.C. B l u e b e r r y Co-op (Richmond, B.C.) were used. Ten samples of 25 g each were stomached i n 225 ml of 0.1% peptone water by a Colworth stomacher. Homogenates were then p l a t e d by the s p i r a l p l a t i n g method ( S p i r a l System Instruments, Inc. Bethesda, Maryland), on PCA ( D i f c o , D e t r o i t , MI) supplemented with 0.01% chloramphenicol and 0.01% c h l o r t e t r a c y c l i n e HC1 (Sigma Chemicals, St. L o u i s , MO). A l l p l a t e s were incubated a t 21°C. Yeast c o l o n i e s were picked from these p l a t e s and r e - s t r e a k e d on Yeast Morphology (YM) agar (Van der Walt and Yarrow, 1984). Only c o l o n i e s with d i f f e r e n t morphology were picked to a v o i d e x c e s s i v e r e p l i c a t i o n s . S t r e a k i n g of s i n g l e c o l o n i e s was repeated s e v e r a l times to o b t a i n pure c u l t u r e s . F i v e yeast s t r a i n s were i s o l a t e d from b l u e b e r r i e s and were t e n t a t i v e l y l a b e l l e d A,B,C,D,E. C u l t u r e s were maintained on YM agar s l a n t s . Al.2 I d e n t i f i c a t i o n of i s o l a t e s I d e n t i f i c a t i o n of i s o l a t e s to the genus l e v e l was based on m i c r o s c o p i c and macroscopic morphology, s e x u a l r e p r o d u c t i o n , p a t t e r n of sugar f e r m e n t a t i o n , a b i l i t y to u t i l i z e v a r i o u s compounds as s o l e carbon source, formation -110- of e x t r a c e l l u l a r amyloid compounds, production of ammonia from urea, and a b i l i t y to grow at 37°C. Test procedures followed those described by Van der Walt and Yarrow (1984) and Barnett et §_i. (1983). Al.2.1 Colonial morphology Cultures were streaked on YM agar plates for isolated colonies (48 hours, 21°C). The macroscopic c h a r a c t e r i s t i c s of isolates are summarized in Table A - l . Al.2 . 2 Characteristics of vegetative c e l l s Yeasts were grown on YM agar for 48 hours (21°C). Single colonies were used to inoculate yeast nitrogen base containing 25 mM D-glucose, which was incubated for 20 hours in a shaking incubator (21<>c, 80 rpm). After incubation, a sample of 10 HL was placed on a microscope s l i d e and covered with a co v e r s l i p . For photography, volume of suspension was adjusted to avoid c e l l movements. Micrographs of yeast isolates are presented in Figures A - l to A-5. A l l photomicrographs had a t o t a l magnification of 1000 x. A Zeiss Photomicroscope and I l f o r d XP1 400 f i l m were used. Vegetative c e l l s of a l l isolates lacked formation of pseudo or true mycelium. Vegetative reproduction of isolates was by m u l t i l a t e r a l budding. - I l l - Table A - l : Colonial morphology of yeast isolates grown on on YM agar plates for 48 hours, at 21°C. Yeast Description of colonies A Pink to orange-red colonies with even edge, convex, d u l l , smooth surface. Average diameter is 4 mm. B Light cream colonies with even edge, convex, shiny surface. Average diameter is 2 mm. C Light pink colonies with even edge, raised elevation, and very d u l l , dry surface. Average diameter is 3 mm. D Cream colonies with slimy appearance, even edge, convex elevation. Average diameter i s 3 mm. E Light cream colonies with f l a t , d u l l surface. Average diameter is 2 mm. - 1 1 2 - F i g u r e A - l : V e g e t a t i v e c e l l s (1000 x ) o f y e a s t i s o l a t e A ( l a t e r i d e n t i f i e d a s a R h o d o t o r u l a s p e c i e s ) , g r o w n o n Y N B - g l u c o s e f o r 20 h o u r s a t 2 1°C. -113- Figure A-2: Vegetative c e l l s (1000 x) of yeast isolate B (later i d e n t i f i e d as a Zygosaccharomyces species), grown on YNB-glucose for 20 hours at 21°C. -114- Figure A-3: Vegetative c e l l s (1000 x) of yeast isolate (later i d e n t i f i e d as a Rhodotorula species), grown on YN glucose for 20 hours at 21 ° C . -115- Figure A -4: Vegetative c e l l s (1000 x) of yeast isolate D (later i d e n t i f i e d as a Cryptococcus species), grown on YNB-glucose for 20 hours at 210C. - 1 1 6 - Flgure A-5:, Vegetative c e l l s (1000 x) of yeast isolate E (later i d e n t i f i e d as a Debaryomyces species), grown on YNB-glucose for 20 hours at 210C. - 1 1 7 - Al.2.3 Sexual r e p r o d u c t i o n Growth from YM agar (48 hours, 21<>C) were t r a n s f e r r e d to Potato Dextrose agar, Saboraud Dextrose agar and 2% agar (growth r e s t r i c t i v e medium). The s p o r u l a t i o n media were incubated a t 21°C f o r 3 days before being examined m i c r o s c o p i c a l l y . Since no growth occurred on 2% agar medium a f t e r three days, t h i s medium was maintained at 21°C, and examined weekly f o r 4 weeks. A l l i s o l a t e s f a i l e d to grow on 2% agar medium. Growth from Saboraud Dextrose and Potato Dextrose agar (3 days) were s t a i n e d and examined m i c r o s c o p i c a l l y f o r ascospores. H e a t - f i x e d smears were flo o d e d with 5% aqueous malachite green (Sigma Chemicals, S t . L o u i s , MO) and heated to steaming (30 s e c ) . The excess s t a i n was washed o f f , and smears were c o u n t e r s t a i n e d with 0.5% S a f r a n i n (Sigma Chemicals, St. L o u i s , MO). The mature ascospores s t a i n e d green and v e g e t a t i v e c e l l s red under l i g h t m i c r o s c o p i c examination (1000 x ) . The r e s u l t s are summarized i n Table A-2. Al.2.4 P r o d u c t i o n of ammonia from urea Yeasts d i f f e r i n t h e i r a b i l i t y to hydrolyze high c o n c e n t r a t i o n s of urea to ammonia i n media c o n t a i n i n g an o r g a n i c n i t r o g e n source. The t e s t medium was C h r i s t e n s e n ' s urea agar. Two-percent agar s o l u t i o n was dispensed i n 4.5 ml a l i q u o t s to t e s t tubes. The tubes -118- Table A-2: Sexual r e p r o d u c t i o n of yeast i s o l a t e s . Yeast D e s c r i p t i o n A no sexual r e p r o d u c t i o n on any media t e s t e d . B C y l i n d r i c a l ascus with obtuse ends (5 um long) c o n t a i n i n g 3 spores. C no s e x u a l r e p r o d u c t i o n on any media t e s t e d . D no sexual r e p r o d u c t i o n on any media t e s t e d . E E l l i p s o i d a s c i (5-7 um long) with 2 ascospores each. -119- were au t o c l a v e d f o r 15 minutes at 121°C. A f t e r a u t o c l a v i n g , 0.5 ml of a f i l t e r - s t e r i l i z e d (20%) urea base ( D i f c o , D e t r o i t , MI) was added, and mixed; the medium was allowed to s e t i n s l a n t e d p o s i t i o n . Yeast c u l t u r e s grown i n YM agar (48 h, 21°C) were used to i n o c u l a t e urea s l a n t s . Tubes were observed d a i l y up to three days on i n c u b a t i o n (21°C), p o s i t i v e r e a c t i o n s were recorded a f t e r appearance of a deep red c o l o u r i n s l a n t s . I s o l a t e s A,C, and D gave p o s i t i v e r e a c t i o n s , B and E responses were n e g a t i v e . Al.2.5 Growth at 37QC Yeasts were grown on YM agar at 37°C f o r 4 days. None of the i s o l a t e s were able to grow a t t h i s temperature. Al.2.6 A s s i m i l a t i o n of carbon compounds The a b i l i t y of yeast i s o l a t e s to u t i l i z e the f o l l o w i n g 13 compounds was t e s t e d : g a l a c t o s e ( D i f c o , D e t r o i t , MI), sucrose (BDH Chemicals, Vancouver, BC), maltose ( D i f c o , D e t r o i t , MI), c e l l o b i o s e (Sigma Chemicals, St. L o u i s , MO), l a c t o s e ( D i f c o , D e t r o i t , MI), m e l i b i o s e (Sigma Chemicals, St. L o u i s , MO), r a f f i n o s e (Sigma Chemicals, S t . L o u i s , MO), xylose (Sigma Chemicals, St. L o u i s , MO), arabinose (Sigma Chemicals, St. L o u i s , MO), a d o n i t o l (Sigma Chemicals, St. L o u i s , MO), i n u l i n (Sigma Chemicals, -120- St. L o u i s , MO), m y o - i n o s i t o l , s a l i c i n ( D i f c o , D e t r o i t , MI). As glucose i s u t i l i z e d by a l l y e a s t s , i t was included as a standard f o r comparing the r a t e s a t which other compounds were u t i l i z e d . The a s s i m i l a t i o n medium was made up by d i s s o l v i n g 0.67 g Yeast N i t r o g e n base ( D i f c o , D e t r o i t , MI) and an a p p r o p r i a t e amount of carbon compound e q u i v a l e n t to 5 g gluc o s e , to 100 ml d i s t i l l e d water. When r a f f i n o s e was used as the carbon source, i t was used at an amount e q u i v a l e n t to 10 g g l u c o s e . Media were s t e r i l i z e d by f i l t r a t i o n (0.45 Jim c e l l u l o s e e s t e r f i l t e r , M i l l i p o r e C o r p o r a t i o n , Bedford, MA), and dispensed i n 10 ml- a l i q u o t s . I n u l i n was d i s s o l v e d by g e n t l y h e a t i n g before f i l t r a t i o n s i n c e u n d i s s o l v e d i n u l i n may be l o s t d u r i n g f i l t r a t i o n . Yeast i s o l a t e s grown i n YM agar (48 hours, 21°C) were i n o c u l a t e d i n t o 5 ml of s t e r i l e d i s t i l l e d water. Two drops of t h i s inoculum was added to each tubes c o n t a i n i n g a s s i m i l a t i o n medium. A l l tubes were incubated at 21°C, and examined every 7 days f o r 3 weeks. The degree of u t i l i z a t i o n was determined by p l a c i n g tubes a g a i n s t a white card b e a r i n g l i n e s drawn approximately 3/4 mm wide. I f growth completely o b l i t e r a t e d the l i n e s , i t was recorded as 3 +; i f the l i n e s appeared as d i f f u s e bands, growth was r a t e d as 2 +; i f the c l e a r l i n e s appeared -121- growth was r a t e d as 1 +. A 3 + or 2 + r e a c t i o n w i t h i n three weeks was c o n s i d e r e d p o s i t i v e , and a 1 + r e a c t i o n as ne g a t i v e . The r e s u l t s of t h i s t e s t are presented i n Table A-3. Al.2 . 6 P r o d u c t i o n of e x t r a c e l l u l a r amyloid compounds A f t e r 21 days, the carbon a s s i m i l a t i o n t e s t c u l t u r e s were examined f o r presence of amyloid compounds. One drop of Gram's io d i n e s o l u t i o n was added per tube. Presence of amyloid compounds was i n d i c a t e d by blue to green c o l o u r i n tubes. Only i s o l a t e D gave a p o s i t i v e r e a c t i o n f o r t h i s t e s t . Al.2.7 Fermentation of sugars A wine yeast Saccharomyces c e r e v i s iae was a l s o t e s t e d i n t h i s procedure to check f o r e f f i c a c y of the media. The ferme n t a t i o n b a s a l medium contained 4.5 g yeast e x t r a c t and 7.5 g peptone i n IL d i s t i l l e d water. S u f f i c i e n t amount of bromothymol blue was added as an i n d i c a t o r . The b a s a l medium (4ml) was p i p e t t e d to Durham tubes ( t e s t tubes c a r r y i n g i n s e r t tubes approximately 50 x 6mm), and the tubes were au t o c l a v e d f o r 15 minutes at 121°C. A f t e r s t e r i l i z a t i o n , 2 m l - a l i q u o t s of v a r i o u s s t e r i l e sugar s o l u t i o n s were added to the tubes. The sugars were d i s s o l v e d i n d i s t i l l e d water to make up 6% s o l u t i o n s , except r a f f i n o s e which was made up i n a 12% s o l u t i o n . A l l -122- T a b l e A - 3 : U t i l i z a t i o n o f c a r b o n c o m p o u n d s b y y e a s t i s o l a t e s . C o m p o u n d s Y e a s t s A B C D E G a l a c t o s e + + + + + S u c r o s e + + + + + M a l t o s e - + - + + C e l l o b i o s e + + + + + L a c t o s e - - - + + M e l l i b i o s e - + - + — R a f f i n o s e + + + + + I n u l i n e - - - - — X y l o s e + + + + I n o s i t o l - + - - + A r a b i n o s e - + + + + S a l i c i n + + + + + A d o n i t o l - + + + + -123- sugar s o l u t i o n s were s t e r i l i z e d by f i l t r a t i o n . The f i n a l c o n c e n t r a t i o n of sugars i n fermentation tubes was 2%, except r a f f i n o s e was t e s t e d a t a 4% f i n a l c o n c e n t r a t i o n . The f e r m e n t a t i o n tubes were i n o c u l a t e d with 0.1ml of a yeast suspension made by d i s p e r s i n g growth on Yeast Morphology agar i n water. A f t e r i n o c u l a t i o n , a l l tubes were t i g h t l y capped and incubated (21°C) and observed every 24 hours f o r gas and a c i d p r o d u c t i o n (Van der Walt & Yarrow, 1984). The above procedure f a i l e d to d e t e c t any gas formation i n fermentation tubes, because the i n v e r t e d i n s e r t tubes can only t r a p gas produced at or near the bottom of t e s t tubes. Carbon d i o x i d e produced by yeast c u l t u r e s fermenting near the s u r f a c e of medium y i e l d e d an apparent negative r e a c t i o n . A new procedure f o r carbon d i o x i d e d e t e c t i o n was adapted. In t h i s procedure, the fermentation medium was prepared as d e s c r i b e d above, and dispensed i n t o tubes capped by a i r - t i g h t rubber s t o p p e r s . At 1 week and 2 week i n t e r v a l s , the headspace of tubes was analyzed by gas chromatography. The instrument and o p e r a t i n g c o n d i t i o n s were i d e n t i c a l to those used i n gas a n a l y s i s of samples i n MA s t o r a g e . Continued p r o d u c t i o n of carbon d i o x i d e i n headspace a f t e r oxygen was d e p l e t e d , and a c i d p r o d u c t i o n was recorded as p o s i t i v e f e r m e n t a t i o n . I s o l a t e s A, C and D lacked the a b i l i t y to ferment any of -124- the sugars t e s t e d . The p a t t e r n s of fermentation of i s o l a t e s B and E are o u t l i n e d i n Table A-4. The yeast i s o l a t e s were i d e n t i f i e d using the c h a r a c t e r i s t i c s of genera compiled by Kregger van R i j ( 1984 ) with c r o s s - r e f e r e n c e to the work of Barnett e_t a l . (1983). I s o l a t e s were i d e n t i f i e d as f o l l o w s : A: a Rhodotorula s p e c i e s B: a Zygosaccharomyces s p e c i e s C: a Rhodotorula s p e c i e s D: a Cryptococcus s p e c i e s E: a Debaryomyces s p e c i e s A l . 3 Standard curves of yeast i s o l a t e s Yeast c u l t u r e s were grown i n YM broth f o r 48 hours, a t 21°C. Yeast counts (colony-forming units/ml) were obtained by p l a t i n g d i l u t i o n s of yeast c u l t u r e s on P l a t e Count agar using the s p i r a l p l a t i n g method ( S p i r a l System Instrument Inc., Bethesda, Maryland). Absorbance (420 nm) of c u l t u r e d i l u t i o n s was obtained u s i n g a Shimadzu UV-160 spectrophotometer. Yeast counts were p l o t t e d a g a i n s t absorbance to prepare standard curves (Figures A-6 to A- 10) . -125- Table A-4: Fermentation of sugars by two i s o l a t e s . Sugars Yeasts B E Glucose + + Galactose - - Sucrose + + Maltose + - Lactose - - Raff inose + + - 1 2 6 - Absorbance (420 nm) F i g u r e A-6: ( i s o l a t e A). (r = 0.97) . Standard curve of a Rhodotorula s p e c i e s The curve was f i t t e d by l i n e a r r e g r e s s i o n -127- Figure A-7: Standard curve of a Zygosaccharomyces species ( i so la te B) . The curve was f i t t e d by l inear regress ion (r = 0.92). -128- 1 1 1 1 1 r~ 0 0.1 0.2 0.3 0.4 0.5 Absorbance (420 nm) Figure A-8: Standard curve of a Rhodotorula species ( i so la te C ) . The curve was f i t t e d by l inear regress ion (r = 0.98). -129- T 1 1 1 1 1 1- 0 02 0.4 0.6 Absorbance (420 nm) F i g u r e A-9: Standard curve of a Crvptococcus s p e c i e s ( i s o l a t e D). The curve was f i t t e d by l i n e a r r e g r e s s i o n (r = 0.96). -130-

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