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UBC Theses and Dissertations

Potential application of lactic acid bacteria to extend the storage life of grey cod (Gadus [morhua]… Jones, Yvonne Marene 1982

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POTENTIAL APPLICATION OF LACTIC ACID BACTERIA TO EXTEND THE STORAGE LIFE OF GREY COD (Gadus [morhua] macrocephalus) MUSCLE YVONNE MARENE JONES A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Food Science Department) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1982 © Yvonne Marene Jones, 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 8* Slfh»L Date 9T ^efik^&tw -lite. DE-6 (3/81) - i i -ABSTRACT Minced grey cod f i l l e t s were i n o c u l a t e d w i th l a c t i c a c i d b a c t e r i a s t a r t e r c u l t u r e s namely, Pediococcus c e r e v i s i a e (Lac tace l 7 5 , No. L750480, M i c r o l i f e Technics ) and a mixture of Pediococcus c e r e v i s i a e and L a c t o b a c i l l u s  p lantarum ( L a c t a c e l MC, No. M12772, M i c r o l i f e T e c h n i c s ) . R e s u l t s showed t h a t when l a c t i c a c i d b a c t e r i a was added to the minced 8 9 product to a f i n a l c e l l p o p u l a t i o n of 10 or 10 /g of mince , growth of p s y c h r o t r o p h i c b a c t e r i a at normal r e f r i g e r a t i o n temperature was i n h i b i t e d . I n h i b i t i o n was more pronounced w i t h the a d d i t i o n of 0.3% d e x t r o s e . This study p o i n t s towards the p o t e n t i a l a p p l i c a t i o n f o r u t i l i z i n g l a c t i c a c i d b a c t e r i a to preserve the q u a l i t y of a r e f r i g e r a t e d minced f i s h p roduct . P o t e n t i a l a p p l i c a t i o n of the use of l a c t i c a c i d b a c t e r i a , through a d i p p i n g procedure to extend the storage l i f e of f i s h f i l l e t s , i s a l s o d i s c u s s e d . - i i i -TABLE OF CONTENTS Paoe A b s t r a c t i i L i s t of Tables v L i s t of F i g u r e s v i i L i s t of P l a t e s i x D e d i c a t i o n x Acknowledgement x i INTRODUCTION 1 LITERATURE REVIEW 6 A. F i s h S p o i l a g e 6 B. L a c t i c A c i d B a c t e r i a 11 C. M i c r o b i a l Antagonism 16 D. T r i methyl amine (TMA) - As A Spo i lage I n d i c a t o r 19 MATERIALS AND METHODS 24 A. Test Organisms 24 B. P r e p a r a t i o n of Minced F i s h Stored 2 -3 Days i n Ice 25 C. F i l l e t P r e p a r a t i o n 26 D. B a c t e r i o l o g i c a l Media 28 E. Determinat ion of V i a b l e Count 28 F. Storage of Samples 29 G. pH Measurement 30 H. D i l u e n t 30 I. I n h i b i t i o n of S p o i l a g e Organisms by P. c e r e v i s i a e and a Mix tu re of F\_ c e r e v i s i a e and L_^_ plantarum 30 J . I n h i b i t i o n Test 30 K. Tr imethylamine A n a l y s i s (TMA) 31 L. Sensory E v a l u a t i o n of Fresh F i l l e t s - Odour 31 RESULTS AND DISCUSSION 34 A. Sensory E v a l u a t i o n of F i l l e t s - Odour 34 B. The E f f e c t of P. c e r e v i s i a e on the Extens ion of Storage L i f e of F i s h Stored at 4C 37 C. The E f f e c t of a Mix tu re of P. c e r e v i s i a e and L. plantarum on Minced F i s h Stored at 4C 46 D. The E f f e c t of P. c e r e v i s i a e and a Mix ture of P. c e r e v i s i a e and L. plantarum on Minced F i s h Stored at iTC and 24C . . 50 E. Tr imethy lamine A n a l y s i s (TMA) 61 F. I n h i b i t i o n Test 76 - iv -TABLE OF CONTENTS (Concluded) Page SUMMARY OF RESULTS 77 CONCLUSIONS 83 BIBLIOGRAPHY 85 APPENDIX 93 LIST OF TABLES Advantages of Minced F i s h C h a r a c t e r i s t i c s of S p o i l i n g F i s h Categor ies of L a c t i c A c i d B a c t e r i a The Odours of Raw and Cooked White F i s h Taken from the Score Sheet of Shewan et aj_, (1953) , i n t h e i r Attempt to Develop a Scor ing System f o r the Sensory Assessment of S p o i l a g e of Wet White F i s h Stored i n Ice Odour Scores f o r Treated and Untreated Cod F i l l e t s Stored at 4C Odour Scores f o r Treated and Untreated Cod F i l l e t s Stored at 4C The pH Values of Untreated and Treated Minced F i s h During Storage at 4C. Treated samples c o n t a i n 1 0 7 c e l l s of P. c e r e v i s i a e / g of mince. The pH Values of Untreated and Treated Minced F i s h During Storage at 4C. Treated samples c o n t a i n 1 0 9 c e l l s of P. c e r e v i s i a e / g of mince. The pH Values of Untreated and Treated Minced F i s h During Storage at 4C. Treated samples c o n t a i n a mixture of 10^ c e l l s of P. c e r e v i s i a e and L. plantarum/g of mince. The pH Values of Untreated and Treated Minced F i s h During Storage at 4C. Treated samples c o n t a i n a mixture of 1 0 9 c e l l s of P. c e r e v i s i a e and L. plantarum/g of mince. The pH Values of Untreated and Treated Minced F i s h During Storage at 11C The pH Values of Untreated and Treated Minced F i s h During Storage at 24C TMA-N Values f o r Untreated and Treated Minced F i s h During Storage at 4C. Treated samples c o n t a i n 1 0 7 c e l l s of P. c e r e v i s i a e / g of mince. TMA-N Values f o r Untreated and Treated Minced F i s h During Storage at 4C. Treated samples c o n t a i n 1 0 9 c e l l s of P. c e r e v i s i a e / g of mince. - v i -LIST OF TABLES (Concluded) Table Page XV TMA-N Values f o r Untreated and Treated Minced F i s h During Storage at 4C. Treated samples c o n t a i n a mix ture of 10^ c e l l s of P. c e r e v i s i a e and L. plantarum/g of mince. 69 XVI TMA-N Values f o r Untreated and Treated Minced F i s h During Storage at 4C. Treated samples c o n t a i n a mix ture of 10^ c e l l s of P. c e r e v i s i a e and L. plantarurn/g of mince. 72 XVII TMA-N Values f o r Untreated and Treated Minced F i s h During Storage at 11C 74 XVII I TMA-N Values f o r Untreated and Treated Minced F i s h During Storage at 24C 75 XIX I n h i b i t i o n of P s y c h r o t r o p h i c Organisms i n Minced F i s h by P. c e r e v i s i a e During Incubat ion ' 76 - v i i -LIST OF FIGURES Page F i g u r e 1 E f f e c t of Storage Per iod on B a c t e r i a l Count and Decomposit ion of Muscle 7 F igure 2 The Rate of Development of TMA and DMA i n Wrapped Cod F i l l e t s Stored i n Ice 23 F i g u r e 3 The Growth of P s y c h r o t r o p h i c Organisms i n the Presence of P. c e r e v i s i a e i n Minced F i s h Stored at 4C. F i n a l p o p u l a t i o n of t e s t organisms was 10? c e l l s / g of mince 39 F igure 4 The Growth of P s y c h r o t r o p h i c Organisms i n the Presence of P. c e r e v i s i a e and 0.3% Dextrose i n Minced F i s h Stored at 4C. F i n a l p o p u l a t i o n of t e s t organism was IO-7 c e l l s / g of mince 40 F i g u r e 5 The Growth of P s y c h r o t r o p h i c Organisms i n the Presence of P. c e r e v i s i a e i n Minced F i s h Stored at 4C. F i n a l p o p u l a t i o n of t e s t organism was 109 c e l l s / g of mince 42 F i g u r e 6 The Growth of P s y c h r o t r o p h i c Organisms i n the Presence of P. c e r e v i s i a e and 0.3% Dextrose i n Minced F i s h Stored at 4C. F i n a l p o p u l a t i o n of t e s t organism was 10^ c e l l s / g of mince • 43 F i g u r e 7 The Growth of P s y c h r o t r o p h i c Organisms i n the Presence of a Mix tu re of P. c e r e v i s i a e and L. p lantarum i n Minced F i s h Stored at 4C. F i n a l p o p u l a t i o n of t e s t organism was 10^ c e l l s / g of mince 47 F i g u r e 8 The Growth of P s y c h r o t r o p h i c Organisms i n the Presence of a Mix tu re of P. c e r e v i s i a e and L. p lantarum and 0.3% Dextrose i n Minced F i s h Stored at 4C. F i n a l p o p u l a t i o n of t e s t organism was 10^ c e l l s / g of mince 48 F i g u r e 9 The Growth of P s y c h r o t r o p h i c Organisms i n the Presence of a Mix ture of P. c e r e v i s i a e and L. plantarum i n Minced F i s h Stored at 4C. F i n a l p o p u l a t i o n of t e s t organism was 10^ c e l l s / g of mince 51 F igure 10 The Growth of P s y c h r o t r o p h i c Organisms in the Presence of a Mix ture of P. c e r e v i s i a e and L. p lantarum i n Minced F i s h and 0 .3% Dextrose Stored at 4C. F i n a l p o p u l a t i o n of t e s t organism was 10^ c e l l s / g of mince 52 - v i i i -LIST OF FIGURES (Concluded) Page F i g u r e 11 The Growth of P s y c h r o t r o p h i c Organisms i n the Presence of P. c e r e v i s i a e and 0.3% Dextrose i n Minced F i s h Stored at 11C. F i n a l p o p u l a t i o n of t e s t organism was 1 0 9 c e l l s / g of mince 55 F igure 12 The Growth of P s y c h r o t r o p h i c Organisms i n the Presence of a Mix tu re of £\_ c e r e v i s i a e and L. p lantarum and 0.3% Dextrose i n Minced F i s h Stored at 11C. F i n a l p o p u l a t i o n of t e s t organism was 1 0 9 c e l l s / g of mince 56 F i g u r e 13 The Growth of P s y c h r o t r o p h i c Organisms i n the Presence of P. c e r e v i s i a e and 0.3% Dextrose i n Minced F i s h Stored at 24C. F i n a l p o p u l a t i o n of t e s t organism was 1 0 9 c e l l s / g of mince 58 F i g u r e 14 The Growth of P s y c h r o t r o p h i c Organisms i n the Presence of a Mix ture of P. c e r e v i s i a e and L. plantarum and 0.3% Dextrose i n Minced F i s h Stored at 24C. F i n a l p o p u l a t i o n of t e s t organism was 1 0 9 c e l l s / g of mince 59 F i g u r e 15 TMA-N Produc t ion i n Untreated and Treated Minced F i s h Stored at 4C. P. c e r e v i s i a e was added to a f i n a l p o p u l a t i o n of 1 0 7 c e l l s per g 62 F i g u r e 16 TMA-N Product ion i n Treated and Untreated Minced F i s h Stored at 4C. P. c e r e v i s i a e was added to a f i n a l p o p u l a t i o n of 10^" c e l l s per g and dext rose to 0.3% 64 F igure 17 TMA-N Product ion i n Treated and Untreated Minced F i s h Stored at 4C. P. c e r e v i s i a e was added to a f i n a l p o p u l a t i o n of T O 9 c e l l s per g 65 F igure 18 TMA-N Product ion i n Treated and Untreated Minced F i s h Stored at 4C. P. c e r e v i s i a e was added to a f i n a l p o p u l a t i o n of TO 9 c e l l s per g and dex t rose t o 0 .3% 67 F i g u r e 19 TMA-N Product ion i n Treated and Untreated Minced F i s h Stored at 4C. Treated product conta ined 1 0 9 c e l l s per g P. c e r e v i s i a e and L. p lantarum 70 F igure 20 TMA-N Product ion i n Treated and Untreated Minced F i s h Stored at 4C. Treated product conta ined 1 0 9 c e l l s per g P. c e r e v i s i a e and L. plantarum and 0.3% dext rose 71 - i x -LIST OF PLATES Page P l a t e 1 A Commercial Trawler from which Grey Cod was Obtained 100 P l a t e 2 F i s h Stored on Ice i n the Hold of the Vessel 101 P l a t e 3 Step One of F i l l e t i n g Process 101 P l a t e 4 Skinned F i l l e t s P r i o r t o Minc ing 102 P l a t e 5 Minc ing of Cod F i l l e t s Using a B i r o Meat Chopper 102 P l a t e 6 Minced Cod P r e p a r a t i o n 103 P l a t e 7 S p i r a l P l a t e r Used i n Enumeration of Microorganisms 103 P l a t e 8 S p i r a l P l a t e Counter Used i n Counting B a c t e r i a l C o l o n i e s 104 P l a t e 9 S p i r a l P l a t e Counting Gr id 104 P l a t e 10 P l a t e s Showing I n h i b i t i o n of P s y c h r o t r o p h i c Organisms i n Minced F i s h Stored at 4C 105 P l a t e 11 P l a t e s Showing I n h i b i t i o n of P s y c h r o t r o p h i c Organisms i n Minced F i s h Stored at 11C 105 P l a t e 12 P l a t e s Showing Growth of L a c t i c Organisms on L a c t o b a c i l l u s S e l e c t i o n Agar 106 - X -DEDICATION This t h e s i s i s ded ica ted to my husband, Sam, f o r h i s h e l p , p a t i e n c e and understanding and to my f a m i l y , e s p e c i a l l y Mom, Dad and s i s t e r s Sonia and P a u l e t t e f o r t h e i r cont inued encouragement, as w e l l as c l o s e f r i e n d s N i c o l e , Joyce , Cathy, Jean , May, E t h y l y n , Akor and Janet . - xi -ACKNOWLEDGEMENTS The author wishes to extend sincere gratitude to my major advisors, Dr. George Strasdine of B.C. Research and Dr. Ph i l l ip Townsley of the University of Bri t i sh Columbia, Department of Food Science, for their undivided concern, guidance and assistance in the preparation and execution of this thesis. A special thanks to Dr. Terry Howard, Head, Division of Fisheries Technology, B.C. Research, for his assistance and cooperation throughout this work and to the B.C. Science Council for partial financial support. Thanks is also extended to the staff of the Fisheries Technology Division for their support and cooperation, the l ibrary staff for their invaluable assistance, and Mr. Gerry O'Neil . A f inal thanks is extended to Mrs. Madelle Quiring, Miss Laurie Lucas and Miss Maggie Ball for their assistance in typing this report. INTRODUCTION A common proverbial expression in the Caribbean i s , "Willful waste make woeful want." It is a well known fact that only a small portion of the total edible resource of the sea is presently being used for human consumption. Different cultures around the world show a characteristic preference for specific species of fish prepared in a particular manner and, as a result , many otherwise edible species of fish are rejected because they do not satisfy traditional tastes or demand. These rejected species taken or landed concurrently with the more desired species are commonly referred to as the 'by-catch.' The term 'trash f i sh ' is also used to characterize these species. The Food and Agriculture Organization (FAO) Yearbook of Fisheries Statist ics for 1978 reported a wide range of f ish by-catch based on areas. Thus, Australia had a by-catch of 21,600 metric tons, the West Central Pacific zone - 4.5 mill ion metric tons, the West Indian Ocean - 3.25 mil l ion metric tons and the Southwest Atlantic - 703,500 metric tons. Vessels operating out of Guyana alone dumped 80,000 tons annually. A conservative estimate of 200,000 metric tons of by-catch is postulated as being dumped annually in the Caribbean Sea (James, 1977). - 2 -With reference to the Caribbean, this wastage is not only depriving Caribbean nations of badly needed protein, but may also serve to contaminate the fishing grounds and enhance the number of carnivorous species. In addition to greater by-catch u t i l i za t ion , technology must be developed to more effectively u t i l i z e processing wastes from tradit ional ly harvested species. One method applicable to both situations and currently receiving wide interest is that of mincing or comminuting the flesh of under-utilized species or waste fractions of tradit ional ly exploited species. This route lends i t se l f to obvious advantages, as shown in Table 1. TABLE I ADVANTAGES OF MINCED FISH Increased use of under- and non-utilized species. Products may be easily formulated. Mincing and preparation is readily mechanized. Easily adapted to on-board processing. The method provides for easy addition of extenders, preservatives or other additives. - 3 -Another factor contributing to the enormous waste of fish in developing areas is the lack of adequate refrigeration f a c i l i t i e s , both aboard vessels at sea and in processing and holding areas on shore. A common alternative to refrigeration is the addition of salt at high concentrations to the prepared product. Although this method is effective in preventing spoilage, i t requires the leaching of the product prior to i ts consumption - therefore detracting from its acceptability. A second disadvantage to the use of high levels of added sal t , particularly with more fatty f i sh , is this agent's ab i l i ty to increase the rate of rancidity formation within the product during storage. The problem of how to more effectively u t i l i ze the enormous quantities of by-catch is not specific to the Caribbean; i t also applies to other developing areas of the world. Therefore, given alternate technologies for extending the storage l i f e of fresh product, better use might be made of the by-catch. In recent years, fishing nations a l l over the world have become more aware of a decline in established fish stocks. This is accompanied by more stringent fishing regulations. The irony of the situation is that the faces of the populus of the world are turned to the resources of the sea as a means of supplying needed protein. In many areas of the developing world, protein malnutrition is said to be a serious problem. It would, therefore, be criminal not to convert such wasted protein into useful products which could be the means of saving a l i f e . - 4 -Uti l izat ion of f ish is considered as requiring less capital investment in terms of increasing protein supply when compared to other agricultural programmes with the same objective. Moreover, fish is more widely accepted by more communities in the world than, for example, beef or pork because of various religious restr ict ions . Any technology that has the objective of improving human health and well being is worth examining. The arguments presented here form the basis for undertaking this study, to examine the potential application of lact ic acid bacteria in extending the storage l i f e of minced f i sh . Various investigators have reported on the antagonistic actions of various species of lact ic acid bacteria to the growth and development of spoilage microorganisms in food products. In f i sh , the major spoilage organisms include psychrotrophic species of the genera Pseudomonas, Moraxella/Acinetobacter and Flavobacter. Various lact ic cultures have been known to produce ant ib iot ic - l ike inhibitory substances with significant effect on Gram-negative spoilage organisms. Price and Lee (1970) (using strains of Lactobacillus plantarum isolated from oysters) caused inhibition of Pseudomonas spp. through the production of an inhibitor which paralleled hydrogen peroxide formation in Lactobacillus cultures. - 5 -Reddy et a_l_. (1970) reported the inoculation of ground beef with mixtures of Streptococcus lact is and Leuconostoc citrovorum resulted in a retardation of the growth of Gram-negative bacteria during storage at 7C. The following investigation is predicated on the premise that the development of a safe and stable fish product, prepared from minced or comminuted f lesh, would allow increased ut i l i zat ion of under- and non-utilized species of f ish in developing nations. - 6 -LITERATURE REVIEW A. Fish Spoilage Spoilage of f ish results primarily from bacterial action. Reay and Shewan (1949) established a population curve for growth of bacteria in f ish muscle. Figure 1 provides a summary of the changes which take place during the spoilage process in gadoid species. Different species display varying rates of spoilage, dependent upon both the rate of bacterial growth and type of organisms present (Liston, 1980). Bacteria are confined i n i t i a l l y to the surface layer and advance slowly into the flesh as spoilage proceeds (Shewan, 1971). Conflicting arguments exist within the l iterature as to the mechanisms involved in the bacterial penetration of the f lesh. Shewan (1971) reported the confinement of bacteria to the surface layers until the advancement of spoilage. According to Beatty and Gibbons (1937), bacterial penetration occurs along the blood vessels within the muscle. Shewan (1977), however, reported confinement of bacterial act iv i ty to the surface slime until spoilage advanced, at which time invasion of the flesh could be demonstrated histological ly . In case of f i l l e t s , penetration is more rapid. S t i l l , the greater part of microbial act ivi ty occurs at the surface due to the oxidative nature of the bacteria involved. - 7 -Figure 1. Effect of storage period on bacterial count and decomposition of f ish muscle. Source: Reay and Shewan, 1949. - 8 -Bacteria primarily responsible for spoilage of marine f ish during the f i r s t two weeks of c h i l l storage include species of Pseudomonas, Moraxel 1a/Acinetobacter and Flavobacter. Shewan (1971) reported that during storage Pseudomonas spp. quickly assume the dominant role , constituting as much as 90-95% of the total f lora after 12 days holding. Similar patterns are reported for meat (Ingram, aj_, 1971; Newton et a}_, 1978) and poultry (Ayres et a l , 1950; Lea et_ jil_, 1969a), suggesting that some common factors are involved. Spoilage is often accompanied by the development of various characteristic odours. Bacteria producing these odours have been implicated as being primarily responsible for spoilage. The Pseudomonas group of organisms are the ones most commonly associated with these odours (Castell and Anderson, 1948; Castell and Greenhough, 1957, 1959; Lerke et al_, 1963, 1965, 1967; Adams et aj_, 1964; Herbert et al_, 1971). These odours are often described as f ru i ty , oniony, musty, or potato-l ike and result from bacterial deamination of amino acids, the production of volat i le esters, and the sulphur compounds from sulphur-containing amino acids. According to Liston (1980), the appearance of hydrogen sulfide producers 6 2 in levels in excess of 10 /cm serve as an indication of spoilage. - 9 -Chung (1968) reported that, in the early stages of spoilage, proteinase production by spoilage bacteria is repressed and becomes de-repressed during the later stages of spoilage, giving rise to an increase in protein hydrolysis and replenishment of the amino acid pool. This action is presumed to influence the production of a l l end products derived from amino acids including hydrogen sulfide and dimethyl sulfide and methyl sulfide from methionine. Shewan (1977) detected these substances after 8 and 10 days storage. Another spoilage feature of some marine f ish is the formation and accumulation of trimethylamine produced by bacteria acting on trimethylamine oxide contained within the f lesh. Certain v is ib le characteristics are associated with spoiling f ish (Table II). Minced f ish is more susceptible to spoilage than whole f i sh . I n i t i a l l y , the bacteriological quality of the mince reflects that of the f ish from which i t was made. In minced product the loss of structural integrity allows the bacterial load to be evenly distributed throughout the mince. The fine particulate nature of the mince provides more favourable conditions for bacterial growth as well as providing intimate contact between bacteria and available nutrients (Cann et a l , 1976). - 10 -TABLE II CHARACTERISTICS OF SPOILING FISH Loss of surface sheen and dullness of colour. Surface slime becomes thick, turbid and dirty with yellowish o brown colours. The eyes shrink and become sunken: pupils become cloudy and c opaque. G i l l s are discoloured to a bleached pink or greyish yellow and covered with thick slime. The flesh becomes softer, easily stripped from the backbone and exudes juice under light pressure. Flesh loses its e las t i c i ty and retains finger indentations. Odour ranges from fishy to sickly sweet, ammoniacal or putrid. Pelagic species may become rancid. - 11 -B. Lactic Acid Bacteria The name, lact ic acid bacteria, was orig inal ly created for bacteria causing fermentation and coagulation in milk. For instance, Weigmann (1899) defined lact ic acid bacteria as those which produce milk acid from milk sugar. At the present time the group is characterized as Gram-positive, non-spore formers, which ferment carbohydrate and produce lact ic acid. The group is acid tolerant, is of non-aerobic habitat and is catalase negative. Although the lact ic acid bacteria are normally considered as catalase negative, there have been several instances of lact ic acid bacteria giving positive reactions, e.g. Pediococci (Felton et al_, 1953) and Lactobaci11 us (Dacre and Sharpe, 1956). Lactic acid bacteria are generally non-motile although again some motile strains exist (Harrison and Hansen, 1950; Diebel and Niven, 1958). Lactic acid bacteria do not as a rule reduce nitrate but Spencer (1969) reported isolating some nitrate reducers from meat. They also produce lact ic acid under anaerobic and microaerophi1ic conditions, resulting in a decline in the medium pH to below 4.0. The amount and isomeric configuration of the lact ic acid produced tends to vary depending on species. The lact ic acid bacteria are subdivided into various genera on the basis of fermentation (end products) and ce l lu lar configuration (Table III). TABLE III CATEGORIES OF LACTIC ACID BACTERIA Homofermehtative cocci in pairs or chains, e.g. Streptococci. Heterofermentative cocci in pairs or chains, e.g. Leuconostoc. Homofermentative cocci dividing into two planes to give tetrads, e.g. Pediococcus. Homofermentative or Heterofermentative rods, e.g. Lactobacillus. Source: Sharpe, Fryer and Smith (1966). - 13 -Homofermentative lact ic acid bacteria produce 1.8 moles of lact ic acid per mole of glucose ut i l ized with minor amounts of acetic acid, ethanol and carbon dioxide. Heterofermentative types produce less than 1.8 moles of lact ic acid as well as acetate, glycerol , mannitol and carbon dioxide (Doelle, 1969). These organisms are used extensively in the food industry, especially in the manufacture of sausage. They are known his tor ica l ly to improve shelf l i f e of fermented dry and semi-dry sausages. In recent years there has been an increased diversif icat ion in the use of these organisms in the area of non-fermented meat products. This increase in act iv i ty is attributed to ris ing costs and an appeal for the use of "natural" food preservation and to government action (Anon, 1980). Their increased use in the food industry results from their ab i l i ty to produce various antimicrobial metabolites during growth, for example, hydrogen peroxide and other unidentified compounds: many of which inhibit the growth of spoilage causing organisms. There are also widely held traditions which relate to the therapeutic value of fermented milk beverages. Lactobaci l l i as well as other lact ic acid bacteria have been intimately involved with man for centuries. This has resulted in not only the manufacture of food products but the promotion of beneficial interactions in various parts of the human body, especially in the - 14 -maintenance of proper microbial balance. One example is the implantation of lactobaci l l i in the intestinal tract (Speck, 1975; Sandine et al_, 1972; Daly et al_, 1972). This is due to the fact that these organisms are capable of growth and survival in the intestinal tract and produce antagonistic actions toward enteric pathogens. There are numerous reports on the inhibitory actions of lact ic acid bacteria towards organisms l ike Staphylococcus aureus (Dahiya et a l , 1968; Iandolo et 1965; Kao et aj_, 1966), Pseudomonas (Pinheiro et al_, 1968; Price etal_, 1970), and Salmonella (Sorrels et a l , 1970). Reddy e_t al_, (1970, 1975) report inhibition of Gram-negative bacteria inherent in ground meat and longissimus muscle steak when meat was inoculated with Streptococcus lactis and Leuconostoc citrovorum. Daly ejt aj_, (1972) observed a similar phenomenon in ground beef, milk, cheese when inoculated with Streptococcus d iacet i lac t i s . G i l l i l and and Speck (1975) were able to induce the same phenomenon in ground meat using Lactobacillus bulgaricus and Pediococcus cerevisiae. G i l l and Newton (1978) reported the inhibition of Enterobacter sp. and Mycobacterium  thermosphactum as being caused by production of antibiotics by Lactobaci1lus spp. Raccach and Baker (1978) reported the use of lact ic acid bacteria as an anti-spoi1 age and safety factor in cooked and mechanically deboned poultry meat. They showed that when a mixture of Lactobaci11 us - 15 -piantarum and Pediococcus cerevisiae was added to the deboned meat, repression of Pseudomonas species (Pseudomonas fluorescens, Pseudomonas  f r a g i , and Pseudomonas putrefaciens) were suppressed by 2.5 to 3.8 log cycles. Salmonella typhimurium and Staphylococcus aureus were tota l ly repressed when grown in association with a mixture of these same lact ic organisms. More recent developments include the use of lact ic acid bacteria to inhibit the growth of Clostridium botulinum in non-fermented meats (Tanaka et aj_, 1980). It is recognized that sodium n i t r i t e , when added to cured meat, provides protection against growth and toxin formation by C. botulinum (Duncan et aj_, 1968). It has also been reported that when n i t r i t e is added to bacon i t reacts with secondary amines present within the bacon to form carcinogenic nitrosamines (Fazio et aj_, 1973; Pensabene e_t _al_, 1974). While decreasing the n i t r i t e content minimizes nitrosamine formation, i t also increases the risk of botulinum toxin production should these organisms be present. Bacus (1979) has provided evidence that added lact ic acid bacteria in the presence of a fermentable carbohydrate source provides effective protection against C.  botulinum. As a result of the lowered pH, residual n i t r i t e also dissipates. It was also indicated that a reduced nitrosamine level was achieved at the time of frying. - 16 -C. Microbial Antagonism The association of microorganisms with each other plays an important role in the spoilage of food. The ab i l i ty to compete for nutrients between the different types of bacteria, yeasts and molds present on a given food wi l l determine which organism wil l outgrow the others. Microorganisms exhibit not only antagonistic behaviour but also exhibit symbiotic, synergistic and metabiotic behaviour to produce a desired outcome as in the case of some fermented products e .g . , pickles, dry sausages and various dairy products. Antagonistic effects of organisms have been recorded as early as the 1800's. Tyndall (1876) reported the struggle for survival between bacteria and penici l l ium. Metchnikoff (1908) postulated the use of lact ic acid bacteria as a repressor of proteolytic bacteria in the intestinal canal. A protocooperative relationship is a form of mutualism in which the survival of the interacting species is not essential . Such relationship exists between Lactobaci1lus piantarum and Streptococcus faecal is where the former produces fo l i c acid which is subsequently ut i l ized by the lat ter . Phenylalanine produced by the Streptococcus faecal is is ut i l i zed by Lactobacillus plantarum (Normiko, 1956). - 17 -Synergism and symbiosis are other forms of mutualism. The former occurs when the production of a given by-product is greater in a mixed rather than a pure population. This may occur where organisms coexist in the same environment and when the growth of one species is highly dependent on the growth of the other. For example, in the manufacture of yoghurt acid production is greater when Lactobacillus bulgaricus and Streptococcus thermophilus are added together than when either organism is present alone. When the growth of one species is enhanced by the presence of a second species with no effect on the lat ter , the process is referred to as commensal ism. Nath and Wagner (1973) demonstrated a synergistic effect as a result of commensal ism. When hydrogen peroxide was produced by some lact ic acid bacteria, acid production was hindered. If, however, a strain of Micrococcus was used to remove the hydrogen peroxide the lactics responded through stimulated growth and acid production. Amensalism, another form of microbial interaction, expresses i t s e l f when the growth of one species is repressed as a result of the presence of toxic substances produced by a second species or to a change in environmental condition. Various forms of this relationship occur in foods. Working with f i s h , Price and Lee (1970) reported that the growth of Gram-negative organisms in spoiling f ish could be inhibited by hydrogen peroxide produced by a lact ic acid bacteria. Pseudomonas organisms were found to decrease in numbers in the presence of - 18 -Lactobacillus plantarum and were undetectable after 48 hours. Further analysis indicated the presence of hydrogen peroxide, the concentration of which paralleled the production of an inhibitory substance produced by the Lactobaci1lus. Dahiya and Speck (1968) also reported strong inhibitory properties of hydrogen peroxide produced by Lactobaci1lus. They found that the concentration of hydrogen peroxide increased during storage of the lactobaci l l i suspension at 5C obtaining a maximum level after 5 days. The amensal relationship can be also attributed to other substances such as antibiotics and bacteriocins. There are various antibiotics produced by lact ic acid bacteria for example, nisin and bulgarican. The antibiotic nisin is produced by Streptococcus lac t i s . This antibiotic was found to be useful in heat processing as a prevention against the outgrowth of bacterial spores especially after a package was subjected to minimum heat (Heineman et al_, 1965). Prescott and Dunn (1959) reported the use of nisin to inhibit Clostridium botulinum and Staphylococcus aureus as well as other food poisoning organisms in certain foods. Reddy and Shahani (1971) reported the production of an antibiotic called bulgarican by Lactobacillus bulgaricus which is active against Gram-positive and Gram-negative bacteria. Other investigators (Tramer, 1966; Bryan, 1965; Deklerk, 1961) have reported antibiotic - 19 -act iv i ty in Lactobacillus acidophilus. Shahani et aj_, (1976) concluded that the inhibitory substance produced by L. acidophilus could be responsible for the therapeutic values attributed to acidophilus milk and yoghurt. D. Trimethylamine (TMA) - As a Spoilage Indicator Shewan et_ a]_, (1971) stated that one of the characteristic features of the chemical changes taking place in fish muscle during spoilage is the production of volat i le bases such as ammonia and short chain aliphatic amines. Volatiles of most species of marine f ish consist of mainly ammonia and trimethyl amine (Shewan et^  ail_, 1971). Trimethyl amine has for a long time been considered to be a valuable tool for evaluating f ish deterioration, especially in the case of groundfish e .g. , cod. Estimation of trimethylamine in fish has been proposed by the Codex Alimentarius Committee on Fish and Fishery Products as the main objective cr i ter ion for assessing quality of f ish such as cod for international trade (Shewan et aj_, 1971). As an indicator of quality, TMA has been and s t i l l is surrounded by much controversy with reference to i ts merits. Some of this controversy wil l be discussed here. TMA formation was f i r s t proposed by Beatty and Gibbons (1937) as an index of freshness of f i sh . They reported that TMA accumulation was dependent on the bacterial population. Beatty, 1938; and Watson, 1939 reported a rapid increase of TMA in cod muscle which - 20 -was correlated to periods of rapid bacterial growth. The production of TMA is attributed to the bacterial reduction of trimethylamine oxide (TMAO) (Beatty, 1938; Tarr, 1940). This compound is found in variable amounts in marine f ish but not in fresh water f ish (Beatty, 1939; Shewan et a l , 1971). Various researchers have reported a number of misgivings associated with TMA as a test, such as: unre l iabi l i ty of data for Pacific coast f ish (Tarr and Ney, 1949) and seasonal fluctuations of TMA content (Shewan and Jones, 1957). Partmann (1951) also reported unreliable results in the use of TMA to study bacterial changes in f i sh . Bose and Chowdhury (1959) obtained overlapping values for both fresh and spoiled f ish using indices of total N, TMA and ammonia in a study of iced, stored f i sh . These inconsistencies have not deterred the use of TMA as a spoilage indicator, as its appl icabi l i ty has been demonstrated in numerous instances. Castell et aj_, (1958) reported obtaining a significant correlation between TMA and assigned grades of fresh and frozen f ish despite differences attributed to species and seasonal fluctuations. Velanhar and Govindan (1960) indicated that TMA and volat i le bases were quite dependable when used as a spoilage indicator for Indian prawns stored in ice. Other investigators have found these compounds to be adequate spoilage indicators as well as providing an index of freshness of raw fishery products stored above freezing temperatures (Shewan and Liston, 1956; - 21 -Farber and Lerke, 1961). Castell (1970), attributes some of the confusion of this test to over-simplification of the relationship of TMA to spoilage. However, not a l l bacteria are capable of reducing TMAO although there is a sufficient number of these organisms present as part of the normal f lora of f ish so that TMA wil l always be formed. If the amount of TMA present in the f ish muscle is determined, a good index as to the extent of bacterial deterioration may be provided. Although the above arguments hold true for some species of f ish e.g. , cod, there are other species to which this theory does not apply. Castell (1970) explains that there are other agents capable of reducing TMAO such as: gamma radiation, trace amounts of heavy metals (e .g. , iron, vanadium), enzymes present in the pyloric caecum of some fish or in the red muscle of pelagic fish e .g . , tuna. Both tissues have been shown to contain enzymes capable of reducing TMAO. Haemoglobin is also capable of reducing TMAO. The importance of making reference to these other mechanisms is to point out their existence; i t is also recognized that they might not be as efficient as bacteria in the conversion of TMAO to TMA. - 22 -TMA is not the only product of conversion: DMA (dimethylamine), MA (methylamine) and formaldehyde are also produced. During frozen storage of most gadoid species, DMA is the main product (Tozawa, 1969 cf . Gould and Peters, 1971). This was also confirmed by Castell (1970). Nevertheless, i f Castel l 's (1970) summation of the status of TMA is to be accepted, i t would appear that TMA is s t i l l considered one of the best tests for indicating the pattern of spoilage in chi l led groundfish and remains a useful mechanism for the grading of such f ish as cod and haddock. In the case of cod, DMA interference appears to be minimal as shown in Figure 2. - 23 -o * • • - — — 1 • •— Q S 10 IS STORAGE PERIOD IN ICE (DAYS) Figure 2. The rate of development of TMA and DMA in wrapped cod f i l l e t s stored in ice. Source: Castel l , 1970. - 24 -MATERIALS; AND METHODS A. Test Organisms The cultures used in this study were obtained as frozen concentrates from Microlife Technics, Sarasota, FL and included: 1. Pediococcus cerevisiae This organism is c lassif ied as Lactacel 75, No. L750480, and is commonly used for the production of dry and semi-dry sausages, e.g. pepperoni, hard salami, thuringer, etc. It is a highly concentrated lact ic culture and possesses the ab i l i ty to produce lact ic acid rapidly at either low or high temperature. 2. Lactobacillus plantarum - P. cerevisiae This culture was obtained in the frozen state in the form of a mixed culture. It is c lass i f ied as Lactacel MC, No. M12772 and is used in the production of summer sausage e .g . , Lebanon bologna, thuringer, Holsteiner cervelat. - 25 -B. Preparation of Minced Fish Stored 2-3 Days in Ice * Dressed, grey cod (2-3 days old) were obtained from a commercial trawler. Fish f i l l e t s were minced using a s ter i le meat chopper, Biro Model 8-22, (Biro Mfg. Co. , Marblehead, OH, U.S.) equipped with a one-quarter inch porosity plate. The minced product was mixed to obtain a uniform distribution of psychrotrophic bacteria. After mincing, the product was dispensed into s ter i le polyethylene bags (Whirlpak) in 100 g quantities. The natural microbial f lora of the f ish served as a source of psychrotrophic organisms. P. cerevisiae (Lactacel 75, No. L750480, Microlife Technics) or a combination of P. cerevisiae and L. plantarum, (Lactacel MC, No. M12772, Microlife Technics) was 7 9 added to a f inal concentration of 10 and 10 organisms per g of mince. Dextrose was added to a f inal concentration of 0.3 percent. Al l samples of inoculated f ish were blended by means of a Colworth 'Stomacher,' Lab-blender 400 (Model BA 6021 240, Seward Laboratory, Blackfriars Road, London) for one minute to ensure distribution of the organisms. Samples containing 50g of minced product were blended with 9 times their weight of 0.1% (w/v) s ter i le peptone (Difco Laboratories, Detroit, MI) water using a Stomacher 400. This homogenate was used for bacteriological analysis. - 26 -C. F i l l e t Preparation Fresh grey cod f i l l e t s were obtained from two local f ish processing plants. Postmortem age varied between four to eight days. F i l l e t s were treated in one of two ways. 1. Dipping Samples were dipped in Suspension 1 or Suspension 2 for a period of one min and allowed to drain for ten sec. 2. Spraying Samples of f i l l e t s were sprayed with 1 ml of Suspension 1 or with Suspension 2. Suspension 1: 10 g Lactacel MC culture of P. cerevisiae and L. plantarum in 1 L of 0.1% dextrose solution. Final ce l l population was 3.0 x 10 9/ml. Suspension 2: 20 g Lactacel MC culture in 1 L of 0.1% dextrose Q solution. Final ce l l population was 5.6 x 10 /ml . - 27 -2 Samples measuring 72 cm and weighing between 73-110 g were used in the preparation of homogenate. Tissue samples were obtained aseptically. Samples were blended for two minutes with 9 x their wei of 0.1% (w/v) s ter i le peptone water (Difco) using a Waring blender. - 28 -D. Bacteriological Media Various bacteriological media were used to determine viable counts. The bacteriological media used in this study were either purchased from BBL (Becton, Dickinson and Co. , Baltimore, MD, U.S.A.) or Difco (Difco Laboratories, Detroit, MI, U.S.A.) or were made up from ingredients supplied by them. The media used were: 1. Standard Methods Agar 2. Lactobacillus Selective Agar 3. Bacto-peptone. E. Determination of Viable Count 1. Spiral Plate Method The method of Gi lchris t (1973) was used. Pre-poured agar plates containing the appropriate agar were placed in a 37C incubator for one hour to dry the agar surface in preparation for plating. The spiral plate method is described in the Appendix. - 29 -2. Pour Plate Method Standard plate count procedures according to the American Public Health Association (APHA), 1976 were used. For both methods mentioned, the plates were prepared in duplicate. Standard Methods Agar was used to enumerate the psychrotrophic organisms and Lactobacillus Selective Agar was used to enumerate the lact ic acid producing organisms. Incubation time and temperature for the psychrotrophic organisms was 48 hours at 24C. Lactic counts were made after 72 hours at 24C. Only plates containing 30-300 colonies were used for estimating bacterial numbers (APHA, 1976). The Colony Forming Units (CFU) counts are expressed as log-jQ per g of sample. F. Storage of Samples Sample storage temperatures used in this study were 4C, 11C and 24C. Test samples of inoculated homogenates and controls were stored for six days at 4 or 11C and four days at 24C. Representative samples for each temperature regime were removed daily for chemical and microbiological analysis. - 30 -pH Measurement pH of minced samples from various treatments was measured dai ly . Measurements were made using a PHM 82 standard meter manufactured by Radiometer, Copenhagen. Diluent Bacto-peptone (0.1% w/v) was used as the diluent for a l l bacteriological examinations and for the preparation of homogenate. Inhibition of Spoilage Organisms by P. cerevisiae and a Mixture of P. cerevisiae and piantarum Minced f ish was inoculated with Pediococci and Lactobaci l l i organisms at 7 9 concentrations of 10 or 10 ce l l s /g of mince. Test samples and control samples were examined chemically and microbiologically to determine the degree of inhibit ion. Inhibition Test To determine i f the observed inhibition of psychrotrophic organisms occurred during storage of the minced fish sample, or subsequently during plating, an aliquot of the culture containing the test organism was ser ia l ly diluted and one ml of each dilution mixed with Standard - 31 -Methods Agar and allowed to so l id i fy . These plates and control plates containing uninoculated media were then streaked with diluted samples of minced f ish by means of a spiral plater. Total counts were determined after 48 h at 24C. K. Trimethylamine Analysis (TMA) Twenty-five grams of frozen samples were blended with 75 ml of 5% (w/v) trichloroacetic acid (TCA) f i l tered through a Whatman #1 f i l t e r , and the clear f i l t r a t e assayed for TMA content. TMA was assayed using the modified Conway diffusion technique of Beatty and Gibbons (1937) as further modified by Bryant et a]_y (1973). A microdiffusion disk (Obrink, 1955) was ut i l ized for this analysis. The releasing agent used was saturated Na^PO^ to which sol id KOH had been added until turbidity persisted. A 3.1% H^BO^ solution containing 1 ml of mixed indicator (Conway, 1958) was used as the trapping agent. Mixed indicator contained a mixture of 0.1% bromocresol green in 95% ethanol and 0.1% methyl red in 95% ethanol. 0.7 ml of 40% formaldehyde was added to fix any ammonia present in the sample. Samples were incubated for one and a half hours at 37C, then t i trated with 0.02N HC1. L. Sensory Evaluation of Fresh F i l l e t s The method used was that developed by Shewan et aj_, (1953) for the sensory assessment of spoilage of wet white fish stored in ice (Table IV). In this method, the f ish sample is given a numerical rating - 32 -of one to ten, as determined by a trained panel, on the basis of odour (10 is best). A 6-member panel of judges was used. Each judge was provided with an odour evaluation sheet. A sample of control and treated f i l l e t s were presented for evaluation on each day of the experiment. Day zero was used to designate the day the fish arrived at the laboratory. - 33 -TABLE IV THE ODOURS OF RAW AND COOKED WHITE FISH TAKEN FROM THE SCORE SHEET OF SHEWAN ET AL, (1953), IN THEIR ATTEMPT TO DEVELOP A SCORING SYSTEM FOR THE SENSORY ASSESSMENT OF SPOILAGE OF WET WHITE FISH STORED IN ICE Odours Score From raw fish Fresh 'seaweedy' 10 Loss of fresh 1seaweediness1, shellfishy 9 None or neutral 8 Slight musty, acetamide-1ike, milky or caprylic acid-l ike 7 'Bready', 'malty', 'yeasty' 6 Lactic acid, 'sour mi lk' , or o i ly 5 Some lower fatty acid (e.g. acetic or butyric acids), 'grassy', s l ight ly sweet, or frui ty 4 Stale, sour, 'cabbage water', ' turnipy', or phosphine-1ike . . . . . . . 3 Ammoniacal (trimethylamine and other lower amines) plus strong o-toluidine-1ike 2 Hydrogen sulphide, other sulphide and strong ammoniacal 1 Nauseating, putrid, faecal; indole, ammoniacal, etc 0 From cooked fish Strong fresh 'seaweedy' 10 Some loss of fresh 'seaweediness' 9 Lack of, or neutral 8 Slight strengthening, but not sour or stale; 'wood shavings', 'woodsap1, van i l l in or terpene-1ike; slight sa l t - f i sh or cold storage-like 7 'Condensed mi lk 1 , caramel or toffee-l ike 6 'Milk j u g ' , 'boiled-potato' or 'boiled clothes', or metallic 5 Lactic acid, 'sour milk' or o-toluidine-1ike 4 Some lower fatty acid (e.g. acetic or butyric acids), 'grassy', 'soapy', 'turnipy' or 'tallowy' 3 Ammoniacal (trimethylamine and lower amines) 2 Strong ammoniacal (trimethylamine, etc.) and some sulphide 1 Strong putrid and faecal (ammonia, indole, etc.) 0 Source: Shewan et a l , (1953). - 34 -RESULTS AND DISCUSSION A. Evaluation of Fresh F i l l e t s At the beginning of this research study, fresh cod f i l l e t s were examined. The objective was to determine the influence of treatment of f ish f i l l e t s with lact ic acid bacteria on the inhibition of growth of spoilage organisms. The test culture used was a mixture of P.  cerevisiae and L. plantarum. The culture suspension was adjusted to give a f inal population of 10 ce l l s per ml. F i l l e t s were either sprayed with or dipped into a suspension of the test organisms. The dipped or sprayed f i l l e t s adsorbed a low level of lact ic culture, giving a population of 106 ce l ls per g. At this low ce l l population samples stored for seven days at 4C showed no apparent extension of storage l i f e . Trimethylamine-N values did show some difference. At the beginning of storage, 5 mg % TMA-N was realized for both treated and untreated samples. By the second day of storage, the untreated sample had surpassed the acceptable l imit of 10 mg % TMA-N and the treated sample one to two days later. Odour evaluations were carried out by a six-member panel according to the method of Shewan, 1953 as discussed in the Methods section. - 35 -A mean score of 7.5-10 indicates good quality; 4.5-7.5, medium quality; and a score of less than 4.5 is considered unacceptable. Table V shows the mean odour scores and standard deviation for treated and untreated samples. TABLE V ODOUR SCORES FOR TREATED AND UNTREATED COD FILLETS STORED AT 4C. TREATED COD FILLETS CONTAINED A MIXTURE OF P. cerevisiae AND L^ plantarum (10 6/g) STORAGE TIME (Days) UNTREATED TREATED Mean s .d .* Range Mean s .d .* Range 1 8.8 0.5 8.0-9.0 8.5 1.0 7.0-9.0 4 6.4 1.4 5.0-8.0 6.5 2.7 3.0-9.0 5 6.0 0.8 5.0-7.0 7.1 2.3 4.0-9.0 6 5.5 2.1 3.0-8.0 4.5 2.1 2.0-7.0 7 4.3 0.5 4.0-5.0 2.8 0.1 2.0-4.0 Standard deviation. From Table V i t can be seen that both treated and untreated f i l l e t s were only considered to be of good quality on the f i r s t day of storage and of medium quality unti l the sixth day of storage. By the seventh day both were considered unacceptable. It was observed that the degree of variation was by far greater in the treated samples than the untreated samples. This could be attributed to the fact that, although the panelists detected an off odour, i t was not similar to the odours that they were accustomed to for fresh or spoiling f i sh . - 36 -Coincidentally, the CFU count per g was IO'' for the treated sample as well as for the untreated on the seventh day of storage, which reflects the low odour scores. F i l l e t s containing more than 10 organisms per g are considered unacceptable. Odour evaluation was also carried out on cod f i l l e t s treated with P. cerevisiae in a similar manner to the previously mentioned samples. The results of these odour scores is shown in Table VI. TABLE VI ODOUR SCORES FOR TREATED AND UNTREATED COD FILLETS STORED AT 4C. COD FILLETS WERE TREATED WITH P. cerevisiae Storage Time (Days) UNTREATED TREATED Mean s .d .* Range Mean s .d .* Range 0 7.9 0.7 7.6-9.0 8.4 0.9 7.5-10.0 1 7.0 0.7 6.0-9.5 7.1 0.6 6.5-8.3 2 6.3 0.6 5.6-7.2 7.8 0.9 7.0-8.0 3 7.0 1.0 6.0-8.0 7.6 0.5 7.6-8.3 6 2.5 1.2 1.0-4.5 3.9 1.5 2.0-6.0 * Standard deviation. Again, no marked differences were observed between treated and untreated f i l l e t s . - 37 -It should be pointed out here that there was no marked inhibit ion of the natural microflora of the f i l l e t s by the lact ic acid bacteria. This could be attributed to the fact that the population of lact ic organism which was adsorbed to the surface of the f i l l e t s was too low. Some 8 9 studies have shown that high levels (10 -10 ) of lact ic acid bacteria/g was successful in extending the storage l i f e of non-fermented refrigerated foods (Gi l l i land and Speck, 1975). The data indicated that extension of storage l i f e of f i l l e t s is not feasible when low levels (10^) of lact ic acid bacteria is used. Inhibition of the microflora of the f i l l e t s might be greatly enhanced i f a larger population of lact ic acid bacteria is used. The data proved quite useful for continuation of the research study which is focussed on the ab i l i t y of the lact ic organisms to suppress the psychrotrophic population responsible for spoilage in minced cod muscle. B. The Effect of P. cerevisiae on the Extension of Storage Life of Minced Fish Stored at 4C Psychrotrophic organisms contribute substantially to the spoilage of flesh type foods, e .g . , f i sh (Reay and Shewan, 1949; Liston, 1980; Shewan, 1971), poultry (Ayres, 1950) and meats (Ingram, 1971). The growth of the psychrotrophic population was therefore monitored to determine the storage l i f e of the minced product. Storage l i f e was used in this study as the length of time (days) over which minced fish can be stored and s t i l l be of acceptable quality. The incubation temperature - 38 -used for the enumeration of psychrotrophic organisms was 24C. Raccach (1977) compared psychrotrophic counts at IOC and 25C and found no significant difference between the two counts. Other workers have also reported using similar temperature for minced f ish (Cann and Taylor, 1976). The addition of P. cerevisiae ce l l s to the minced product at a population of 10^/g provided no repressive act iv i ty on the developing spoilage f lora naturally occurring within the minced fish (Figure 3). The addition of dextrose showed no marked repression of the psychrotrophic bacterial population (Figure 4). When minced fish was treated with P. cerevisiae at a concentration of 9 / \ 10 ce l l s per g mince (Figure 5), repression of the psychrotrophic f lora was observed and a storage l i f e extension 1.5 days was realized over a storage period of six days. One mill ion ce l l s per g is the recommended bacteriological l imit for minced fish (International Commission on Microbiological Specification for Foods [ICMSF], 1975). 9 Samples containing 10 P. cerevisiae per g showed a psychrotrophic count of 10 per g on the sixth day of storage, whereas the control sample attained a count of 10 ce l l s per g on the fourth day of storage. Samples treated with the same population of P. cerevisiae but with the addition of 0.3% dextrose prolonged the storage l i f e by greater than two days since the treated samples did not attain a psychrotrophic count of 10 /g throughout the storage period. - 39 -• o SPC Test O O SPC Control A * pH Test & A pH Control TEST: P. cerevisiae (107/g) -, 1 — 1 1 1 — — r 1 2 3 4 5 6 STORAGE TIME (days) F igure 3. The growth of p s y c h r o t r o p h i c organisms i n the presence of P. c e r e v i s i a e i n minced f i s h s to red at 4C. F i n a l p o p u l a t i o n of t e s t organisms was IO 7 c e l l s / g of mince. - 40 -• • SPC Test O — - o SPC Control A ~A pH Test A A pH Control TEST: P. cerevisae (10?g ) 4-0.3% Dextrose 9.0 \-8.0 r-7.0 L6.0 5.0 STORAGE TIME (days) Figure 4. The growth of psychrotrophic organisms in the presence of P. cerevisiae and 0.3% dextrose in minced fish stored at 4C. Final population of test organism was 107 ce l l s /g of mince. - 41 -A large decrease in bacterial counts was evident, approaching 1 log cycle in the absence of added dextrose (Figure 5) and 3 log cycles for samples containing 0.3% added dextrose (Figure 6). The pH values of control and test samples containing 10^ cel ls of P. cerevisiae per g of mince with or without 0.3% dextrose showed no marked differences. The lowest value was only 0.1 unit lower than the control (Table VII). With an increase in the concentration of inoculum Q to 10 ce l l s the pH decline was more pronounced (Table VIII). It would appear from this data that the pH of the treated mince was dependent on the population of lact ic organisms and on the presence of a carbohydrate source. A decrease in the pH of the mince during storage cannot be used solely to explain the repressive act iv i ty of the test organisms since similar populations of spoilage f lora were observed at both higher and lower pH values. The lower pH values could, however, contribute by providing a lower pH environment which could greatly stimulate the lact ic organisms to produce anti-metabolites. Various researchers have implicated lower pH as a contributing factor in bacterial inhibition by lact ic acid bacteria with respect to the metabolic products which they produce (Daly et aj_, 1972; Sorre l s»and Speck, 1970; Pinheiro et aj_, 1968; Kao and Frazier, 1966). - 42 -• • SPC Test O O SPC Control A • pH Test A - A pH Control TEST: P. cerevisiae <109/g) 1 2 3 4 5 6 STORAGE TIME (days) Figure 5. The growth of psychrotrophic organisms in the presence of P. cerevisiae in minced f ish stored at 4C. Final population of test organism was 10 9 ce l l s /g of mince., * No growth on lowest dilution (1(H). - 43 -• • SPC Test O -O SPC Control A A pH Test A- pH Control STORAGE TIME (days) Figure 6. The growth of psychrotrophic organisms in the presence of P. cerevisiae and 0.3% dextrose in minced fish stored at 4C. The f inal population of test organism was 109 ce l l s /g of mince. * No growth on lowest dilution (10~^). - 44 -TABLE VII THE pH VALUES OF UNTREATED AND TREATED MINCED FISH DURING STORAGE AT 4C Treatment Storage Time (Days) 7 10 /g Pediococci Untreated lp ' /g Pediococci Cells + 0.3% Cells dextrose 0 6.94 6.82 6.92 1 6.87 6.89 6.89 2 6.93 6.92 6.90 3 6.85 6.87 6.85 4 6.85 6.84 6.82 5 6.89 6.86 6.80 6 6.91 6.86 6.81 - 45 -TABLE VIII THE pH VALUES OF UNTREATED AND TREATED MINCED FISH DURING STORAGE AT 4C Storage Time (Days) Untreated Treatment 10 /g Pediococci Cells 10 /g Pediococci Cells + 0.3% dextrose 0 1 2 3 4 6 6.87 6.87 6.86 6.88 6.86 7.00 6.76 6.75 6.70 6.65 6.55 6.38 6.67 6.22 6.05 5.89 6.25 6.10 - 46 -C. The Effect of a Mixture of Pediococcus cerevisiae and Lactobacillus plantarum on Minced Fish During Storage at 4C o Samples of minced fish were inoculated with 10 ce l l s of a mixture of P. cerevisiae and L. plantarum per g of mince and stored for 6 days at 4C. A storage l i f e extension of two days was achieved in the treated minced samples (Figure 7). This was based on the fact that the control samples attained a ce l l population of 10 cel ls per g of mince on the fourth day of storage. In contrast, the test samples did not attain this level unti l the sixth day of storage. Unlike samples treated with dextrose and P. cerevisiae alone, the addition of dextrose to the mixed culture system did not markedly change the results appreciably with respect to either total bacterial numbers or pH of the minced product after 6 days of storage (Figure 8, Table IX). There did, however, appear to be a sl ight extension of the lag phase prior to the onset of logarithmic growth of spoilage organisms in the presence of added dextrose. Whether the less pronounced effect of added dextrose observed with the mixed culture t r i a l s was the result of strain differences between the two sets of commercial cultures or in fact due to a lower population of P. cerevisiae in the mixed culture system, is not known. In this respect, i t is interesting to note that the single P. cerevisiae culture (Lactacel 75) is designated by the supplier as one showing enhanced fermentative capacity over a broad temperature range. - 47 -• • S P C Test O O SPC Control -A pH Test - - A pH Control Mixture of P. cerevisiae &L. plantarum d 0 8 / g ) 9.0 r-8.0 L7.0 r-6.0 r 5 . 0 x a STORAGE TIME (days) Figure 7, The growth of psychrotrophic organisms in the presence of mixture of P. cerevisiae and L. plantarum in minced fish stored at 4C . Final population of test organisms was IO8 cells/g of mince. - 48 -• • SPC Test O o S P C Control -A pH Test A pH Control TEST: Mixture of P. cerevisiae & L. plantarum (10 8 /g) +0.3% Dextrose JO" L.9.0 r-8.0 I 7.0 \-6.0 X 2H r-5.0 1^ 2 T" 3 -r-4 1 5 T-6 STORAGE TIME (days) Figure 8. The growth of psychrotrophic organisms in the presence of a mixture of P. cerevisiae and L. plantarum and 0.3% dextrose in minced fish stored at 4C. Final population of test organisms was 108 ce l l s /g of mince. - 49 -TABLE IX THE pH VALUES OF UNTREATED AND TREATED MINCED FISH DURING STORAGE AT 4C Treatment Mixture of Q 10 /g Pj_ cerevisiae and L^ pi antarum + dextrose 0 6.45 6.60 6.60 1 6.50 6.35 6.50 2 6.49 6.40 6.35 3 6.49 6.25 6.23 4 6.54 6.24 6.37 5 6.55 6.39 6.57 6 6.90 6.75 6.70 Storage Time Mixture of 10 /g cerev and L. piantarum ( D a y s ) Untreated 8 P. erevisiae - 50 -When the mixed lact ic inoculum was increased to 10 ce l l s per g of minced f i s h , an even greater reduction in spoilage f lora was evident (Figure 9); standard plate counts of spoilage f lora remaining below the 10 per g level . Again, the stimulatory effect observed with P. cerevisiae alone with added dextrose was not observed with the mixed culture system (Figure 10). The odour of spoiling f ish detectable in the control about the fourth day of storage, became stronger as storage progressed. The samples containing lact ic organisms did not exhibit this off odour. It would appear that the lact ic organisms, when added to minced f i s h , inhibit those organisms which are responsible for-producing off odours. pH decline was also evidenced during storage with lower values occurring in samples with added dextrose (Table X). D. The Effect of f\_ cerevisiae and a Mixture of Pj_ cerevisiae and L. plantarum- on Minced Fish Stored at 11C and 24C Temperatures of 11 and 24C were chosen to determine i f lact ic organisms were able to inhibit those organisms responsible for spoilage at non-refrigeration temperatures. Such information would be of practical value in areas where refrigeration is not readily available. g Samples with 0.3% dextrose were inoculated with 10 cel ls of either a mixture of cerevisiae and piantarum or P_^  cerevisiae alone per g - 51 -• • S P C Test O O SPC Control A * pH Test A- A pH Control TEST: Mixture of P.cerevisiae & L. plantarum (109/g) STORAGE TIME (days* igure 9. The growth of psychrotrophic organisms in the presence of mixture of P. cerevisiae and L . plantarum in minced fish stored at 4C." Final population of test organisms was IO 9 eel ls /g of mince. * No growth on lowest di lution ( l (H) . - 52 -9 H 8H 7H • • SPC Test 0-----0 SPC Control A A pH Test A-—-A pH Control TEST: Mixture of P. cerevisiae & t . plantarum (10 9/g) + 0.3% Dextrose \-9.0 r-8.0 L7.0 !-6.0 5.0 X a STORAGE TIME (days) Figure 10. The growth of psychrotrophic mixture of f\ cerevisiae and dextrose 0.3% stored at 4C. organisms was 109 ce l l s /g of No growth on lowest di lution ( 1 0 _ l ) . organisms in the presence of a L. plantarum in minced fish and Final population of test mince. - 53 -TABLE X THE pH VALUES OF UNTREATED AND TREATED MINCED FISH DURING STORAGE AT 4C Treatment Storage Time (Days) 0 1 2 3 4 5 6 Untreated 6.45 6.50 6.49 6.49 6.54 6.55 6.90 Mixture of 9 10 /g cerevisiae & L. piantarum 6.55 6.40 6.30 6.20 6.19 6.27 6.45 Mixture of 9 10 /q Pj_ cerevisiae & L_^  pi antarum .+ 0.3% dextrose 6.50 6.30 6.10 6.00 6.04 6.15 6.20 - 54 -of mince. Treatment of minced fish with P. cerevisiae suppressed fish microflora responsible for spoilage (Figure 11). In the f i r s t three days of storage, repressive act ivi ty was greater than in the latter portion of the storage period. In this storage period, bacterial reduction approaching one log cycle was observed for the treated sample as compared to the untreated sample. In terms of bacteriological quality, a much better product was obtained for the treated sample within the f i r s t two days of storage. A similar pattern was observed in samples treated with a mixture of P. cerevisiae and L. plantarum (Figure 12). pH values for the treated samples declined throughout the storage period as compared to those values for untreated samples (Table XI). In samples stored at 24C, a storage l i f e extension of two days was observed for the treated samples containing dextrose and either a mixture of Pj_ cerevisiae and I L ^ pi antarum (Figure 14) or P^ cerevisiae alone (Figure 13). Within the f i r s t day of storage, the psychrotrophic Q population in the untreated samples attained a level of 10 ce l l s per g whereas the treated samples increased to an acceptable l imit of 10^ per g which was maintained for an additional day. The untreated samples exhibited an offensive ammonia-like odour after only one day at 24C. Treated samples, on the other hand, displayed only a slight lact ic culture odour. pH analysis of the minced product related well with the observed odour characteristics for the treated and untreated samples; the latter showing values of seven or higher after only one day storage (Table XII). - 55 -9A • • SPC Test O O SPC Control • • pH Test A a pH Control TEST: P. cerevisiae ( 10%) + 0.3% Dextrose P" 19.0 r-8.0 h7.0 U6.0 r-5.0 X a STORAGE TIME (days) Figure 11. The growth of psychrotrophic organisms in the presence of P. cerevisiae and 0.3% dextrose in minced fish stored at l l C . Final population of test organism was 109 ce l l s /q of mince. - 56 -• • SPC Test O — - O SPC Control A A PH Test £, ^ PH Control T E S T : Mixture of P. cerevisiae & L. plantarum (K)9/g ) + 0.3% Dextrose P" 2 " T " 3 6 r-9.0 r8 .0 r-7.0 r-6.0 r-5.0 X a STORAGE TIME (days) Figure 12. The growth of psychrotrophic organisms in the presence of P. cerevisiae and L . plantarum and 0.3% dextrose in minced fish stored at 11C. Final population of test organisms was 109 ce l l s /g of mince. - 57 -TABLE XI THE pH VALUES OF UNTREATED AND TREATED MINCED FISH DURING STORAGE AT 11C Treatment Storage Time (Days) Untreated cerevisiae ( lOVg cel ls ) + 0.3% dextrose Mixture of 10 /g P. cerevisiae & Lj_ plantarum + 0.3% dextrose 0 6.55 6.60 6.60 1 6.65 6.60 6.00 2 7.02 6.80 6.60 3 7.25 6.72 6.60 4 7.21 6.60 6.87 5 7.40 6.61 7.46 6 7.43 6.63 6.83 - 58 -• • SPC Test O-- - -O SPC Control -A pH Test - A pH Control T E S T : P. cerevisiae (109/g) + 0.3% Dextrose 4 T -6 9.0 h8.0 r7.0 \-6.0 h5.0 Z a STORAGE TIME (days'! Figure 13. The growth of psychrotrophic organisms in the presence of P. cerevisiae and 0.3% dextrose in minced fish stored at 24C. Final population of test organism was 109 ce l l s /g of mince. - 59 -• • SPC Test O O SPC Control A • p H Test A ^ p H Control TEST: Mixture of P. cerevisiae & L. plantarum (109/g) + 0.3% Dextrose h9 .0 h8.0 r-7.0 h6.0 h-5.0 -I 1 1 ! — r -3 4 5 6 STORAGE TIME (days) Figure 14. The growth of psychrotrophic organisms in the presence of a mixture of cerevisiae and plantarum and 0.3% dextrose in minced fish stored at 24C. Final population of test organisms was 109 ce l l s /g of mince. - 60 -TABLE XII THE pH VALUES OF UNTREATED AND TREATED MINCED FISH DURING STORAGE AT 24C Treatment Storage Time (Days) 0 1 2 3 Untreated 6.70 7.00 7.30 7.04 P. cerevisiae (10 9/g cel ls) + 0.3% dextrose 6.50 6.00 5.78 5.84 Mixture of 10 /g P. cerevisiae & L^ plantarum + 0.3% dextrose 6.50 5.80 5.80 5.76 - 61 -E. Trimethylamine Analysis (TMA) Ten mg % is the maximum acceptable l imit of TMA-N (trimethylamine nitrogen) allowed for fresh cod (Codex Alimentarius Committee on Fish and Fishery Products, cf . Tozawa et aj_., 1971). Above this l imi t , appreciable spoilage is considered to have taken place. Minced f ish treated with io / cel ls of P. cerevisiae per g of mince obtained a level of 11 mg % on the sixth day of storage similar to that observed for the control sample (Figure 15 and Table XIII). However, when dextrose was added to the treated sample, TMA-N was less than 10 mg % on the sixth day, supposedly providing an extension in storage l i f e of one day. This result , however, is not comparable with the bacteriological data which shows the product to be spoiled after only 3 to 4 days storage on the basis of containing greater than 10 organism per g (Figure 16). Increasing the population of P. cerevisiae to 10 ce l l s per g of minced fish gave TMA-N values within the acceptable l imit throughout the storage period (Figure 17 and Table XIV). With the addition of dextrose (Figure 18), TMA-N on the sixth day of storage was 1.52 mg %. In comparison, the untreated sample contained 17.1 mg % TMA-N. - 62 -• - • SPC Test a -O SPC Control • • TMA-N Test o D TMA-N Control TEST: P. cerevisiae OoVg) 2 3 4 STORAGE TIME fdays) 5 6 He 1 4 M2 o> o c "55 8 E < S Figure 15. TMA-N production in untreated and treated minced fish stored at 4C. P. cerevisiae was added to a f inal population of 107 c e l l s / g . - 63 -TABLE XIII TMA-N VALUES FOR UNTREATED AND TREATED MINCED FISH DURING STORAGE AT 4C TMA-N mg/100 g Sample 10 7/g cel ls 10 7/g cel ls P. cerevisiae P. cerevisiae +0.3% dextrose 0 1 - -2 0.57 0.57 0.57 3 0.57 0.38 0.38 4 1.14 0.57 0.57 5 1.52 0.76 1.14 6 11.40 11.02 9.50 (Days) Untreated - 64 -• • SPC Test t> O SPC Control • • TMA-N Test • -o TMA-N Control -18 9 TEST: P. cerevisiae (K) 7/g) 4 0.3% Dextrose 16 —r 2 —r 3 4 5 ~i 6 f-14 -12 -10 O) o o O) r-8 E h4 h2 STORAGE TIME (days) Figure 16. TMA-N production in treated and untreated minced fish stored at 4C. P. cerevisiae was added to a f inal population of 107 cel ls per g and dextrose to 0.3%. - 65 -STORAGE TIME (days) Figure 17. TMA-N production in untreated and treated minced fish stored at 4C. P. cerevisiae was added to a f inal population of 109 c e l l s /g . * No growth on lowest di lution ( I O - 1 ) . - 66 -TABLE XIV TMA-N VALUES FOR UNTREATED AND TREATED MINCED FISH DURING STORAGE AT 4C TMA-N mg/100 g Sample Storage Time Z Z ( D a y s ) Untreated 1 0 '9 C e l l s 1 0 ^ C e l l s P. cerevisiae P. cerevisiae +.0.3% dextrose 0 1 2 0.57 0.38 0.38 3 0.95 0.57 0.57 4 0.95 0.57 0.57 6 17.10 11.40 1.52 - 67 -• • SPC Test O O SPC Control • • TMA-N Test D- a TMA-N Control E z S 1 2 3 4 5 6 STORAGE TIME (days) Figure 18. TMA-N production in untreated and treated minced fish stored at 4C. P. cerevisiae was added to a f inal population of 109 ce l l s /g and dextrose to 0.3%. * No growth on lowest di lution (10"^). - 68 -8 9 Trimethylamine values for minced f ish treated with 10 or 10 cel ls per g of a mixture of P. cerevisiae and L. plantarum per g mince were also measured (Table XV). A similar trend was observed in that samples with added dextrose exhibited lower TMA-N values. Table XV shows that TMA-N values exceeded the acceptable l imit for the untreated samples by the fourth day of storage, coinciding to a count of 10^ spoilage o organisms per g (Figure 8). On the basis of TMA content, the 10 cel ls of mixed culture provided a one day extension in acceptable quality without dextrose and a two day extension with dextrose. With higher populations of mixed lact ic organisms (10 ), an even greater suppression of TMA formation was evident: 7.84 and 9.52 mg % TMA-N with and without dextrose, respectively, after 6 days storage. Untreated product accumulated 44 mg % TMA-N after the same storage period (Figure 19 and 20, Table XVI). When minced fish samples were stored at 11C, the increase in TMA-N was accelerated. Although lact ic culture addition (Table XVII) suppressed TMA formation, high values were observed after only two days of storage for both the treated and untreated product coincident with rapid increases in total numbers of spoilage organisms. - 69 -TABLE XV TMA-N VALUES FOR UNTREATED AND TREATED MINCED FISH DURING STORAGE AT 4C TMA-N mg/100 g Sample Storage Time Q R (Days) Mixture of 10° /g Mixture of 10° /g Untreated P. cerevisiae P. cerevisiae and L. plantarum and L . plantarum +0.3% dextrose 0 1 2 3 4 5 6 0.22 0.22 0.22 0.22 0.22 0.22 3.14 0.34 1.68 15.68 7.94 5.60 16.80 15.68 8.51 44.24 22.40 27.40 - 70 -• • SPC Test O o SPC Control • • TMA-N Test D- - • TMA-N Control TEST: Mixture of P. cerevisiae & L. plantarum (109/g ) STORAGE TIME (days) Figure 19. TMA-N production in untreated and treated minced fish stored at 4C. Treated product contained 109 ce l l s /g P. cerevisiae and U_ plantarum. * No growth on lowest di lution (10~1). - 71 -• • SPC Test O—-O SPC Control • • TMA-N Test 0-----0 TMA-N Control STORAGE TIME (days) Figure 20. TMA-N production in untreated and treated minced fish stored at 4C. Treated product contained 109 ce l l s /g P. cerevisiae, L. plantarum and 0.3% dextrose. * No growth on lowest di lution (10" 1). - 72 -TABLE XVI TMA-N VALUES FOR UNTREATED AND TREATED MINCED FISH DURING STORAGE AT 4C TMA-N mg/100 g Sample Mixture of 10 /g P. cerevisiae and L. piantarum + 0.3% dextrose 0 - - -1 0.22 0.22 0.22 2 0.22 0.22 0.22 3 3.14 0.56 0.56 4 15.68 3.92 3.36 5 16.80 6.90 5.38 6 44.24 9.52 7.84 Storage Time M . . , , J , (Days) „ . . . S 1 X t U r e ° f - 1 0 / 9 v J Untreated F\_ cerevisiae and L. piantarum - 73 -The lack of relationship between TMA values and total spoilage f lora might be attributed to the presence of an increased proportion of spoilage organisms capable of reducing TMA-oxide at this temperature. This would not be evident i f only total numbers of cel ls were estimated. Alternately, endogenous, non-microbial TMA-oxide reductase(s) could contribute to the increased levels of TMA (Castel l , 1970). Interestingly, at 24C, suppressions of TMA-N formation due to the presence of lact ic acid bacteria was less pronounced than at 11C (Tables XVII and XVIII). One explanation for this apparent anomaly might be that the developing spoilage f lora associated with marine f ish are better adapted to growth at the lower temperature (11C) than are the added lact ic acid bacteria. The early rapid decline in pH observed at 24C in comparison to 11C would tend to support this explanation (Figures 11 and 13). - 74 -TABLE XVII TMA-N VALUES FOR UNTREATED AND TREATED MINCED FISH DURING STORAGE AT 11C TMA-N mg/100 g Sample Mixture of 10 /g n . . Untreated cerevisiae ^ cerevisiae and L. plantarum (10 9/g) +.0.3% dextrose +0.3% dextrose 0 1 10.98 6.16 4.26 2 49.28 52.08 40.32 3 58.91 56.00 58.24 4 60.48 60.26 53.76 5 62.16 62.72 62.16 6 67.20 63.84 61.40 Storage Time (Days) - 75 -TABLE XVIII TMA-N VALUES FOR UNTREATED AND TREATED MINCED FISH DURING STORAGE AT 24C TMA-N mg/100 g Sample Storage Time Mixture of 10^/ ( D a y s ) Untreated P^cerevisTae 9 ^ cerevjsiae •>9 and L . plantarum (10 /g) + 0.31. dextrose + 0.3% dextrose 0 1 53.80 16.24 3.58 2 58.24 42.33 10.08 3 75.09 43.46 19.04 - 76 -F. Inhibition Test Comparison between untreated minced f ish samples plated on Standard Methods Agar containing P. cerevisiae and untreated minced samples without the test organism was made to determine i f inhibit ion of the spoilage f lora occurred during incubation of plated samples. At lower dilutions of the test organism, inhibit ion was observed, evidenced by slow growth or non-apparent growth. At higher di lut ions, however, total number of viable organisms compared favourably with total numbers obtained from plates without the test organism (Table XIX). TABLE XIX INHIBITION OF PSYCHROTROPHIC ORGANISMS IN MINCED FISH BY P. cerevisiae DURING INCUBATION Sample Dilution of Test Organism (P. cerevisiae) Colony Forming Unitsa/g Test organism added 10-1 <1.00 x 10*1 Test organism added 10-2 2.77 x IO 2 Test organism added 10-3 4.51 x 10* Test organism added 10-4 5.95 x 104 Test organism added 10-5 5.03 x 10* No test organism added 4.65 x IO* a Total counts of psychrotrophic organisms in minced f ish sample. The data demonstrate that the counts do, in fact , ref lect the microbial status of the stored minced product. - 77 -SUMMARY OF RESULTS The addition of P. cerevisiae 10 7/g to minced cod with or without added dextrose resulted in very l i t t l e or no extension of product storage l i f e at 4C. This was evident on the basis of no reduction in the numbers of developing spoilage f lora , no repression of increasing pH on storage, and no significant suppression in the formation of TMA-N relative to untreated product. When the population of added Q P. cerevisiae cel ls was increased to 10 per g of the mince, there was a marked difference in the quality of the products when stored at 4C. These included; a reduction in the rate of growth of spoilage organisms, reduced levels of trimethylamine formation, and lower pH values of the mince. Addition of low levels of fermentable sugar (dextrose) to the inoculated product further enhanced the quality of the product - based on the above parameters. On the basis of accepted standards for total plate counts, TMA, and pH of fresh f ish products, an extension of product storage l i f e of one day in the absence of added carbohydrate and greater than two days for product containing 0.3% added dextrose at a storage temperature of 4C. The f u l l extent of storage l i f e is not known since, at six days storage, the total counts had not approached 10^  per g of mince. Although the mechanism involved in the inhibit ion observed in this study is not known, the ab i l i ty of the lact ic organisms to inhibit the spoilage f lora in f ish muscle is quite pronounced. - 78 -Many investigators have documented'simi1ar observations in various food products. Price and Lee (1970) identified hydrogen peroxide as the inhibitory substance produced by Lactobacillus plantarum and was effective in inhibit ing Pseudomonas spp. Oahiya and Speck (1968) also identified hydrogen peroxide as the inhibitory compound found in Lactobaci11 us lact is and Lactobaci1lus bulgaricus culture f i l t r a t e s , which inhibited S. aureus. They postulated that hydrogen peroxide produced by lactobaci l l i would be an important element in the inhibition of undesirable microorganisms in food. Other investigators have implicated that the acids produced by lact ic organisms could be the causative agents in bacterial inhibit ion. Antibiotics produced by lact ic acid bacteria have also been reported causative agents of inhibition (Reddy and Shahani, 1971; Baribo and Foster, 1951). Whatever the mode of action involved, which could be any of the aforementioned poss ibi l i t ies as well as unknown inhibitors present, this tremendous ab i l i t y of lact ic organisms to repress unwanted bacteria is of great importance for extending the storage l i f e of perishable flesh food products. A similar extension in storage l i f e 4C was observed for minced product inoculated with a mixture of P. cerevisiae and L. plantarum at a level of 10 ce l l s per g, again based on these same indexes of quality. A - 79 -further extension of storage l i f e was observed when the mixed inoculum 9 was increased to 10 cel ls per g of mince. The bacterial data do not provide an indication as to what extent storage l i f e was increased, since even at six days' storage total counts had not approached 10 / g . Based on TMA content, however, this would appear to be about 2.5 days in the absence and about 3.5 days in the presence of the sugar. Raccach and Baker (1978) reported that in cooked mechanically deboned poultry meat a mixture of L. plantarum and P. cerevisiae repressed the growth, of Pseudomonas species, resulting in a delay of greater than three days, which is the time required for the Pseudomonas population to reach a level of IO'7 c e l l s / g . When the Pseudomonas species were grown with each organism, a delay of one to two days was observed. They postulated that lact ic acid bacteria, especially when used as a mixture, has a potential anti-spoilage factor which could decrease the economic loss due to action of bacteria. When stored at 11C, the untreated product deteriorated rapidly and became unacceptable at two days' storage. Based on total plate counts only, product inoculated with P. cerevisiae alone or a mixed culture of g P. cerevisiae and L. plantarum (10 /g) and containing 0.3% dextrose was acceptable for an additional 24 h. Trimethylamine content, on the other hand, revealed no extension in storage l i f e . At 24C, samples containing either P. cerevisiae or a mixture of P. cerevisiae and L. plantarum showed similar patterns with respect to total counts and - 80 -pH values. P. cerevisiae alone was more effective in inhibit ing TMA formation, allowing a level of only 10 mg% TMA on the second day of storage as compared to 42 mg% for samples containing a mixture of P. cerevisiae and piantarum. TMA-N values were found to increase during storage quite rapidly after a two to three day lag phase in the untreated sample and to a lesser extent in the treated sample. This period of rapid TMA production also appears to correspond somewhat to rapid bacterial growth and subsequent spoilage. This is not unusual and has been well documented. However, this is not always the case. In this study, high TMA-N values have also been obtained in both treated and untreated samples containing lower bacterial numbers than 10 /g which is the c r i t i c a l l imit of acceptance, after which spoilage is considered to have taken place. For the major part of this study, the untreated samples were consistently higher than the treated samples which support the observation that a definite inhibit ion has taken place in samples containing the lact ic organisms. TMA-N values were lower in samples treated with P.  cerevi si ae than in samples containing a mixture of P. cerevisiae and L.  pi antarum. At higher temperatures of 11C and 24C, TMA-N values increased quite rapidly for a l l treatments. At 24C, however, samples containing P. cerevisiae TMA-N values increased at a much slower rate. In fact , - 81 -the product was quite acceptable at day 2 of the storage period since 10 mg % TMA-N was observed as compared to 42 mg % for sample containing P. cerevisiae and plantarum and 58 mg % for control . Laycock and Reiger (1971) also noted a significant lag phase in TMA-N production in haddock (Melanogrammus aeglefinus) f i l l e t s and then a rapid increase which coincided with significant numbers of pseudomonads. They indicated that Pseudomonas putrefaciens was predominant in TMA production, emphasizing this previously-mentioned link between bacterial spoilage and TMA production, since this organism is considered one of the avid contributors to spoilage of f ish during refrigerated storage (Castell et aj_, 1949; Chai et al_, 1968). The lact ic organism used in this study with a population of 10 and g 10 ce l l s /g of mince would therefore appear to have inhibited the major organisms responsible for spoilage which is reflected in lower TMA-N values. It has been reported that, in cases where preservatives such as EDTA (ethylenediaminetetracetic acid) and gamma irradiation which inhibit the growth of Pseudomonas groups on f ish are used, TMA production was also depressed (Pelroy and Seman, 1969; Power et a l , 1964). Reddy et aj_ (1970) reported no i n i t i a l significant difference in volat i le nitrogen content between ground beef samples treated with lact ic cultures and control samples. However, marked'differences were - 82 -observed on 3, 5 and 7-day control sample. Volat i le nitrogen content was s ignif icantly lower in the cultured samples which indicated a definite inhibitory action of lact ic cultures on Gram-negative spoilage f lora in ground beef. Similar observations were made in this research study with minced f i sh . Spraying or dipping of f i l l e t s in solutions containing the test organisms produced no apparent extension of storage l i f e . It would appear that a much greater concentration of inoculum would be required for this procedure to be successful. Moon et al_ (1982) also reported 1 5 the use of 10 and 10 viable lact ic acid bacteria/g on shrimp through a dipping procedure. Their data indicated very l i t t l e inhibition of Pseudomonas species by lact ic acid bacteria. From this , they concluded that the use of these organisms as a dip to increase the shelf l i f e of shrimp was not feasible. Incorporating the lact ic organisms into a minced product would appear to be better than coating the surface of the f i l l e t . - 83 -CONCLUSIONS This study demonstrates the potential application of lact ic acid bacteria for extending the storage l i f e of minced f ish products. Depending on the species of lact ic organism used, their population, and the storage temperature of the product, storage l i f e extension could range from one to four days. With respect to choice of cultures, the single strain culture of P. cerevisiae (Lactacel 75) used in this study appeared to be as effective as the mixed culture of P. cerevisiae and L. plantarum in extending the storage l i f e of minced f i sh . Regardless of the culture used, a f inal population of 10 ce l l s /g of mince is the minimum population required for extending storage l i f e . The presence of a fermentable carbohydrate source greatly enhances the act iv i ty of the lact ic organisms. A storage temperature of 4C appears to be most suitable for both organisms in extending storage l i f e . However, even in the event of temperature abuse, the addition of the lact ic organisms doubles the storage l i f e of minced f i sh . - 84 -Dipping and spraying of f i l l e t s resulted in no apparent extension of storage l i f e , notably due to the low level of attachment of lact ic organisms to the surface of the f i l l e t s . Incorporation of the lact ic organisms into the minced product appears to be more effective and far less cumbersome than coating the surface of the f i l l e t s . The apparent lack of relationship in TMA-N values relative to bacterial numbers, especially at higher storage temperature, warrants further investigation. At 24C, samples containing either P. cerevisiae or a mixture of P. cerevisiae and L. plantarum showed similar patterns with respect to total counts and pH values. P. cerevisiae alone was more effective in inhibiting TMA formation, allowing a level of only 10 mg % TMA on the second day of'storage as compared to 42 mg % TMA for the samples containing a mixture of F\_ cerevisiae and piantarum. - 85 -BIBLIOGRAPHY 1. Adams, R. , Farber, L . , Lerke, P. 1964. Bacteriology of spoilage of f ish muscle. II. Incidence of spoilers during spoilage. Appl. Microbiol . 12:277. 2. Andrews, W.H., Wilson, C.R. , Poelma, P . L . , Romero, A . , Rude, R .A . , Duran, A . P . , McClure, F . D . , Gentile, D.E. 1978. Usefulness of the Stomacher in a microbiological regulatory laboratory. Appl. and  Environ. Microbiol . 35(1): 89. 3. Anonymous, 1980. Fermentation may answer needs for natural foods, low-energy processes. Food Product Development 14(9): 48. 4. APHA. 1976. Compendium of Methods for the Microbiological Examination  of Foods. (Ed.) Marvin L . Speck. American Public Health Association, Washington, D.C. .5. Ayres, J . C , Ogilvy, W.S.., Steward, G.F. 1950. Postmortem changes in stored meats. 1. Microorganisms associated with development of slime on eviscerated cut up poultry. Food Technology 2: 199. 6. Bacus, J . N . , Brown, W.L. 1981. Use of microbial cultures: meat products. Food Technology 35(1): 74. • 7. Bacus, J . N . 1979. Reduces nitrosamines. Food Eng. 51(5): 24. 8. Baribo, L . E . , Foster, M.E. 1951. Some characteristics of growth inhibitor produced by lact ic Streptococcus. J . Dairy Sc i . 34: 1128. 9. Beatty, S.A. 1938. Studies of f ish spoilage. II. The origin of trimethylamine produced during spoilage of cod muscle press juice. J . Fish Res. Bd. Can. 4: 63. 10. Beatty, S.A. 1939. Studies of fish spoilage. III. The TMAO content of the muscles of Nova Scotia f i sh . J . Fish. Res. Bd. Can. 4: 229. 11. Beatty, S .A. , Gibbons, N.E. 1937. The measurement of spoilage in f i sh . J . B i o l . Can. 3:77. 12. Bose, A . N . , Chowdhury, D.R. 1959. Changes in chemical composition of f ish tissue during storage. J . Proc. Inst. Chem (India) 31: 171; Chem Abstr. 54: 124 6b. 13. Brown, W.L. 1980. Starter cultures - new versus old techniques. Presented at Meat Ind. Res. Conf. American Meat Inst. Foundation, Washington, D.C. pp. 1. - 86 -14. Bryan, A.H. 1965. Lactobacil l i for enteric infections. Drug Cosmet.  Ind. 96: 474. 15. Bryant, F . , Alaniz , I . , Thompson, C.A. 1973. Biochemical and microbial studies on shrimp: volati le nitrogen and amino nitrogen analysis. J . Food Sc i . 38: 431. 16. Cann, D . C , Taylor, L .Y. 1976. The bacteriology of minced fish prepared and stored under experimental conditions. In: Conference  Proceedings: The Production and Ut i l i sat ion of Mechanically Recovered  Fish Flesh (Minced Fish). Ministry of A g r i c , Fisheries and Food. Tory Res. S ta . , Aberdeen, pp. 39. 17. Chung, J . 1968. PhD Thesis, University of Washington, Seattle, Washington. 18. Caste l l , C.H. 1970. Current status of the TMA test as a measure of spoilage in f i sh . Fisheries of Canada 22(19): 16. 19. Caste l l , C . H . , Greenhough, M.F. 1959. The action of pseudomonas on fish muscle. 4. Relation between substrate composition and development of odours by Pseudomonas t r a g i . J . Fish . Res. Bd. Can. 16: 21. 20. Caste l l , C . H . , Greenhough, M . F . , Rodgers, R .S . , MacFarlane, A.S. 1958. Grading fish for quality. 1. TMA values of f i l l e t s from graded f i s h . J . Fish. Res. Bd. Can. 15: 701. 21. Caste l l , C H . , Greenhough, M.F. 1957. The action of pseudomonas on fish muscle. 1. Organisms responsible for odours produced during incipient spoilage of chi l led fish muscle. J . Fish. Red. Bd. Can. 14: 617. 22. Caste l l , C . H . , Richards, J . F . , Wilmot, I. 1949. Pseudomonas  putrefaciens from cod f i l l e t s . J . Fish Res. Bd. Can. 7: 430. 23. Caste l l , C . H . , Anderson G.W. 1948. Bacteria associated with spoilage of cod f i l l e t s . J . Fish. Res. Bd. Can. 7: 370. 24. Conway, E . J . 1958. Microdiffusion Analysis and Volumetric Error. The MacMillan Co. , New York. pp. 199. 25. Dacre, J . C , Sharpe, M.E. 1956. Catalase production by l ac tobac i l l i . Nature 178: 700. 26. Dahiya, R .S . , Speck, M.L. 1968. Hydrogen peroxide formation by lactobaci l l i and its effects on Staphylococcus aureus. J . Dairy Sci 51:1568. - 87 -D a l y , ' C , Sandine, W.E. , E l l i k e r , P.R. 1972. Interactions of food starter cultures and food-borne pathogens: Streptococcus diacet i lact is versus food pathogens. J . Milk Food Techno!. 35: 349. DeKlerk, H . C . , Coetzee, J .N. 1961. Antibiosis among lactobaci11i. Nature 192: 340. Diebel, R . H . , Niven, C F . J r . 1958. Microbiology of meat curing 1. The occurrence and significance of motile microorganism of the genus Lactobacillus in curing brines. Appl. Microbiol . 6: 323. Doelle, H. 1969. Bacterial Metabolism. Academic Press. New York. Duncan, C . L . , Foster, E.M. 1968. Role of curing agents in the preservation of shelf-stable canned meat products. Appl. Microbiol . 16: 401. FAO. 1978. Yearbook of Fishery Stat is t ics : Catches and Landing. Vol. 46. FAO, Rome. Farber, L . , Lerke, P.A. 1961. Studies on evaluation of freshness and on the estimation of storage l i f e of raw fishery products. Food  Technol. 15: 191. Fazio, T . , White, R . H . , Dusold, L . R . , Howard, J.W. 1973. Nitrosopyrolidine in cooked bacon. J . Assoc. Off. Anal. Chem. 56: 401. Felton, E . A . , Evans, J . B . , Niven, C F . J r . 1953. Production of catalase by the pediococci. J . Bacteriol . 65: 481. G i l l , C O . , Newton, K.G. 1978. The ecology of bacterial spoilage of fresh meat at c h i l l temperatures. Meat Science 2: 207. Gi l chr i s t , J . E . , Campbell, J . E . , Donnelly, C . B . , Peeler, J . T . , Delaney, J .M. 1973. Spiral plate method for bacterial determination Appl. Microbiol . 25(2): 244. G i l l i l a n d , S .E. 1979. Beneficial interrelationship between certain microorganisms and humans: candillate microorganisms for use as dietary adjuncts. J . Food Protection 42: 164. G i l l i l a n d , S . E . , Speck, M.L. 1975. Inhibition of psychrotrophic bacteria by lactobaci l l i in non-fermented, refrigerated foods. J . Food  Protection: 40: 903. Gould, E . , Peters, J .A . 1971. On Testing the Freshness of Frozen  Fish. Fishing News Ltd. Fleet Street, London. Harrisson, A.P. J r . , Hansen, P.A. 1950. A motile lactobacillus from caeca! faeces of turkeys. J . Bacteriol . 59: 444. - 88 -42. Heineman, B . , Voris, L . , Stembo, C R . 1965. Use of nisin in processing food poducts. Food Techno!. 19: 592. 43. Herbert, R .A . , Hendrie, M.S. , Gibson, D.M., Shewan, J .M. 1971. Bacteria active in spoilage of certain seafoods. J . Appl. Bacteriol . 34(1): 41. 44. Hurst, A. 1973. Microbial antagonisms in foods. Can. Inst. Food Sc i .  Techno!. Journal 6: 80. 45. Iandolo, J . J . , Clark, C.W., Bluhn, L. Ordad, Z . J . 1965. Repression of Staphylococcus aureus in associative culture. Appl. Microbiol . 13: 646. 46. Ingram, M. 1975. The lact ic acid bacteria - a broadview. In: Carr, J . G . , Cutting, C . V . , Whiting, G . C (Eds.), Lactic Acid Bacteria in  Beverages and Food. Proceedings of a symposium held at Long Ashton Research Station, University of B r i s t o l . Academic Press, London, pp. 1. 47. Ingram, M . , Dainty, R.H. 1971. Changes caused by microbes in the spoilage of meats. J . Appl. Bacteriol . 34: 21. 48. International Commission on Microbiological Specifications for Foods. 1974. Microorganisms in Foods 2. Sampling for microbiological analysis^ Principles and specific applications. University of Toronto Press, Toronto and Buffalo, Canada, pp. 100. 49. James, Carlton. 1977. The by-catch story. In: Proceedings of Seminar  on Potential Resources - The By-Catch of Shrimp Industry. Guyana Printers L t d . , Greater Georgetown, Guyana, pp. 1. 50. Johnston, M.A. , Piunick, H . , Samson, J .M. 1969. Inhibition of Clostridium botulinum by sodium n i tr i t e in bacteriological medium and in meat. Can. Inst. Food Techno!. J . 2: 52. 51. Kao, C . T . , Frazier, W.C 1966. Effect of lact ic acid bacteria on growth of Staphylococcus aureus. Appl. Microbiol . 14: 251. 52. Laycock, R .A. , Reiger, L.W. 1971. Trimethylamine-producing bacteria on haddock (Melanogrammus alglefinus) f i l l e t s during refrigerated storage. J . Fish. res. Bd. Canada 28(3):305. 53. Lea, C . H . , Stevens, B . J . H . , Smith, M.J . 1969a. Chemical and Organoleptic changes in poultry meat resulting from the growth of psychrophylic spoilage bacteria at IC. ! . Introduction and changes in free amino acids. Br. Poult. Sc i . 10: 203. 54. Lerke, P . , Adams, R., Farber, L . 1967. Bacteriology of spoilage of f ish muscle. IV. Role of protein. Appl. Microbiol . 15: 770. - 89 -55. Lerke, P. Adams, R., Farber, L. 1965. Bacteriology of spoilage of fish muscle. III. Characterization of spoilers. Appl. Microbiol . 13: 625. 56. Lerke, P. Adams, R., Farber, L. 1963. Bacteriology of spoilage of fish muscle. 1. Steri le press juice as a suitable experimental medium. Appl. Microbiol . 11: 458. 57. Liston, J . 1980. Microbiology in fishery science. In: Advances in  Fish Science and Technology. (Ed.) J . J . Connell. Tory Research Station. Publ. Fishing News L t d . , pp. 138. 58. Metchnikoff, E . 1908. The Prolongation of L i f e . N.Y. Putnam. 59. Moon, N . J . , Beuchat, L . R . , Kinkaid, D . T . , Hayo, E.R. 1982. Evaluation of lact ic acid bacteria for extending the shelf l i f e of shrimp. J . Food Sc i . 47: 897. 60. Nath, K . R . , Wagner, B . J . , 1973. Stimulation of lact ic acid bacteria by micrococcus isolate: Effects for multiple effects. Appl. Microbiol . 26: 49. 61. Newton, K . G . , Harrison, J . C . C . , Wauters, A.M. 1978. Sources psychrotrophic bacteria on meat at the abbatoir. J . Appl. Bacteriol . 45(1): 75. 62. Normikko, V. 1956. Biochemical factors affecting symbiosis among bacteria. Experientia 12: 245. 63. Obrink, K . J . 1955. A modified Conway unit for microdiffusion analysis. Biochem. J . 59:134. 64. Partmann, W. 1951. cf . Determining Freshness of Fish. Z. Lebensen -Untersuch u - Forsch 93: 341. 65. Pelroy, G.A. , Seman, J . P . 1969. Effect of EDTA treatment on spoilage characteristics of petrale sole and ocean perch f i l l e t s . J . Fish. Res. Bd. Canada 26: 2651. 66. Pensabene, J.W., Fiddler, W., Grates, R .A. , Fagan, J . C , Wasserman, A . E . 1974. Effect of frying and other cooking conditions on nitrosopyrolidine formation in bacon. J . Food Sc i . 39:314. 67. Pinheiro, A . J . R . , Liska, B . J . , Parmelee, C E . 1968. Properties of substances inhibitory to Pseudomonas fragi produced by Streptococcus  citrovorum and Streptococcus d iacet i lac t i c . J . Dairy Sc i . 51: 183. 68. Price, R . J . , Lee, J . S . 1970. Inhibition of Pseudomonas species by hydrogen peroxide producing l a c t o b a c i l l i . J . Milk Food Techno!. 33: 13. - 90 -69. Prescott, S . C , Dunn, C.G. 1959. Industrial Microbiology. 8th ed. McGraw-Hill Book Co. N.Y. 70. Power, H . E . , Fraser, W., Neal, W., Dyer, J . , Caste l l , C H . 1964. Gamma irradiation as a means of extending the storage l i f e of haddock f i l l e t s . J . Fish . Res. Bd. Canada 21: 827. 71. Raccach, M. 1977. "The Control of Spoilage and Pathogenic Microorganisms in Poultry Meat by Lactic Acid Bacteria", Diss. Cornell University, New York. pp. 61. 72. Raccach, M . , Baker, R.C. 1978. Lactic acid bacteria as an antispoilage and safety factor in cooked and mechanically deboned poultry meat. J . Food Protection 41: 703. 73. Reay, G.A. , Shewan, J .M. 1949. The spoilage of f ish and preservation by c h i l l i n g . In: Advances in Food Research 2: 343. 74. Reddy, S .G . , Chen, M . L . , Patel, P . J . 1975. Influence of lact ic cultures on the biochemical bacterrial and organoleptic changes in beef. J . Food Sc i . 40: 314. 75. Reddy, S . G . , Hendrickson, R . L . , Olson, H . C 1970. The influence of lact ic cultures on ground beef quality. J . Food Sc i . 35: 787. 76. Reddy, G .V. , Shahani, K.M. 1971. Isolation of an antibiotic from Lactobacillus bulgaricus. J . Dairy Sc i . 54: 748. 77. Sandine, W.E. 1979. Role of Lactobacillus in the intestinal tract . J . Food Protection 42: 259. 78. Sandine, W.E. , Muradlihara, K . S . , E l l i k e r , P .R. , England, D.C. 1972. Lactic acid bacteria in food and health: a review with special emphasis to enteropathogenic escherachia co l i as well as certain enteric diseases and their treatment with antibiot ics . J . Milk Food Technol. 35: 691. 79. Shahani, K . M . , Vaki l , J.R. Kilana, A. 1976. Natural antibiotic act ivi ty of Lactobaci1lus acidophilus and Lactobacillus bulgaricus. 1. Cultural conditions for the production of antibiosis . Cultured Dairy  Products Journal 11(4): 14. 80. Sharpe, A . N . , Jackson, A.K. 1972. Stomaching a new concept in bacteriological sample preparation. Appl. Microbiol . 24: 175. 81. Sharpe, M . E . , Fryer, T . F . , Smith, D.G. 1966. In: Identification  Methods for Microbiologists. Part A. Academic Press, New York., pp. 65. - 91 -82. Shewan, J.M. 1977. The bacteriology of fresh and spoiling f ish and the biochemical changes induced by bacterial action. In: Hand!ing  Processsing and Marketing of Tropical Fish. Tropical Products Institute, London, pp. 51. 83. Shewan, J .M. 1971. The microbiology of fish and fishery products -progress report. J . Appl. Bacteriol . 34(2): 299. 84. Shewan, J . M . , Liston, J . 1956. Objective and subjective assessments of f ish quality. Bu l l . International, Refr ig . , Suppl. Annex. 1: 137. 85. Shewan, J . M . , Jones, N.R. 1957. Chemical changes occurring in cod muscle during c h i l l storage and their possible use as objective indices of quality. J . Sc i . Food Agric. 8: 491. 86. Shewan, J . M . , Macintosh, R . G . , Tucker, C . G . , Ehrenberg, A .S .C . 1953. The development of a numerical scoring system for the sensory assessment of the spoilage of wet white fish stored in ice. J . S c i . Food Agric. 4: 283. 87. Shewan, J . M . , Gibson, D.M., Murray, C K . 1971. The estimation of trimethylamine in f ish muscle. In: Fish Inspection and Quality  Control. (Ed.) Rudolf Kreuzer. Publ. Fishing News (Books) L t d . , London, pp. 183. 88. Sorrels, K . M . , Speck, M.L. 1970. Inhibition of Salmonella gallinarum by culture f i l t rates of Leuconostoc citrovorum. J . Dairy Sc i . 53: 239. 89. Speck, M.L. 1981. Use of microbial cultures: Dairy Products. Food Technol. 35(1): 71. 90. Speck, M.L. 1975. Interactions among lactobaci l l i and man. J . Dairy Sc i . 59: 338. 91. Speck, M.L. 1972. Control of food-borne pathogens by starter cultures. J . Dairy Sc i . 55: 1019. 92. Spencer, R. 1969. New procedure for determining the ab i l i ty of microorganisms to reduce nitrate and n i t r i t e . Lab Pract. 18: 1286. 93. Tanaka, N . , Traisman, E . , Lee, M.H. , Cassens, R . G . , Foster, E.M. 1980. Inhibition of botulinum toxin formation in bacon by acid development. J . Food Protection 43(6): 450. 94. Tarr, H.L .A. 1940. The bacterial reduction of TMAO to TMA. J . Fish.  Res. Bd. Can. 4: 367. 95. Tarr, H . L . A . , Ney, P.W. 1949. Effect of flesh acidity on bacterial numbers and TMA in spoiling f i sh . Prog. Rep. Pacific Coast Sta. Fish Res. Bd. Can. No. 78: 11. - 92 -96. Tozawa, H . , Enokihara, K . , Amano, K. 1971. Proposed modification of Dyer's method for Trimethylamine determination in cod f i s h . In: Fish Inspection and Quality Control, ed. Rudolf Kreuzer. Fishing News Book Limited, London, England. ppT 187. 97. Tozawa, H . , Enokihara, K. Amano, K. 1969. Proposed modification of Dyer's method for trimethylamine determination in cod f i l l e t s . FAD Halifax, FE: FIC/69/0/48, Topic III. 98. Tramer, J . 1966. Inhibitory effect of Lactobacillus acidophilus. Nature 211: 204. 99. Tyndall, J . 1876. The optical deportment of the atmosphere in relation to the phenomenon of putrefaction and infection. P h i l . Trans. Roy. Soc. 166: 27. 100. Uprett i , G . C , Hinsdell , R.D. 1975. Production and mode of action of lactocin 27: bacterocin from a homofermentative Lactobacillus. Antimicrob. Agents Chemother. 7: 139. 101. Velankar, N.K. , Govindan, T.K. 1960. Preservation of prawns in ice and the assessment of their quality by objective standards. Indian J .  World Fish 6: 306; World Fisheries Abstr. 11: 25, 26. 102. Watson, D.W. 1939a. Studies of f ish spoilage. IV. The bacterial reduction of trimethylamine oxide. J . Fish Res. Bd. Can. 4: 252. 103. Weigmann, H. 1899a. cf. Versuch einer Einteilung der Milchsaurebakterien des Molkereigewerbes. Zentbl. Bakt. ParasitKde. Abt. II, 5, 825. - 93 -APPENDIX - 94 -Spiral Plating The principle of spiral plating wi l l be discussed here since i t was used as the main method of determining viable organisms rather than more conventional plating methods. This method is based on the continuous deposition of a small amount of sample on the surface of a rotating agar plate in an ever decreasing amount in the form of an Archimedes s p i r a l . The amount of sample deposited is controlled and decreases while the dispensing stylus is moved from the centre to the edge of the rotating agar plate. After incubation of the plates, colonies wil l develop along the lines where the l iquid was original ly deposited. The number of colonies per unit length of line or per unit area of the agar surface is dependent on the bacterial concentration in the deposited l iquid (Gi lchr is t , 1973). As a result of the differing densities from the centre to the edge of the plate there is considerable ease in counting some areas of the plate. Mode of operation At the onset of plating, l iquid sample is' introduced into the t ip of a teflon tubing or stylus by means of a vacuum to a two-way valve which pulls the l iquid through the tubing and syringe. After f i l l i n g the tubing and syringe with the sample, the valve to the syringe is closed. - 95 -A plate containing the agar is then placed on the platform with the teflon tip touching the agar surface and the motor is switched on. As the, platform moves, gravity causes the arm to follow the contour of the cam. Due to the fact that the arm is connected to the rack and pinion which controls the a r m , the subsequent fall of the arm is proportional to the movement of the plunger in the syringe. The initial drop of the arm is quite rapid but becomes slower in its path over the agar surface in such a way that there is more deposition of the sample in the centre of the plate which decreases as i t moves towards the edge of the plate. At the end of the dispensing, the stylus arm is elevated and the valve is opened to remove residual sample from the system before the next sample is plated. The direction of the motor is reversed and the carriage returns to its original position after which the system is flushed with 5% hypochlorite and rinsed with sterile disti l led water. 2. Counting of plates Colonies were counted by placing the agar plate on a colony counting grid which relates the colonies on the spiral plate to the volume in which they are contained. The grid (Plate 8) itself comprises of a 13.2 cm circle which is divided into five areas by four concentric rings placed at equidistant along the diameter with 4 being the nearest to the centre and 1 the furthest to the edge. It is further divided into eight forty-five degree wedges or octant and each octant further subdivided by - 96 -four arcs linearly equidistant from each other (Gilchrist et al_, 1977). The outer ring of the opposite octants, for example, A and E is further subdivided in half an arc in the middle (marked 1/2) while the outer ring is subdivided in half by a line through the centre. On this grid there are other lines to accommodate a 10 cm plate (Plate 9) which is the size plate used in this study. In counting the plates placed on the grid, one can choose any octant sector and count the colonies from the outer edge to the centre until twenty colonies are obtained. Counting is continued until a segment is reached in which the 20th colony is obtained. The octant sector opposite to this is also counted in like manner. The counts are then recorded together with the segment in which they were counted and, subsequently, added together. If twenty colonies are not obtained in an octant, then the total plate should be counted. If, however, the count in the segment exceeds 75 colonies, the count is likely to be too low due to the incidence of overcrowding. An alternative means is to count circumferentially adjacent commencing with sector (1) until greater than or equal to f i fty colonies are counted, then the remaining colonies in which the fift ieth colony was observed are counted. The number of the colonies (or the number of the two sector counts) is divided by the corresponding volume of the sector counted in ml to obtain the bacteria count per ml. - 97 -L o g 1 Q of the arithmetic average of the Colony Forming Unit (CFU) counts on both plates corrected to the dilution used per g is reported. This method is accepted by APHA (1976); AOAC (1980; and the FDA Bureau of Bacteriological Methods (1978). - 98 -8. Stomacher - Concept The technique of stomaching is used throughout this study as a means of blending. The principle of stomaching involves placing the sample and diluent into an inexpensive s ter i le plastic bag, the outer surface of which is pounded upon quite vigorously when placed inside the machine. The bag i t s e l f is sealed near the top by being trapped by the bevelled edge of the case as the Stomacher door is tightened. The handle or knob (depending on type) on the top of the machine serves to pull the door firmly into contact with the bevel (Sharpe and Jackson, 1972). When the machine is switched on, the resulting compression and shearing forces effectively remove the deep-seated bacteria. The removal of the bacteria is thought to be due to the shearing forces experienced as the l iquid is swept from side to side and partly by a series of rapid compression as the sample becomes trapped beneath the paddles. The "sponging action" (Sharpe and Jackson, 1972) experienced by the sample is also thought to be responsible for the effective removal of bacteria from fissures or crevices. There are, however, various advantages as well as disadvantages associated with this technique. F i r s t , the advantages: (a) Very l i t t l e heat is generated as compared to other blenders, e .g . , Waring Blenders. - 99 -(b) Noise is low. (c) Capital outlay is low. (d) There is a saving of time since there is no glassware to wash. (e) Higher counts are realized in some than when a Waring blender is used. Disadvantages: (a) Decreased recovery of bacteria from high fat containing foods. However, addition of Tween 80 could increase recovery (Sharpe and Hershman, 1976). (b) Occasionally the bag breaks inside the machine. The use of more than one bag can help to reduce the r i sk . Andrews et al_, (1978) reported that efficiency of the Stomacher, e.g. Models 400 & 3500, varied according to the type of food being examined, but advocated that the Stomacher could be used quite advantageously in quality control laboratories especially those analyzing large volumes of foods. As a result of the advantages given above, the Stomacher was the main method used in the homogenization of the f ish samples used in this study. - TOO -Photographs Taken During Experimental Period Plate 1. A commercial trawler from which fresh grey cod was obtained. - 101 -Plate 3. Step 1 of the f i l l e t i n g process. - 102 -P l a t e 7. S p i r a l P l a t e r used in the enumeration of micro -organ isms - 104 -Plate 9. Plate after 48 h incubation at 24C. Plate is on top of Spiral Plate counting gr id . - 105 -Plate 10. Plates showing inhibition of psychrotrophic organisms in minced fish stored at 4C. (IA-I) Untreated sample. (IB-I) Treated with a mixture of P^ _ cerevisiae and L. plantarum. Plated on Standard Methods Agar. Plate 11. Plates showing inhibition of psychrotrophic organism in minced cod f i l l e t s stored at 11C. (IIA-I) Untreated sample. (IID-1) Treated with P. cerevisiae. Plated on Standard Methods Agar. - 106 -Plate 12. Plates showing growth of Lactic organism on Lactobacillus Selection Agar. 

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