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Effect of brine- and plate-freezing at sea on chemical, physical, and organoleptic properties of three… Botta, Joseph Richard 1971

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THE EFFECT OF BRINE- AND PLATE-FREEZING AT SEA ON CHEMICAL, PHYSICAL, AND ORGANOLEPTIC PROPERTIES OF THREE SPECIES OF FISH by JOSEPH RICHARD BOTTA B.Sc.(Agr.), University of B r i t i s h Columbia, 1969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Food Science We accept th i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July, 1971. In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of FOOD SCIENCE The University of British Columbia Vancouver 8, Canada Date J u l y 23, 1971. ABSTRACT The e f f e c t of b r i n e - and p l a t e - f r e e z i n g and l e n g t h o f subsequent f r o z e n storage upon f l e s h pH, thaw d r i p , c o l o r , f l a v o r , TBA ( 2 - t h i o b a r b i t u r i c a c i d ) v a l u e s , and l o n g c h a i n f r e e f a t t y a c i d s o f P a c i f i c h a l i b u t , Chinook and Coho slamon was determined. The e f f e c t of f r e e z i n g method upon sodium, potassium, and c h l o r i d e c o n c e n t r a t i o n was a l s o determined. F l e s h pH o f a l l t h r e e s p e c i e s g e n e r a l l y d e c l i n e d s i g n i f i c a n t l y (P - 0.05) w i t h l e n g t h o f s t o r a g e . The thaw d r i p of P a c i f i c h a l i b u t and Chinook salmon was l e s s f o r the b r i n e - than the p l a t e - f r o z e n samples a f t e r s t o r a g e f o r 9 to 31 weeks whereas subsequently the b r i n e -f r o z e n samples had approximately equal o r g r e a t e r thaw d r i p than the p l a t e - f r o z e n . The thaw d r i p of a l l samples, except those from p l a t e - f r o z e n h a l i b u t , tended to i n c r e a s e with l e n g t h o f s t o r a g e . The Hunter 'a' and a/b values of Chinook and Coho salmon g e n e r a l l y i n c r e a s e d d u r i n g s t o r a g e . The d i f f e r e n c e i n f l a v o r between b r i n e - and p l a t e -f r o z e n o u t s i d e muscle o f h a l i b u t and Chinook salmon reached a maximum at 31 and 26 weeks of storage r e s p e c t i v e l y , and then s t e a d i l y decreased. In c o n t r a s t , the d i f f e r e n c e i n f l a v o r between b r i n e - and p l a t e - f r o z e n Coho salmon o u t s i d e muscle s t e a d i l y i n c r e a s e d d u r i n g s t o r a g e . The d i f f e r e n c e i n f l a v o r between b r i n e - and p l a t e -f r o z e n i n s i d e muscle o f a l l s p e c i e s , except f o r the Coho salmon at 10 weeks and h a l i b u t a t 31, 62 and 81 weeks o f s t o r a g e , was not s i g n i f i c a n t . The d i f f e r e n c e i n TBA val u e s (an index of o x i d a t i v e r a n c i d i t y ) between b r i n e - and p l a t e - f r o z e n o u t s i d e muscle samples r a p i d l y i n c r e a s e d and reached a maximum at 45, 26, o r 27 weeks (the b r i n e - f r o z e n samples having the h i g h e r v a l u e s ) then decreased u n t i l t h e r e was approximately no d i f f e r e n c e at 81, 77 and 78 weeks of storage f o r h a l i b u t , Chinook and Coho salmon, r e s p e c t i v e l y . Method o f f r e e z i n g o r l e n g t h o f storage had l i t t l e e f f e c t on the TBA values o f i n s i d e muscle f o r a l l s p e c i e s . Method o f f r e e z i n g had l i t t l e e f f e c t on the c o n c e n t r a t i o n of i n d i v i d u a l f r e e f a t t y a c i d s (percentage o f t o t a l f r e e f a t t y a c i d s a n a l y z e d ) . The c o n c e n t r a t i o n s of s e v e r a l f r e e f a t t y a c i d s was a f f e c t e d by l e n g t h of storage but the p a t t e r n o f change d u r i n g s t o r a g e was e r r a t i c . F r e e z i n g method had an e f f e c t on the c o n c e n t r a t i o n o f some i n d i v i d u a l f r e e f a t t y a c i d s (_u.g per gram o f n e u t r a l l i p i d ) o f h a l i b u t and Chinook salmon but not o f Coho salmon. In g e n e r a l , w i t h a l l s p e c i e s , the c o n c e n t r a t i o n o f the i n d i v i d u a l f r e e f a t t y a c i d s was g r e a t e s t i n the i n s i d e muscle. A l s o f o r h a l i b u t and Chinook salmon, p a r t i c u l a r l y where th e r e was a s i g n i f i c a n t d i f f e r e n c e among storage t i m e s , the c o n c e n t r a t i o n of the f r e e f a t t y a c i d s r a p i d l y i n c r e a s e d d u r i n g the f i r s t 26 t o 31 weeks of s t o r a g e . Method o f f r e e z i n g and l e n g t h of f r o z e n storage had a s i g n i f i c a n t e f f e c t on t o t a l f r e e f a t t y a c i d s analyzed f o r o n l y Chinook salmon. T o t a l f r e e f a t t y a c i d s s i g n i f i c a n t l y (P ^ 0 . 0 5 ) d i f f e r e d between i n s i d e and o u t s i d e muscle o f h a l i b u t and Chinook salmon but not of Coho salmon. The e f f e c t o f method of f r e e z i n g upon potassium c o n c e n t r a t i o n was s m a l l and v a r i e d w i t h s p e c i e s . The e f f e c t o f b r i n e - f r e e z i n g upon most v a r i a b l e s measured was e i t h e r s m a l l and/or complex. For a l l t h r e e s p e c i e s the sodium and c h l o r i d e c o n c e n t r a t i o n was two to t h r e e times g r e a t e r i n the b r i n e - f r o z e n o u t s i d e muscle than i n a l l o t h e r samples. The t a s t e panel r e s u l t s and the TBA values i n d i c a t e t h a t b r i n e - f r e e z i n g does impair the q u a l i t y of the o u t s i d e muscle o f h a l i b u t and Chinook salmon d u r i n g the e a r l y stages o f f r o z e n s t o r a g e . - i v -TABLE OF CONTENTS Page L i s t of Tables i x L i s t of Figures x x Acknowledgement s x i i i INTRODUCTION ' . 1 LITERATURE REVIEW 3 A. Freezing Fish At Sea ' 3 1) General Considerations 3 2) Plate-Freezing 5 3) Brine-Freezing 8 B. Frozen Storage 16 Changes Occurring During Frozen Storage 16 a) L i p i d Hydrolysis 16 b) Changes i n Protein E x t r a c t a b i l i t y 20 c) Changes i n Thaw Drip 2k d) Oxidative Rancidity 25 •METHODS AND MATERIALS" 31 A. Catching and Freezing the Fish 31 B. Sampling and Analysis 32 a) Determination of Thaw Drip 33 b) Determination of pH 33 c) Color Determination 35 d) Determination of Flavor Differences 35 e) Determination of 2-Thiobarbituric Acid Reactive Substances 37 - V-Page f) L i p i d Extraction 38 g) Removal of the Phospholipids 39 h) Isolation of Neutral Lipids 40 i ) Removal of Free Fatty Acids 41 j) E s t e r i f i c a t i o n of Free Fatty Acids 41 k) Determination of the Weight of the Neutral Lipids 42 1) Gas Chromatographic Analysis of the Free Fatty Acids m) Determination of the Mineral Concentration in the Flesh 4 3 C. S t a t i s t i c a l Analysis 44 RESULTS A. Analysis of Variance 46 a) Flesh pH 46 i ) P a c i f i c Halibut 46 i i ) Chinook Salmon 46 i i i ) Coho Salmon 51 b) Thaw Drip .-- 51 i ) P a c i f i c Halibut 51 i i ) Chinook Salmon 51 i i i ) Coho Salmon 55 c) Color 1) Hunter Rd Values 55 i ) Chinook and Coho Salmon 55 2) Hunter 'a» Values 5 8 i ) Chinook Salmon 58 i i ) Coho Salmon 58 - v i -Page 3) Hunter 'b» V a l u e s 6 3 i ) Chinook and Coho Salmon 6 3 4) Hunter a/b Pxatios 6 3 i ) Chinook Salmon 6 3 i i ) Coho Salmon 6 3 d) F l a v o r D i f f e r e n c e s 67 i ) P a c i f i c H a l i b u t 67 i i ) Chinook Salmon 70 i i i ) Coho Salmon ^ 70 e) M i n e r a l C o n c e n t r a t i o n , 7 2 i ) P a c i f i c H a l i b u t 72 i i ) Chinook Salmon 75 i i i ) Coho Salmon 78 f ) TBA V a l u e s 81 i ) P a c i f i c H a l i b u t 81 i i ) Chinook Salmon 81 i i i ) Coho Salmon 87 g) Long Chain Free F a t t y A c i d s 87 i ) P a c i f i c H a l i b u t 87 1) Free F a t t y A c i d s E x p r e s s e d as P e r c e n t o f T o t a l F r e e F a t t y A c i d s A n a l y z e d 87 2) Free F a t t y A c i d s E x p r e s s e d as )xg F a t t y A c i d per Gram o f N e u t r a l L i p i d 88 i i ) Chinook Salmon 94 1) Free F a t t y A c i d s E x p r e s s e d as P e r c e n t o f T o t a l Free F a t t y A c i d s A n a l y z e d 94 - v i i -Page 2) Free F a t t y A c i d s E x p r e s s e d as jag F a t t y A c i d p e r Gram o f N e u t r a l L i p i d 9 8 i i i ) Coho Salmon 101 1) Free F a t t y A c i d s E x p r e s s e d as P e r c e n t o f T o t a l Free F a t t y A c i d s A n a l y z e d 101 2) Free F a t t y A c i d s E x p r e s s e d as jUg F a t t y A c i d p e r Gram o f N e u t r a l L i p i d 102 h) T o t a l Free F a t t y A c i d s ( e x p r e s s e d as ju.g Free F a t t y A c i d p e r Gram o f N e u t r a l L i p i d . 107 i ) P a c i f i c H a l i b u t 107 i i ) Chinook Salmon 10 7 i i i ) Coho Salmon 107 B. C o r r e l a t i o n s 109 a) C o r r e l a t i o n s o f pH, Thaw D r i p and C o l o r w i t h Each Other 109 i ) P a c i f i c H a l i b u t 109 i i ) Chinook Salmon 109 i i i ) Coho Salmon 10 9 b) C o r r e l a t i o n s o f pH, Thaw D r i p , C o l o r , TBA V a l u e s and F r e e F a t t y A c i d s w i t h F l a v o r 109 i ) P a c i f i c H a l i b u t . 109 i i ) Chinook Salmon 110 i i i ) Coho Salmon 110 DISCUSSION 111 a) F l e s h pH - 111 b) Thaw D r i p 112 c) C o l o r 113 - v i i i -Page d) Flavor Differences 114 e) Mineral Concentration 116 f) TBA Values 117 g) Free Fatty Acids 119 SUMMARY AND CONCLUSIONS 124 LITERATURE CITED 129 APPENDIX - i x -LIST OF TABLES TABLE Page I Analyses of variance of pH values of P a c i f i c h a l i b u t , Chinook salmon and Coho salmon 4 7 . II Duncan's new multiple range test on the s i g n i f i c a n t time e f f e c t s from the analyses of variance on the pH values of P a c i f i c halibut, Chinook salmon and Coho salmon 4 8 III Analyses of variance of the thaw drip data of P a c i f i c h a l i b u t , Chinook salmon and Coho salmon 53 IV Duncan's new multiple range te s t on the s i g n i f i c a n t time means from the analyses of variance on thaw drip 56 V Analyses of variance of color readings f o r Chinook salmon 59 VI Analyses of variance of color readings for Coho salmon 60 VII Duncan's new multiple range test on the s i g n i f i c a n t time effects from the analyses of variance on the Rd, a, b and a/b values of Chinook salmon and Coho salmon 68 VIII A c c e p t a b i l i t y preferences f o r P a c i f i c Halibut, Chinook salmon and Coho salmon 71 IX Analyses of variance of mineral concen-t r a t i o n of P a c i f i c halibut muscle 7 3 X Analyses of variance of mineral concen-t r a t i o n of Chinook salmon muscle 7 6 XI Analyses of variance of mineral concen-t r a t i o n i n Coho salmon muscle 79 XII Analyses of variance of TBA values of P a c i f i c h a l i b u t , Chinook salmon and Coho salmon 82 XIII Duncan's new multiple range test on s i g n i f i c a n t time means from the analyses of variance of the TBA values of Chinook salmon 83 Method, l o c a t i o n , and time means from t h e a n a l y s e s o f v a r i a n c e o f f r e e f a t t y a c i d s ( e x p r e s s e d as p e r c e n t o f t o t a l f r e e f a t t y a c i d s a n a l y z e d ) o f P a c i f i c h a l i b u t Method, l o c a t i o n , and t i m e means from t h e a n a l y s e s o f v a r i a n c e o f f r e e f a t t y a c i d s ( e x p r e s s e d as jAg f r e e f a t t y a c i d p e r gram o f n e u t r a l l i p i d ) o f P a c i f i c h a l i b u t Method, l o c a t i o n , and time means from t h e a n a l y s e s o f v a r i a n c e o f f r e e f a t t y a c i d s ( e x p r e s s e d as p e r c e n t o f t o t a l f r e e f a t t y a c i d s a n a l y z e d ) o f Chinook salmon Method, l o c a t i o n , and t i m e means from the a n a l y s e s o f v a r i a n c e o f f r e e f a t t y a c i d s ( e x p r e s s e d as jug f r e e f a t t y a c i d p e r gram o f n e u t r a l l i p i d ) o f Chinook salmon Method, l o c a t i o n , and t i m e means from t h e a n a l y s e s o f v a r i a n c e o f f r e e f a t t y a c i d s ( e x p r e s s e d as p e r c e n t o f t o t a l f r e e f a t t y a c i d s a n a l y z e d ) o f Coho salmon Method, l o c a t i o n , and t i m e means from t h e a n a l y s e s o f v a r i a n c e o f f r e e f a t t y a c i d s ( e x p r e s s e d as jag f r e e f a t t y a c i d p e r gram o f n e u t r a l l i p i d ) o f Coho Salmon Method, l o c a t i o n , and- ti m e means from t h e a n a l y s e s o f v a r i a n c e o f t o t a l f r e e f a t t y a c i d s ( e x p r e s s e d as jog f r e e f a t t y a c i d p e r gram o f n e u t r a l l i p i d ) o f P a c i f i c h a l i b u t , Chinook salmon and Coho salmon LIST OF FIGURES A diagramatic representation of a cross-section of a f i s h showing the areas sampled as inside and outside muscle Taste panel questionnaire used to determine f l a v o r differences between frozen stored plate-frozen and brine-frozen P a c i f i c h a l i b u t , Chinook salmon and Coho salmon Average f l e s h pH of P a c i f i c halibut stored at -30°C Average f l e s h pH of Chinook salmon stored at -30°C Average f l e s h pH of Coho salmon stored at -30°C Average thaw drip of P a c i f i c halibut stored at -30°C Average thaw drip of Chinook salmon stored at -30°C Average thaw drip of Coho salmon stored at -30.°C Average Hunter Rd values of Chinook salmon stored at -30°C Average Hunter 'a' values of Chinook salmon stored at -30°C Average Hunter Rd values of Coho salmon stored at -30°C Average Hunter 'a' values of Chinook salmon stored at -30°C Average Hunter b values of Chinook salmon stored at -30°C Average Hunter b values of Coho salmon stored at -30°C - X l l -LIST OF FIGURES Average Hunter a/b r a t i o s of Coho salmon stored at -30°C Average Hunter a/b r a t i o s of Chinook salmon stored at -30°C Taste panel (triangle test) r e s u l t s of P a c i f i c h a l i b u t , Chinook salmon, and Coho salmon Average sodium, potassium, and chloride concentration i n the f l e s h of P a c i f i c halibut Average sodium, potassium, and chloride concentration i n the f l e s h of Chinook salmon Average sodium, potassium, and chloride concentration i n the f l e s h of Coho salmon Average TBA values of P a c i f i c halibut stored at -30°C Average TBA values of Chinook salmon stored at -30°C Average TBA values of Coho salmon stored at -30°C ACKNOWLEDGEMENTS The author i s indebted to and expresses thanks to both Dr. J.P. Richards, Associate Professor, Department of Food Science, and Dr. N. Tomlinson, Acting Director, Vancouver Laboratory, Fisheries Research Board of Canada, and Honorary Lecturer, Department of Food Science, for t h e i r advice, encouragement and help throughout the course of t h i s study. The author also wishes to thank Miss Lynne Robinson, Department of Food Science, for her assistance with the s t a t i s t i c a l analyses and Miss Shirley Geiger and Mr. Bob Hurst, Vancouver Laboratory, Fisheries Research Board of Canada, for t h e i r h e l p f u l c r i t i c i s m s and suggestions. The author i s extremely grat e f u l to the Vancouver Laboratory, Fisheries Research Board of Canada, for providing laboratory space, chemicals and equipment which made, th i s study possible. The writer also g r a t e f u l l y acknowledges the Fisheries Research Board of Canada for providing f i n a n c i a l assistance. INTRODUCTION The n u t r i t i o n a l importance of f i s h has long been recognized (Geiger and Borgstrom, 1 9 6 2 ) . As a protein source Borgstrom ( 1962) claims f i s h i s superior to a l l other major food products whether the protein content i s calculated on the basis of grams of protein per 100 calories or as percent protein of the dry matter i n food. Many people i n c o r r e c t l y believe that food supply from the sea i s "unlimited". Pood production from the sea probably can be increased to an eventual t o t a l of only 150 -160 m i l l i o n metric tons of f i s h annually (about 2 . 5 times that produced i n 1 9 6 8 ) . Although world production of f i s h has increased from 1 9 . 6 m i l l i o n metric tons i n 19^8 to 5 7 . 3 m i l l i o n metric tons i n 1966 (Bligh, 1969) there have been major declines i n annual yields of certain commercially important species. Production of Northwest P a c i f i c Salmon began declining about 1950 and there are as yet no clear signs of recovery (Ricker, 1 9 6 9 ) . One possible way of compensating f o r t h i s decline i n catches i s to provide better means of preservation. Freezing f i s h at sea instead of preserving them with flake ice or ref r i g e r a t e d sea water (RSW) would allow the boats ( p a r t i c u l a r -ly the salmon t r o l l e r s ) to.remain out at sea f i s h i n g longer and thus increase the number of productive days i n a year i n addition to improving the quality of the catch (Eddie, 1 9 6 2 ) . - 2 -Freezing f i s h at sea would also reduce the quantity of spoiled f i s h sent to reduction plants. However, the method used to freeze f i s h at sea may affect the quality of the frozen stored product (Harrison and Roach, 1 9 5 3 ; Kuprianoff, 1 9 5 6 ) . The f l e s h of chinook salmon (Oncorhynchus tschawytscha), coho salmon (Oncorhynchus kisutch), and P a c i f i c halibut (Hippoglossus stenolepis) i s used primarily for the fresh and frozen market. Depending upon the species, the f l e s h may be kept i n frozen storage for up to two years before marketing. The purpose of the present study was to determine the e f f e c t s of brine- and plate-freezing at sea and the length of subsequent frozen storage on f l e s h pH, thaw d r i p , color, TBA value, f l e s h content of various long chain free f a t t y acids, mineral concentration (sodium, potassium, and chloride) and f i n a l l y f l a v o r of P a c i f i c halibut, chinook salmon, and coho salmon. Evaluations were conducted on inside and outside muscle where appropriate. -3-LITERATURE REVIEW A. Freezing Fish At Sea 1) General Considerations Fish are either k i l l e d i n catching or shortly thereafter, and as dead tis s u e , are subject to enzymatic breakdown the rate of which i s approximately doubled for each 10°F increase i n temperature. In order that a frozen pack of the highest quality can be obtained, i t i s necessary to freeze f i s h immediately after they are taken from the water, (Lemon and Carleson, 1 9 ^ 8 ) . Advantages of freezing f i s h at sea, as compared to preserving them i n flake ice are: (a) extension of f i s h i n g to more distant grounds, (b) landing f i s h of high and uniform q u a l i t y , (c) landing capacity loads, and (d) l e v e l i n g out supplies of raw materials for the processing plant through frozen storage (Oldershaw, 1 9 5 5 ; Eddie, 1 9 5 9 ) . Dassow ( 1 9 6 3 ) stated that an obvious solution to the l i m i t e d storage l i f e of c h i l l e d (Iced) P a c i f i c halibut i s to freeze i t at sea. Also Merritt ( 1 9 6 9 ) stated that, i n the B r i t i s h f i s h e r y , i t i s conceivable that eventually freezing at sea w i l l be employed o n . a l l vessels i n which the bulk of the catch must be stored on board for more than seven days before landing. The d i f f e r e n t methods of freezing f i s h at sea as practised by several countries f i s h i n g for d i f f e r e n t or s i m i l a r f i s h were reviewed by Keen and K a r s t i , 1 9 6 5 ; Banks and Waterman, 1 9 6 8 ; and S l a v i n , 1 9 6 8 . The t h r e e p r i n c i p a l methods used t o f r e e z e f i s h at sea are a i r - b l a s t , c o n t a c t p l a t e and b r i n e i mmersion. U s i n g a consumer t a s t e p a n e l L a n t z and C a r t e r ( 1 9 5 1 ) found t h a t h a l i b u t a i r - b l a s t f r o z e n a t sea a t - 1 7 . 8°C (0°P) and s t o r e d at - 2 3 . 3 ° C ( - 1 0°F) were p r e f e r r e d t o h a l i b u t i c e d f o r p e r i o d s o f 12 t o 18 days. However, L a n t z ( 1 9 5 2 ) r e p o r t e d t h a t t h e consumer t a s t e p a n e l p r e f e r r e d h a l i b u t which were f r o z e n a f t e r b e i n g s t o r e d i n i c e f o r t h r e e t o f i v e days o v e r h a l i b u t a i r - b l a s t f r o z e n a t s e a . Depending upon the r a t e o f c a t c h i n g and the r a t e o f f r e e z i n g o r i f f r e e z i n g i s p u r p o s e l y d e l a y e d , f r e e z i n g f i s h a t sea can i n v o l v e f r e e z i n g f i s h i n v a r i o u s s t a g e s o f r i g o r m o r t i s . F r e e z i n g o f p r e - r i g o r and t o a much l e s s e r e x t e n t , i n - r i g o r f i s h , may p r e s e n t c e r t a i n problems. Jones ( 1 9 6 5 ) s t a t e d t h a t changes I n appearance a s s o c i a t e d w i t h t e x t u a l changes r e s u l t i n g from p r e - r i g o r f r e e z i n g o f f i s h ( A t l a n t i c cod) "on t h e bone" are uncommon. T h i s i s because t h e m u s c u l a -t u r e i s a t t a c h e d t o the s k e l e t o n and the "thaw r i g o r " e f f e c t s ( s e v e r e c o n t r a c t u r e and e x c e s s i v e d r i p ) can o c c u r o n l y i f the c o n n e c t i v e s t r u c t u r e f a i l s . Partman and G utschmidt ( 1 9 6 3 ) r e p o r t e d t h a t the c o n c e n t r a t i o n o f ATP r e m a i n i n g i n f i s h muscle a f t e r c o m m e r c i a l f r e e z i n g p r o c e d u r e s are u n l i k e l y t o s u p p o r t "thaw" c o n t r a c t u r e . These r e s u l t s t e n d t o s u p p o r t t h o s e o f T o r r y r e s e a r c h s c i e n t i s t s (Anon, 1 9 6 l ) , However, T o m l i n s o n e_t a l . ( 1 9 6 9 ) o b s e r v e d s e v e r e thaw c o n t r a c t u r e and h i g h f r e e d r i p i n p r e - r i g o r f r o z e n f i l l e t s o f l i n g c o d and -5-P a c i f i c cod. Jones (1969) s t a t e d that the f l e s h of e v i s c e r a t e d f i s h i n r i g o r can, upon subsequent thawing, present a 'broken appearance' i f handled r o u g h l y . However, t h i s appearance i s commonly the r e s u l t of passage i n t o and through r i g o r at h i g h temperatures or the r e s u l t of undue delay i n f r e e z i n g post r i g o r . With f i s h i t i s important to e v i s c e r a t e the f i s h immediately and wash the cut s u r f a c e s w e l l . I f t h i s i s not done the f l e s h w i l l be d i s c o l o r e d by blood that has not escaped the muscle. A l s o " b e l l y - b u r n " , p r o t e o l y s i s of the f l e s h adjacent to the v i s c e r a l c a v i t y , may develop (Jones, 1965; 1969). 2) P l a t e - F r e e z i n g Most B r i t i s h v e s s e l s that f r e e z e f i s h at sea use p l a t e f r e e z e r s , e i t h e r the h o r i z o n t a l or more commonly the v e r t i c a l t y pe. P l a t e f r e e z e r s are p r e f e r r e d over a i r - b l a s t f r e e z e r s f o r s e v e r a l reasons. The a i r - b l a s t f r e e z e r occupies 2 - 3 times the space and i s n e a r l y twice the weight. A i r -b l a s t f r e e z e r s are c o n s i d e r a b l y more complicated i n c o n s t r u c t i o n than p l a t e f r e e z e r s . The r e f r i g e r a t i o n demand i s c o n s i d e r a b l y g r e a t e r than f o r p l a t e "freezers because of the forced-draught f a n s , and lower r e f r i g e r a n t e v a p o r a t i n g temperatures t h a t must be used. D e s i c c a t i o n and o x i d a t i o n of the s u r f a c e of the f i s h may occur w i t h f i s h f r o z e n i n an a i r - b l a s t f r e e z e r . Heavy m e c h a n i c a l l y operated doors are r e q u i r e d i n a i r - b l a s t f r e e z e r s . . -6-A l l the above disadvantages are obviated with plate freezers (Ranken, 1 9 5 8 ) . It has been reported (Anon, 1 9 5 2 ) that A t l a n t i c cod which were plate-frozen immediately aft e r death and kept i n frozen storage for one month possessed abnormal q u a l i t i e s which adversely affected a c c e p t a b i l i t y . The texture of the cooked f i s h was rather soft and 'short', and the smoke cure had a rather poor 'gloss' and 'cut'. Also abnormally large amounts of expressible f l u i d were obtained. However, a f t e r a further two months storage these defects had largely disappear-ed. It was also reported that cod which had been iced one to three days, and then .stored at - 3 0°C ( - 2 2°P) for three months, were of very good q u a l i t y . They had an a t t r a c t i v e appearance, f i l l e t e d well, and yielded excellent smoke cures. Investigations using a consumer taste panel at the Torry Research Station, (Anon, 1 9 5 * 0 , showed that cod which had been iced for four days then plate-frozen to - 3 0°C ( - 2 2°P) was comparable with good quality fresh f i s h . Also cod which had been iced for one day then plate-frozen to - 3 0°C ( - 2 2°F) was judged superior to good quality fresh f i s h . It was con-cluded that the duration of storage i n ice before freezing should not exceed three days. It has also been reported (White Pish Authority, 1 9 5 7 ) that A t l a n t i c cod plate-frozen i n - r i g o r or immediately post-r i g o r yielded a s a t i s f a c t o r y product. The thawed f i s h were firm enough to produce smoothly cut f i l l e t s . However, the f i l l e t s lacked the sheen of those obtained from iced f i s h . Apart from the dullness, the sea-frozen f i s h possessed a 'sea fresh' f l a v o r , good texture, and made sa t i s f a c t o r y smoke cures. The f i s h could be stored without appreciable deterioration for a period of eight to nine months at - 2 0°P. Dyer e_ a_. ( 1 9 6 2 ) observed that when Newfoundland trap-caught cod were plate-frozen at sea, thawed and refrozen at - 1 8°C or - 2 3°C there was a rapid decrease i n taste panel scores and protein e x t r a c t a b i l i t y as well as an increase i n free f a t t y acid formation during the f i r s t two months afte r refreezing. MacCallum et_ al_. ( 1 9 6 4 ) showed that once-frozen Newfoundland trap-caught cod frozen i n - r i g o r i n a horizontal plate freezer yielded an acceptable product. Treating f i l l e t s with sodium tripolyphosphate s i g n i f i c a n t l y improved the texture of the frozen - thawed product but had no e f f e c t upon the taste of the f i s h . It has also been shown that the quality of cod frozen at sea then thawed, f i l l e t e d , and refrozen ashore, varies with time' and place of catching. However, i n a l l cases an accept-able or better twice-frozen product was obtained (MacCallum et a l . , 1 9 6 6 ) . Tomlinson et a l . (1969) studied the e f f e c t of the stage of r i g o r at freezing on the keeping quality of lingcod, P a c i f i c cod, ocean perch, red snapper, orange spotted r o c k f i s h , rock sole, s a b l e f i s h , and P a c i f i c halibut, plate-frozen at sea i n the northeastern P a c i f i c Ocean. In general, the thaw contracture of the white muscle of pre-rigor halibut steaks - 8 -was very s l i g h t (not measurable) while that of the red muscle was quite severe. However, the thaw contracture of the red muscle decreased with time of frozen storage. After nine days of frozen storage the thaw contracture was 35%; a f t e r - 4 5 5 days, 9%. The thaw contracture of the red muscle of the post-rigor steaks was s l i g h t i n comparison to that of the pre-rigor samples. The i n - r i g o r steaks had a thaw contracture between those frozen pre- and post-rigor. The thaw contracture of the i n - r i g o r steaks decreased from 10% a f t e r eight days of frozen storage to 3% a f t e r 45*J days of frozen storage. The free drip of the post-rigor samples increased more than that of the i n -r i g o r or pre-rigor samples. During frozen storage the pH of the pre-rigor samples decreased the most while the pH of the post-rigor samples decreased the l e a s t . At the beginning of the f i r s t sampling period the f l a v o r was rated as 'very good' and the texture rated as 'good', while at the l a s t sampling period the fl a v o r of a l l samples was s t i l l good, with no r a n c i d i t y . However, the pre-rigor samples were preferred over the i n - r i g o r and post-rigor samples, the l a t t e r two samples being d r i e r . The magnitude and changes i n pH, free drip and thaw contracture of lingcod, P a c i f i c cod, ocean perch, red snapper, orange spotted r o c k f i s h , rock sole, and sa b l e f i s h varied with the species. 3) Brine-Freezing The majority of tuna harvested i n the U.S. are brine-- 9 -frozen aboard the tuna c l i p p e r s . The hold of a tuna clip p e r i s divided into s t e e l wells or tanks on both sides of the shaft a l l e y . The wells are f i l l e d with sea water, the water cooled to +29°F, and the warm tuna are loaded into the well. It requires 24 to 72 hours to bring the temperature of the tuna down to +29°F. After the tuna are precooled, s a l t i s added gradually and mixed by means of the brine c i r c u l a t i o n pumps. The brine and tuna are then cooled to about +15°F at which time the c h i l l e d brine i s pumped to another well or overboard. The tuna are then held at from +10°F to +20°F i n the dry r e f r i g e r a t e d well. The f i s h may be unloaded frozen, and thawed at the cannery; or i f they are to be processed promptly at the cannery, the r e f r i g e r a t i o n i n the well i s shut of f and the f i s h thawed by means of c i r c u l a t i n g sea water during the l a s t few days of the t r i p . Salt must be added to the sea water during i n i t i a l thawing to avoid freezing a s o l i d mass of tuna and ice i n the well (Hendrickson, 1 9 5 9 ) . There are several advantages of brine immersion freezing i n tuna clippers (Slavin, 1 9 5 6 ) ; i t requires minimal product handling and also has a lov; maintenance cost; at rated capacity, i t produces a good quality frozen f i s h i n large volume and requires a minimum amount of space because f i s h are frozen, stored, and thawed i n the same tank. There are also several disadvantages. It freezes the product very slowly and requires careful control of temperature because, i f proper temperatures are not maintained within very close l i m i t s , s p o i l -age of the product may r e s u l t . This control requires careful loading to avoid overloading the well and thus exceeding i t s freezing capacity. It i s not v e r s a t i l e , as t h i s freezer i s not suitable for freezing ground f i s h , mackerel, or s h e l l f i s h . The system requires large amounts of s a l t for both freezing and thawing and requires a high capacity r e f r i g e r a t i o n system and considerable a u x i l i a r y power for large volume brine pumps. With small tuna, l i k e skipjack, a slow rate of freezing i n brine often leads to excessive absorption of s a l t into the f l e s h (Slavin, 1956). Some of the above mentioned advantages and disadvan-tages apply only to the way brine-freezing i s used on tuna c l i p p e r s . Research conducted by the U. S. Fish and W i l d l i f e Service i n the New England area, using the experimental trawler Delaware, has shown that groundfish can be s a t i s f a c t o r i l y frozen i n a 23$ sodium chloride brine, thawed i n fresh water, f i l l e t e d and refrozen and marketed as packaged f i l l e t s (Slavin, 1968). The procedure used i n brine-freezing ground f i s h i s somewhat d i f f e r e n t than that used to brine-freeze tuna aboard tuna c l i p p e r s . Uneviscerated groundfish are put into c y l i n d r i -c a l baskets located i n the brine tank (+10°F), the freezer-tank doors are closed, and the basket-drive motor i s started, causing the baskets to rotate through the brine. The movement of the baskets through the brine provides adequate brine c i r c u l a t i o n around each f i s h , thereby insuring uniform, quick and e f f i c i e n t freezing. Fish of approximately the same weight are put together in. the same basket. After the proper freezing - 1 1 -time elapses, the f i s h are removed from t h e i r baskets, glazed and conveyed by aluminum chutes to the cold-storage hold. The f i s h are held i n cold storage u n t i l the a r r i v a l of the trawler at port, when they are discharged, thawed, f i l l e t e d , and refrozen (Slavin, 1 9 5 6 ) . This type of brine-freezing has several advantages. The f i s h are frozen quickly and e f f i c i e n t l y . The system i s v e r s a t i l e , as i t can also freeze tuna or shrimp, quickly and e f f i c i e n t l y . It requires a minimum of handling, uses a sodium chloride brine, which i s r e l a t i v e l y inexpensive, maintenance cost i s low and i t produces a high-quality frozen f i s h . However, i n the Delaware experiments the brine-frozen f i s h were only compared with f i s h preserved with flake ice and then frozen a f t e r landing at port and not with other methods of freezing f i s h at sea. There are also several disadvantages. Careful tem-perature regulation i s required i n the brine cooler to eliminate the p o s s i b i l i t y of the brine "freezing out" at - 6°F, which might res u l t i n bursting tubes within the brine cooler. Also the penetration of s a l t into the f l e s h w i l l be excessive i f f i s h are l e f t i n the brine considerably longer than the required freezing time (Slavin, 1 9 5 6 ) . When a large run of Sockeye salmon enter B r i s t o l Bay, Alaska, the catch often exceeds the l o c a l cannery capacity. Consequently part of the B r i s t o l Bay catch i s delivered to brine-freezer packers. This brine-frozen salmon i s then - 1 2 -delivered to canneries i n Alaska, Washington, or Oregon for processing. Salmon contains highly unsaturated o i l s , and thus the brine-freezing operation may present serious problems from the salt-catalyzed oxidation of these o i l s during frozen storage p r i o r to canning (Yonker, 1 9 6 3 ) . Dassow ( 1 9 5 6 ) stated that salmon, brine-frozen for l a t e r canning, should be handled, frozen, and stored v/ith even greater care than that practiced with tuna, because of d i f f e r -ences i n the subsequent canning process. Oxidative r a n c i d i t y may occur i n the surface f a t t y f l e s h of both tuna and salmon. However,, v/ith tuna, but not with salmon, the skin and dark f l e s h are scraped o f f and not packed with the l i g h t meat. Thus oxidative r a n c i d i t y may be more of a problem with salmon, p a r t i c u l a r l y i f i t i s kept i n frozen storage for a prolonged period before being marketed or canned. Tomlinson and Geiger ( 1 9 6 3 ) reported that with brine-spray frozen tuna, penetration of sodium into the f l e s h was quite high i n the outer layers (outer 3 / 8 inch) of muscle. Hov/ever, sodium penetration into the inner layers of muscle was n e g l i g i b l e . It was also found that water-thawing reduced the sodium concentration i n the outer 1/8 inch of muscle by approximately 50%. Thus the sodium content of the f i s h a f t e r water-thawing i s acceptable for canning when a suitable reduction i n the s a l t added i s made to compensate for that present i n the muscle, Butler et a l . ( 1 9 5 2 ) reported that round brine-frozen scrod haddock, when compared to iced_ haddock, offered no - 1 3 -complications for s c a l i n g and f i l l e t i n g . The y i e l d of f i l l e t s obtained from the round brine-frozen f i s h was as high as that from control l o t s of iced, dressed f i s h . The appearance of the f i l l e t s from brine-frozen haddock was i n a l l instances comparable with that of good quality f i l l e t s from iced f i s h . The appearance, f l a v o r , odor, and texture of the f i l l e t s from round brine-frozen f i s h , thawed In fresh water at + 5 3 ° F or +72 ° F , were quite acceptable. Pottinger ( 1 9 5 2 ) reported that the free drip from f i l l e t s of f i s h brine-frozen at sea was about the same as that from iced f i s h (3 to k%). Also p a l a t a b i l i t y tests revealed no objections to the s l i g h t l y more salty f l a v o r of f i l l e t s prepared from f i s h frozen i n c i r c u l a t i n g brine at + 5 ° F to + 1 0 ° F and then air-thawed i n comparison with f i l l e t s prepared from f i s h frozen on cold plates ashore and then air-thawed. However, when the brine-frozen f i s h were water-thawed the s a l t content of the f l e s h was reduced to pre-freezing l e v e l s . Studies on s a l t content of haddock which were brine-frozen and water-thawed showed that s a l t penetration into the meat of f i s h during immersion freezing varied d i r e c t l y with the temperature of the brine. The increased penetration reached serious proportions from the standpoint of p a l a t a b i l i t y when the brine temperature was + 1 5 ° F or above. It was also shown that an increase i n brine concentration caused a propor-tionate increase i n the penetration of s a l t into f i s h during brine-freezing. However, the addition of small quantities of calcium (1%) and of potassium ( 0 . 6 % ) s a l t s to a sodium chloride brine retarded the penetration of s a l t . It was also reported that the s a l t content of commercial f i l l e t s prepared from f i s h that were eviscerated p r i o r to brine-freezing was below the range of 'optimum p a l a t a b i l i t y ' for s a l t {0.9% to 1.2%). Also, water-thawing of the f i s h p r i o r to f i l l e t i n g re-duced the s a l t content to a l e v e l below the taste threshold for s a l t ( 0 . 5 $ to 0.6%) i n f i s h . Excessive s a l t penetration occurred only i n the nape of the f i s h , a portion which i s not normally incorporated into the commercial f i l l e t (Holston and Pottinger, 195*0 . The r e s u l t s of Peters ( 1 9 5 9 ) , who investigated the s a l t content of large eviscerated haddock frozen i n brine at +5°F, +10°F, or +15°F, were si m i l a r to those of Holston and Pottinger (195*0. Miyauchi and Heerdt (195**) investigated the s a l t content of sockeye salmon frozen by immersion for 12 hours i n brine, cooled to about + 5°F, then held i n dry storage at +5°F. After thawing i n running water, i n s t i l l water, or i n s t i l l a i r the f i s h were canned. The amount of s a l t added to each can varied according to the thawing method used. It was concluded that the amount of s a l t retained by sockeye salmon was not excessive, and that the s a l t retained from brine-freezing can be compensated for by the reduction of the s a l t usually added i n canning by 20 to 50%. The s a l t content of canned chum salmon stored i n brine at 5°F for approximately - 1 5 -two weeks p r i o r to canning was also determined. When the f i s h were thawed i n running water less than 0.5% s a l t was present i n the canned product. This retained s a l t could e a s i l y be compensated for by decreasing the amount of s a l t added during the canning process. Harrison and Roach ( 1 9 5 3 ) froze chinook and chum salmon, and grey cod i n an eutectic solution of sodium chloride then rinsed them i n fresh water immediately aft e r freezing. The f l e s h , even the f i r s t layer under the skin, had s a l t concentrations well below the generally acceptable l e v e l for p a l a t a b i l i t y . MIyauchi ( 1 9 5 3 ) observed that the s a l t absorbed by brine-frozen sockeye salmon i n t e r f e r e s with ice glazing of f i s h at.storage temperatures of 0°P to +10°F. The glaze taken by brine-frozen sockeye salmon i n t h i s temperature range was not considered s a t i s f a c t o r y . However, the glaze taken at -20°F was considered fgood'. Peters et a l . ( 1 9 6 8 ) while investigating the effects of stage of r i g o r , method of freezing (brine-freezing vs plate-f r e e z i n g ) , and the method of thawing (microwave vs water) on refrozen cod showed that neither the average taste panel scores nor the chemical tests for moisture, t o t a l l i p i d , t i t r a t a b l e free f a t t y acids, and extractable protein nitrogen showed any difference a t t r i b u t a b l e to state of r i g o r , freezing method, or thawing method. - 1 6 -B* Frozen Storage Changes Occurring During Frozen Storage a) L i p i d Hydrolysis In contrast to oxidative changes i n f i s h l i p i d s , hydrolysis by i t s e l f has no obvious n u t r i t i o n a l s i g n i f i c a n c e (Lovern, 1 9 6 2 ) . In f i s h t i s s u e , as such, any effects of l i p i d hydrolysis on product quality are l i k e l y to be due to secondary changes, e.g., possible increased s u s c e p t i b i l i t y to oxidation and development of o f f - f l a v o r s . Brocklesby ( 1 9 3 3 ) observed a gradual increase i n free f a t t y acids during the frozen storage of chinook and coho salmon. Dyer et a_l. ( 1 9 5 8 ) reported that there was almost no hydrolysis i n r o s e f i s h stored at either +10°F or - 1 0°F but i n A t l a n t i c halibut while there was no hydrolysis at - 1 0°F, some free f a t t y acid formation did occur at 0°F (up to about 10$ i n 6 months) and when the halibut were stored at +10°F hydrolysis was more rapid (about .20$ free fatty acids i n 6 months). With pla i c e there was an increase i n free f a t t y acids to about 3 2 $ i n 6 months at +10°F. Fresh A t l a n t i c cod had free f a t t y acid values of about 1 5 $ , however, when stored at - 1 0°F the values increased to about 50$ i n 16 months and when stored at +10°F hydrolysis was very rapid, the free f a t t y acids reaching values of about 6 0 $ i n one month and eventually reaching values of 80 to 9 0 $ i n 16 months. -17-Wood and Haqq ( 1 9 6 2 ) o b s e r v e d l i p i d h y d r o l y s i s and f r e e f a t t y a c i d f o r m a t i o n i n l i n g c o d and P a c i f i c gray cod s t o r e d a t +10°P. F r e s h l i n g c o d and P a c i f i c gray cod c o n t a i n e d 3 . 4 and 5 . 2 $ f r e e f a t t y a c i d ( $ o f t o t a l l i p i d ) , r e s p e c t i v e l y . A f t e r 15 weeks o f s t o r a g e at +10°F t h e s e v a l u e s i n c r e a s e d t o 28 and 42%, r e s p e c t i v e l y . Gray cod resembled A t l a n t i c cod i n t h a t t h e r e was a p e r i o d o f r a p i d h y d r o l y s i s f o l l o w e d by a s l o w e r more u n i f o r m r a t e o f h y d r o l y s i s . T h i s p e r i o d o f r a p i d h y d r o l y s i s was not o b s e r v e d w i t h l i n g c o d . O l l e y e_t a _ l . ( 1 9 6 2 ) showed t h a t w i t h A t l a n t i c c o d , lemon s o l e , A t l a n t i c h a l i b u t , d o g f i s h and e l e v e n o t h e r s p e c i e s t h e r e was a c o n s i d e r a b l e i n c r e a s e i n t i t r a t a b l e f r e e f a t t y a c i d s a f t e r 16 weeks at -l4°C. The f o r m a t i o n o f f r e e f a t t y a c i d s , e x p r e s s e d as a p e r c e n t a g e o f t h e t o t a l l i p i d , was v e r y s i m i l a r i n c o d , lemon s o l e , and h a l i b u t but was much l e s s i n d o g f i s h . P h o s p h o l i p a s e a c t i v i t y appeared t o be n e g l i g i b l e i n t h e Elasmobranchs s t u d i e d , but i n a l l o t h e r s p e c i e s , p h o s p h o l -i p a s e was a t l e a s t as i m p o r t a n t as l i p a s e i n p r o d u c i n g f r e e f a t t y a c i d s , and i n t h e Gadoids and r e l a t e d s p e c i e s almost a l l t h e f r e e f a t t y a c i d s came from h y d r o l y s i s o f p h o s p h o l i p i d s . I t was a l s o o b s e r v e d t h a t the average l i p i d c o n t e n t ( $ o f wet m uscle) o f h a l i b u t v a r i e d from 0 . 7 5 $ i n June t o 1 . 0 5 % i n December, lemon s o l e v a r i e d from 0 . 7 8 $ i n J u l y t o 1.04$ i n J a n u a r y w h i l e d o g f i s h v a r i e d from 4 . 2 7 $ i n J u l y t o 1 4 . 0 $ i n F e b r u a r y . The c o u r s e o f f r e e f a t t y a c i d f o r m a t i o n i n rainbow t r o u t s t o r e d a t -4°C has been found t o be s i m i l a r t o t h a t i n cod (Jonas and B i l i n s k i , 1 9 6 7 ) . Free fa t t y acids of fresh-water whitefish muscle, stored at - 1C°C for sixteen weeks, have been reported to increase from 4 . 6 $ of the t o t a l l i p i d to 2 1 . 4 $ (Awad et a l . 1 9 6 9 ) . About 61% of the t o t a l free fatty acid increase was derived from phospholipid, and the remainder was probably derived from t r i g l y c e r i d e s * , Olley e_t al_. ( 1 9 6 9 ) showed that the extent of the i n i t i a l rapid, f i r s t order hydrolysis reaction, appeared to be limited by the amount of free water available i n the frozen state. Other re s u l t s of Olley _t_ al_. ( 1 9 6 9 ) showed that with haddock there was a p r e f e r e n t i a l hydrolysis of ci^.Qt ^ 1 8 * 0 a n c ^ ^ 2 0 * 5 P n o sP n°lipids, and that the rates of hydrolysis of phosphatidylcholine and phosphatidylethanolamine were s i m i l a r . Previously, Bligh ( 1 9 6 1 ) had shown that phosphat-idylethanolamine and phosphatidylcholine hydrolysis were mainly responsible for free fatty acid increase i n frozen stored A t l a n t i c cod. Also Bligh and Scott ( 1 9 6 6 ) showed that with cod stored at - 1 2°C the rate of breakdown of phosphatidylcholine was faster than that of phosphatidylethanolamine. Bosund and Ganrot ( 1 9 6 9 ) , while studying l i p i d hydrolysis i n frozen B a l t i c herring, also observed that phosphatidylcholine was hydrolyzed faster than cephalin (phosphatidylethanolamine + phosphatidylserine). They also observed considerably more phospholipid breakdown and free fatty acid formation i n the red muscle than i n the white muscle. They estimated that only 45$ of the free f a t t y acids i n the red muscle and 75$ of those - 1 9 -In the white muscle are formed by hydrolysis of phospholipids, the remainder being formed by hydrolysis of t r i g l y c e r i d e s . U n t i l recently f i s h muscle was known only to contain a lipase able to catalyze the hydrolysis of short chain t r i g l y -cerides ( B i l i n s k i , 1 9 6 9 ) . However, the results of B i l i n s k i and Lau ( 1 9 6 9 ) indicate that rainbow trout muscle also possesses l i p o l y t i c a c t i v i t y capable of hydrolyzing depot fat which, In f i s h , i s composed of t r i g l y c e r i d e s containing predominantly f a t t y acids with 12 - 2H carbons. Thus, i t i s now known that hydrolysis of long-chain t r i g l y c e r i d e s can occur i n herring (Bosund and Ganrot, 1 9 6 9 ) and i n rainbow trout ( B i l i n s k i and Lau, 1 9 6 9 ) . Whether other species of f i s h , other than A t l a n t i c cod, also possess t h i s a b i l i t y i s not yet known. The work of Yurkowski and Brockerhoff ( 1 9 6 5 ) indicated that the phospholipids of frozen stored cod are broken down by two enzymes, phospholipase and lysophospholipase. Also t h e i r studies on lys o l e c i t h i n a s e showed that o l e i c acid had a strong i n h i b i t o r y e f f e c t . Although many researchers have investigated the increase i n free fatty acid formation during frozen storage of various species of f i s h , l i t t l e work has been done to determine i f t h i s free fatty acid Increase relates to organ-o l e p t i c changes during frozen storage. Fraser and Dyer ( 1 9 5 9 ) stated that with A t l a n t i c cod the fact that taste panel scores were s t i l l high af t e r a year's storage, although the percent free fatty acids had increased to approximately 50% and S0% - 2 0 -when the f i s h were stored at - 1 0°F and +10°F, respectively, showed that free fatty acids probably do not affe c t taste. Peters et al_. ( 1 9 6 8 ) , while investigating the eff e c t of stage.of r i g o r , method of freezing and method of thawing on the storage of refrozen cod stored at - l 8 ° C , observed that the c o r r e l a t i o n c o e f f i c i e n t of free fatty acid production with the average taste panel scores (average of odor, f l a v o r , texture and o v e r a l l quality scores) of the frozen stored samples was - 0 . 9 7 4 (P ^ 0 . 0 1 ) . However, Olley e t ' a l . ( 1 9 6 9 ) stated that i f Peters had attempted the c o r r e l a t i o n with samples stored at a d i f f e r e n t temperature the significance might not have been so high. Olley's r e s u l t s indicated that free fatty acid production does not go to completion at a l l temperatures or, i f i t does so, i t i s at two d i s t i n c t rates, an i n i t i a l rapid reaction followed by a much slower one. Thus she i s of the opinion that free f a t t y acid production and actomyosin i n s o l u b i l i s a t i o n cannot both equate to a taste panel for texture at a l l temperatures of frozen storage. b) Changes i n Protein E x t r a c t a b i l i t y Some workers have observed that, depending upon the species, protein e x t r a c t a b i l i t y ( i n 5$ NaCl) tends to decrease during frozen storage. This phenomenon has been observed i n frozen plaice f i l l e t s (Dyer and Morton, 1 9 5 6 ) ; i n ro s e f i s h (Dyer ei_ a l , , 1 9 5 6 ) ; i n At l a n t i c cod (Dyer and Fraser, 1 9 5 9 ; Olley and Lovern, i 9 6 0 ) ; i n A t l a n t i c halibut, lemon sole, and dogfish (Olley et a l . , 1 9 6 2 ) ; i n saithe, haddock, whiting, and - 2 1 -mackerel (Olley e_t a l . , 1 9 6 7 ) ; i n P a c i f i c cod, and P a c i f i c halibut (Tomlinson et a l . , 1 9 6 9 ) ; and i n fresh water whitefish (Awad et a l . , I 9 6 9 ) . No detectable change i n the extractable protein nitrogen of lingcod, ocean perch, red snapper, orange spotted r o c k f i s h , and rock sole stored for 8 1/2 months at - 3 0°C was noticed by Tomlinson e_t a l . , ( 1 9 6 9 ) . The decrease i n protein e x t r a c t a b i l i t y was also quite slow for lemon sole and dogfish (Olley et a l . , 1 9 6 2 ) . The r e l a t i o n s h i p between the^increase i n free f a t t y acid formation and the decrease i n protein e x t r a c t a b i l i t y that occurs during frozen storage appears to vary among species. Olley et a_l. ( 1 9 6 2 ) while studying frozen stored A t l a n t i c cod, lemon sole, A t l a n t i c halibut, and dogfish observed that the rate of increase of free f a t t y acids was twice as high for dogfish as for the other species, but the rate of decrease i n protein e x t r a c t a b i l i t y was less for dogfish than i t was for cod. Also lemon sole, which was si m i l a r to cod i n free fatty-acid production, showed much less protein denaturation than cod. However, Hanson and Olley ( 1 9 6 5 ) hypothesized that neutral l i p i d s protect protein from free fatty acid denaturation i n s i t u and not only at the homogenization stage of the soluble protein determination. This hypothesis was supported by the work of Olley et a l . ( 1 9 6 7 ) who showed that small quantities of neutral l i p i d may have a protective effect on f i s h muscle proteins. Using model systems King e_t a l . ( 1 9 6 2 ) showed that the addition of "small amounts of l i n o l e i c or l i n o l e n i c acid - 2 2 -caused cod actomyosin to p r e c i p i t a t e from a solution of i s o l a t e d cod actomyosin. Also using model systems, Anderson e_t 'al. ( 1 9 6 5 ) concluded that the i n t e r a c t i o n of protein with fa t t y acid r e s u l t i n g i n i n s o l u b i l i z a t i o n of the protein i s optimal at an i o n i c strength of 0 . 5 at pH 7 . 2 and that 5 u i s the i o n i c strength that should exist i n the c e l l u l a r f l u i d of cod muscle as a r e s u l t of freezing to - 1 . 5°C. In his recent review Connell ( 1 9 6 8 ) stated that the protein e x t r a c t a b i l i t y of r o s e f i s h stored at +10°F and of skate and nursehound stored at - 7°C, declined considerably with-out the formation of free fatty acid. He refers to unpublished data and concludes that free f a t t y acid production i s merely one change which coincides i n some species with the decline i n protein e x t r a c t a b i l i t y . Dyer and Morton ( 1 9 5 6 ) observed that the texture ratings of p l a i c e f i l l e t s stored at - 1 2°C, showed an increase i n tough-ness p a r a l l e l to the decrease i n protein e x t r a c t a b i l i t y . Also Dyer et_ a l . ( 1 9 5 6 ) found that with r o s e f i s h stored at - 1 2°C taste panel r e s u l t s correlated reasonably well with protein e x t r a c t a b i l i t y . However, with r o s e f i s h stored at - 2 3°C (Dyer £ _ . _ _ • » 1 9 5 6 ) and A t l a n t i c cod stored at - 3 0°C (Love, 1 9 5 6 ) marked increases i n toughness occurred before any appreciable decrease i n protein e x t r a c t a b i l i t y had taken place, Luippen ( 1 9 5 7 ) observed a consistent close r e l a t i o n s h i p between the increase i n toughness aft e r b o i l i n g and the decrease i n the r a t i o of soluble nitrogen to t o t a l nitrogen i n cod samples that had been stored at -lQ°C. However, no c o r r e l a t i o n was observed with the samples that had been stored at - 2 0°C and at - 3 0°C. Moorjani e_ al. ( i 9 6 0 ) reported that with f i l l e t e d morwong packed i n evacuated cans or sealed i n cellophane bags and stored at -l8°C, the differences In protein e x t r a c t a b i l i t y were associated with texture differences a f t e r storage times of 2 to 6 months. Cowie and L i t t l e ( 1 9 6 6 ) studied toughness and protein s o l u b i l i t y of cod f i l l e t s during storage at - 2 9°C for 82 months. There was a steady decrease i n protein s o l u b i l i t y from 72% to ^5% but the development of toughness during frozen storage was extremely var i a b l e . In fact cod which had been stored for 82 months were more tender than some of the control f i l l e t s which had been freshly frozen but not stored. Also Cowie and L i t t l e ( 1 9 6 7 ) investigated the r e l a t i o n s h i p between toughness and protein e x t r a c t a b i l i t y with cod stored at -7°C and at -14°C. There was a poor c o r r e l a t i o n between toughness and protein e x t r a c t a b i l i t y . They concluded that protein e x t r a c t a b i l i t y alone cannot accurately describe toughness and suggested that pH must also be considered. Tomlinson e_t a l , ( 1 9 6 5 a ) observed that.protein e x t r a c t a b i l i t y can change markedly during the thawing of frozen f l e s h . The a l t e r a t i o n appeared to be related to f l e s h pH as e x t r a c t a b i l i t y decreased to a greater extent at lower pH. Thus one possible explanation of the lack of c o r r e l a t i o n between protein e x t r a c t a b i l i t y and toughness of cooked f l e s h observed by Cowie and L i t t l e ( 1 9 6 7 ) and others i s that the protein e x t r a c t a b i l i t y was measured by homogenization of frozen rather than thawed f l e s h . - 2 4 -In contrast, Peters et a l . ( 1 9 6 8 ) found that taste panel scores of cod stored at - 18°C for 12 months correlated (r = + 0 . 9 2 ) s i g n i f i c a n t l y ( P - 0 . 0 5 ) with extractable protein. Connell, ( 1 9 6 9 ) while investigating changes i n the eating quality of frozen stored cod, observed that protein e x t r a c t a b i l i t y correlated much better with fla v o r (r = - 0 . 7 3 0 ) or firmness (r = - 0 . 6 6 4 ) of cold stored f i s h than did either color r a t i o or c e l l f r a g i l i t y methods. °) Changes in Thaw Drip The amount of thaw drip or l i q u i d that exudes when frozen f i s h tissue thaws i s affected by several factors. The amount of drip formed i s d i r e c t l y related to the r a t i o of cut surface area to the weight of f l e s h . With chinook, coho, and chum salmon and P a c i f i c halibut the rate of freezing influences the amount of drip; rapid freezing r e s u l t s i n the least d r i p . Drip increases with length of frozen storage and i s greater at higher temperatures. Brining of f i s h f l e s h appears to reduce the amount of drip formed. Increases i n drip are probably related to decreased protein e x t r a c t a b i l i t y (Miyauchi, 1 9 6 3 ) . Young ( 1 9 4 1 ) observed that with P a c i f i c halibut, over 40 pounds i n weight, the amount of drip increased from head to t a i l but with smaller f i s h the drip was less and did not always increase i n the same sequence. Tomlinson e_t a l . ( 1 9 6 9 ) reported that the free (thaw) drip of P a c i f i c h a l i b u t , frozen pre-rigor, increased during - 2 5 -frozen storage from 5% a f t e r nine days to 7 . 6 $ a f t e r 455 days, while that of the i n - r i g o r samples, increased from 5$ to 7.8% and the free drip of the post-rigor samples Increased from 6 . 6 $ to 1 0 . 8 $ . d) Oxidative Rancidity One reason ran c i d i t y i n foods i s undesirable i s that o f f - f l a v o r s and off-odors develop making the food unsuit-able for consumption. It i s known that oxidized fats cause the destruction of several fat-soluble .vitamins and carotene. It i s also claimed that oxidized fats are carcinogenic or i n other ways seriously harmful, as very highly oxidized and oxidatively polymerized fats have been shown to produce toxic e f f e c t s i n animals (Lundberg, 1 9 6 1 ) . The muscle of most f i s h i s not uniform i n color. That part located just beneath the skin, the so c a l l e d l a t e r a l l i n e muscle, i s often brown or reddish i n color. This red muscle has a high l i p i d concentration and the li p i d s ' are highly unsaturated. A t l a n t i c halibut (Hippoglossus  hippoglossus) contains 2 3 . 7 $ l i p i d (wet weight basis) i n the red muscle while the white or ordinary muscle ( F i g . l ) contains 7 . 0 $ l i p i d (Love, 1 9 7 0 ) . The rate at which oxidative r a n c i d i f i c a t i o n occurs i s affected by (a) the amount of oxygen present, (b) the degree of unsaturation of the l i p i d components, (c) a n t i -oxidants, (d) metals such as copper, (e) organic catalysts such -26-as hematin compounds and lipoxidases, (f) certain s a l t s , (g) processing treatments, (h) packaging, ( i ) exposure to l i g h t , and (j) storage temperatures. In most types of l i p i d oxidation i t i s e s s e n t i a l that some oxygen be present. The more highly unsaturated the fatty acids i n the l i p i d s , the faster the rate of oxidation. Contamination of foods with inorganic ox-i d a t i v e catalysts such as copper from equipment or other sources, often leads to rapid development of r a n c i d i t y ( M i t c h e l l and Henick, 1 9 6 1 ) . It has also been shown that certain products, such as frozen meats, become rancid at a slower rate i f stored without added s a l t . Autoxidation takes place at increasing rates as the storage temperature of a food i s increased, provided oxygen i s present. Thus storage at low temperatures i s advantageous i n those instances where ra n c i d i t y may be a problem. Lipids i n foods that have been exposed to conditions promoting oxidation may have already passed through the induc-t i o n period of oxidative r a n c i d i t y at the i n i t i a l time of storage and naturally w i l l not have a shelf l i f e as long as that optimally possible (Mitchell and Henick, 1 9 6 1 ) . the presence of oxygen, i s the general chain mechanism outlined below (Lundberg, 1 9 6 2 ; Ingold, 1 9 6 8 ) . I n i t i a t i o n The mechanism of autoxidation of l i p i d s , stored i n RH + 0 2 ->free r a d i c a l s -=>free r a d i c a l s (e.g.,R*, RO*, ROC, • HO*, etc.) -27-Propagation R ' + 0 _ — •ROO* R 0 0 ' + RH-Branching R 0 0 * - ^ R O O H + R ' ROOH + M-- £ ^ R O # + HO* -s»-free r a d i c a l s Termination R ' + R* R* + R O C " ROO* + ROO" -^.stable (non-radical inactive end products) ROO" and ROOH represent a peroxy r a d i c a l and a hydro-peroxide, respectively. M represents a metal catalyst which increases the rate of oxidation of the substrate by increasing the rate of decomposition of the hydroperoxide to free r a d i c a l s . Heavy metals, p a r t i c u l a r l y those possessing two or more valency states with a suitable oxidation-reduction p o t e n t i a l between them, e.g., cobalt, copper, i r o n , manganese, n i c k e l , are the most powerful catalysts (Ingold, 1968). C a s t e l l and MacLean (1964, a) i n a study of copper-catalyzed r a n c i d i t y of cod f i l l e t s , observed that muscle from the t a i l section becomes rancid more rapidly than muscle from the head or centre sections. They also observed that cod caught i n the winter and early spring develops r a n c i d i t y at a greater rate than cod caught i n the summer and f a l l . The seasonal differences were primarily concerned with the induction period i n the development of r a n c i d i t y . Consequently -28-they thought the seasonal differences could be the r e s u l t of differences i n the natural antioxidants i n the muscle, p a r t i c u l a r l y tocopherol, which are known to fluctuate with the feeding cycle of cod. In contrast, Bailey e_t a l . ( 1 9 5 2 ) re-ported that with chinook salmon the o i l from the white muscle i s generally more unsaturated than o i l from the red muscle and that o i l from the f l e s h near the head i s more unsaturated than o i l from the f l e s h near the t a i l . Thus, with chinook salmon one would expect that muscle near the head would become rancid f a s t e r than muscle near the t a i l although no d i r e c t evidence to support t h i s suggestion has been reported i n the l i t e r a t u r e . C a s t e l l and MacLean ( 1 9 6 4 ,b) showed that a c t i v e l y growing bacteria exert an antioxidant e f f e c t and suppress the development of copper-catalyzed r a n c i d i t y i n cod muscle. C a s t e l l e_t a l . ( 1 9 6 6 Ja) reported that the addition of free aromatic, het e r o c y c l i c , and sulphur containing amino acids retarded copper-catalyzed r a n c i d i t y as measured by the TBA t e s t . In the absence of added metallic ions, however, the a l i p h a t i c amino acids and cysteine showed strong pro-oxidant a c t i v i t y . Also those amino acids which i n h i b i t e d metal-induced r a n c i d i t i e s did not retard r a n c i d i t y induced by the addition of sodium chloride. C a s t e l l and Spears ( 1 9 6 8 ) added from 1 to 50 ppm of ten d i f f e r e n t heavy metal ions to blended muscle taken from freshly k i l l e d cod, haddock, flounder, r e d f i s h , herring, mackeral, scallops, and lobster stored for 24 hours at 0°C. - 2 9 -The r e s u l t i n g r a n c i d i t i e s were determined by TBA values and 2+ 2+ 2+ by odours. With some exceptions Fe , V , and Cu were the 2+ most active c a t a l y s t s . Fe was always more e f f e c t i v e than 3+ 2+ 2+ 2+ Fe while Cd , Co , and Zn produced r a n c i d i t y with the fa t t y species but not with any of the other species while 2+ 2+ 2+ Ni , Cr , and Mn did not accelerate r a n c i d i t y i n any of the muscles. There was considerable difference i n the r e l a t i v e s u s c e p t i b i l i t y to ra n c i d i t y induced by s p e c i f i c metals i n muscle from d i f f e r e n t species. Banks ( 1 9 3 7 ) found that s a l t .accelerated r a n c i d i t y i n raw herring but not after i t was cooked. Tarr ( 1 9 4 4 , 1 9 4 7 ) reported that immersion of chinook salmon, pink salmon, and chum salmon f i l l e t s i n NaCl solutions increased the r a n c i d i t y (peroxide values) during frozen storage. Also C a s t e l l et a l . ( 1 9 6 5 ) reported that sodium chloride accelerated r a n c i d i t y (TBA values) i n blended cod muscle at 0°C and that the active agent appeared to be Na + ions rather than whole sa l t or C l ~ ions. In addition, E l l i s et a l . ( 1 9 7 0 ) observed that sodium chloride had a d i r e c t pro-oxidant action on the lard of freezer-stored and dehydrated gels while hydrated gels con-tai n i n g sodium chloride, when stored at 20°C, had an " i n h i b i t -ing autoxidation pattern" somewhat sim i l a r to the quantitative influence of NaCl on pH, Also sodium chloride accelerated heme c a t a l y s i s regardless of the presence of antioxidants or chelators. The oxidative r a n c i d i t y of frozen red salmon (chinook -30-and coho) has been found to be accompanied by a fading of the red and yellow pigments of the f l e s h (Tarr, 1 9 ^ 7 ; 1 9 5 5 ] Boyd et a l . 1 9 5 7 ) . - 3 1 -METHODS AND MATERIALS A) Catching and Freezing the Fish P a c i f i c halibut (Hippoglossus stenolepis) were caught by long l i n e i n Queen Charlotte Sound, Northeast of Cape Scott, on June 7 , 1 9 6 9 . The f i s h were eviscerated and placed on ice immediately a f t e r being caught. Approximately six hours l a t e r three of the f i s h were frozen to - 3 0°C i n a v e r t i c a l plate freezer during a period of about 3 . 5 hours. Another three f i s h were frozen to - 5 . 6°C ( 2 2°F) i n a 13% NaCl solution. In order to simulate the anticipated worst possible brine-freezing times aboard commercial halibut long l i n e r s , the halibut were brine-frozen over approximately a 47 hour period. Chinook salmon (Oncorhynchus tsawytscha) and coho salmon (Oncorhynchus kisutch) were caught by seining i n Goletas Channel and Queen Charlotte S t r a i t near Duval Point on the Northeast coast of Vancouver Island on July 2 1 , 1 9 6 9. The f i s h were obtained from commercial salmon seiners on the day of the catch. Three f i s h of each species were frozen to approximately - 3 0°C ( - 2 2°F) i n a v e r t i c a l plate freezer during ~ a period of about 3 . 5 hours. Another three f i s h of each species were brine-frozen to - 5 . 6°C (22°F) i n a 13% NaCl solution over a 9 . 5 hour period. After freezing (whether by brine or plate) the halibut were stored i n a home-type chest freezer and the salmon (chinook and coho) were stored i n a plate freezer u n t i l a r r i v a l at Vancouver. The f i s h were then glazed twice, using a water and ice mixture, put In p l a s t i c bags, and stored at - 3 0°C (-22°F) u n t i l required. B) Sampling and Analysis Depending on species, the f i s h were sampled and an-alyzed 3 or 5 d i f f e r e n t times during the maximum frozen storage time for that species. 'The maximum frozen storage times for the three d i f f e r e n t species were: P a c i f i c halibut 8 l weeks, chinook salmon 77 weeks, and coho salmon 78 weeks. Sampling consisted of sawing one-half inch thick steaks from the anterior end of the f i s h , immediately reglazing the remainder and replacing It i n a p l a s t i c bag for further frozen storage. P r i o r to the f i r s t analysis of each species, the six f i s h of that species were randomly divided into three p a i r s , each pair consisting of one brine-frozen and one pla t e -frozen f i s h . The pairs remained constant throughout' the experiment, and provided t r i p l i c a t e determinations per species per time period. Analysis of one species (six f i s h ) required one week (three days for organoleptic analysis at one pa i r per day, three days for chemical analysis at one pair per day and one day to prepare the samples and reagents for the next six days). Consequently the steaks were sawn from each of the six f i s h at the beginning of the week, placed i n p l a s t i c bags and stored over dry ice u n t i l needed for analysis. The -33-2 - t h i o b a r b i t u r i c acid (TBA) te s t , long chain free f a t t y acid analysis, and taste panel evaluation were conducted on both inside and outside muscle (Fig. 1 ) . Color and pH determinations were made only on white muscle (inside muscle plus the white muscle part of the outside muscle), The whole steak(s) or cross-section(s) of a f i s h was used to determine free d r i p . Immediately p r i o r to a l l analyses, the glaze was removed and the skin torn from the frozen steak leaving the red muscle inta c t on the steak. a) Determination of Thaw Drip Thaw drip from the frozen muscle was determined by a s l i g h t modification of the U. S. standard method for frozen cod f i l l e t s (Anon., i 9 6 0 ) . The time of thawing at 20°'C ( 6 8°F) was fixed at 2 hours for a l l samples. The samples were drained on paper toweling instead of using a U. S. Standard No, 8 c i r c u l a r sieve. The drip was expressed as percent by weight of the frozen f i s h , b) Determination of pH Measurements of pH were made using the method of Tomlinson, e_t al. ( 1 9 6 6 ) . A combination glass electrode was inserted d i r e c t l y into the thawed f l e s h . A Metrohm Model E 2 8 0 A portable pH meter was used. ' - 3 4 -INS IDE M U S C L E (white) O U T S I D E M U S C L E (white) M U S C L E (red) OUTS IDE M U S C L E (white) INS IDE M U S C L E (white) Figure 1. A diagramatic representation of a cross-section of a f i s h showing the areas sampled as inside and outside muscle. - 3 5 -c) Color Determination Hunter Rd, a, and b values were determined on the thawed f l e s h of chinook and coho salmon using the method of Schmidt and Idler ( 1 9 5 8 ) . The thawed f l e s h was placed i n a 3 . 2 cm diameter p l a s t i c p e t r i dish and the average of t r i p l i c a t e measurements of Rd, a, and b were taken using a Model C Gardner Color - Difference Meter. The sample i n the p e t r i dish was turned twice af t e r the i n i t i a l measurement. The reference stan-dard was i d e n t i c a l to that used by Schmidt and Idler ( 1 9 5 8 ) . The reference t i l e had Rd, a, and b readings of 8 . 7 3 , 3 3 . 8 , and 2 0 . 7 r e s p e c t i v e l y . d) Determination of Flavor Differences Differences i n f l a v o r between f i s h brine-frozen and plate-frozen at sea were determined using a t r i a n g l e test (Fi g . 2 ) as outlined by Larmond ( 1 9 6 7 ) . The samples were prepared for organoleptic evaluation by wrapping the'steaks i n aluminum f o i l and steaming them for 12 minutes. The steaks were then unwrapped and divided into samples of inside and outside muscle ( F i g . 1 ) . The f l a v o r of the samples was evaluated by a taste panel consisting of 5 experienced panel members. Each panel member was instructed to taste the red muscle o n . a l l of the outside muscle samples. Taste panels were conducted d a i l y for three days per week. Each session was devoted to the evaluation of one pair of f i s h . Thus -36-TRIANGLE TEST DIFFERENCE ANALYSIS DATE TASTER PRODUCT Instructions: Here are three samples for. evaluation. Two of these samples are duplicates. Separate the odd sample for flavour difference only. (1) (2) Sample Check odd sample (3) Indicate the degree of difference between the duplicate samples and the odd sample. Sl i g h t Much Moderate Extreme_ (4) A c c e p t a b i l i t y : Odd sample more acceptable Duplicate samples more acceptable (5) Comments including texture and odor abnormalities: Figure 2. Taste panel questionaire used to determine f l a v o r differences between frozen stored plate-frozen and brine-frozen P a c i f i c halibut, chinook salmon, and coho salmon. - 3 7 -a f t e r 3 sessions (days) the organoleptic evaluation of one species was completed for that time period giving 30 observations on the outside muscle ( 1 0 observations per pa i r of f i s h ) and 30 observations on the inside muscle. e ) Determination of 2-Thiobarbituric Acid Reactive  Substances The t h i o b a r b i t u r i c acid (TBA) test was conducted using the procedure of C a s t e l l et a l . ( 1 9 6 6 ) with some modifications. Ten grams of frozen tissue and 1 . 1 0 grams of the disodium s a l t of ethylenediaminetetraacetic acid (EDTA) were homogenized with 50 ml. d i s t i l l e d water for 90 seconds i n a Serval Onmi-Mixer. The homogenate was transferred to a 2 5 0 ml. beaker and the mixer rinsed with two 20 ml. portions of d i s t i l l e d water giving a t o t a l volume of approximately 100 ml. The homogenate was s t i r r e d well af t e r the addition of the water and was then a c i d i f i e d to pH 1 . 5 - 1 . 6 by the addition of 4N HC1. The homo-genate was then transferred to a 5 0 0 ml. round bottom flask and the beaker rinsed.with 10 ml. of d i s t i l l e d water. Dow Corning Antifoam A was sprayed into the flask and the mixture was then d i s t i l l e d at a rate to give a t o t a l of 50 ml. d i s t i l l a t e i n 18 - 20 min. Duplicate 5 ml. aliquots of the d i s t i l l a t e were combined with 5 m l . of 0 . 0 2 M TBA solution i n 90% acetic acid, and heated i n a stoppered test tube i n a b o i l i n g water bath for 35 min. The solution was then cooled for 10 min. i n running tap water and the absorbance of the solution was read - 3 8 -i n a Bausch and Lomb double beam Precision Spectrophotometer at 5 3 8 nm. Blank determinations, using d i s t i l l e d water, were run at the same time as the samples. f) L i p i d Extraction The l i p i d s were extracted from 20 g. of frozen f l e s h by a procedure si m i l a r to that of Bligh and Dyer ( 1 9 5 9 ) . Twenty grams of f l e s h , 40 ml. methanol and 20 ml. chloroform were homogenized for 2 min. i n a Serval Omni-Mixer, to give a chloroform: methanol: water r a t i o of 1 : 2 : 0 . 8 . Twenty ml. of chloroform was then added and homogenization continued for another 30 sec. when 20 ml. of water was added and the mixture homogenized for a further 30 sec. This gave a homogenate with a chloroform: methanol: water r a t i o of 2 : 2 : 1 . 8 . The homogenate v/as then suction f i l t e r e d through Whatman No. 1 f i l t e r paper on a No. 2 Buchner funnel into a 250 ml. side-arm f l a s k . The tissue residue, f i l t e r paper and 40 ml. of chloroform were homogenized for 1 min. and suction f i l t e r e d as before. The mixer cup and the extracted tissue residue were rinsed with 20 ml. of a ( 1 : 1 ) chloroform: methanol mixture. The combined f i l t r a t e s were then transferred to a 2 5 0 ml. stoppered graduated cylinder and the side-arm flask was rinsed with 20 ml. of a ( 1 : 1 ) chloroform: methanol mixture. After the chloroform and alcoholic layers completely separated the volume of the chloroform layer was adjusted to - 3 9 -105 ml. and the alco h o l i c layer was removed by aspir a t i o n . Anhydrous sodium sulphate was then added to dry the chloroform layer. The water free l i p i d chloroform solution was then gravity f i l t e r e d through Whatman No. 1 f i l t e r paper into a 250 ml. round bottom f l a s k . The sodium sulphate was rinsed with 15 ml. of reagent grade chloroform. The l i p i d -chloroform solution was then evaporated under vacuum to approximately 10 ml. using a Buchler Plash-Evaporator, the water bath temperature being 30°C. The concentrated l i p i d -chloi'Oform sol u t i o n was then transferred to a 75 ml. culture tube; the 250 ml. round bottom flask was rinsed with a t o t a l of 25 ml. chloroform. g) Removal of the Phospholipids The phospholipids were separated from the l i p i d -chloroform solution using a procedure s i m i l a r to that of Hornstein £t_ a l . ( 1 9 6 7 ) . Mallinckrodt 1 0 0 mesh s i l i c i c acid was activated by heating i t overnight at 120°C. Four grams of activated s i l i c i c acid was added to the chloroform-lipid s o l u t i o n . The tube was then stoppered and the contents mixed on a Vortex mixer for 1 minute, allowed to stand for 5 minutes and then suction f i l t e r e d through Whatman No. 1 f i l t e r paper on a No. 0 Buchner funnel into a 125 ml, side-arm f l a s k . The s i l i c i c acid residue was washed three times with successive 8 ml. portions of chloroform. The f i l t r a t e was then transferred to a 1 0 0 ml. round bottom fl a s k . The 125 ml. side-arm flask - 4 0 -was rinsed with 15 ml. chloroform. The phospholipid free-l i p i d chloroform f r a c t i o n was then evaporated under vacuum to approximately 2 - 5 ml. using a Buchler Plash-Evaporator with, a water bath temperature of 30°C. n ) Is o l a t i o n of Neutral Lipids An isopropanol-KOH solution was prepared by adding 6 . 2 5 gm. KOH to 100 ml. isopropanol then heating and shaking the mixture u n t i l the KOH was dissolved. The solution was allowed to cool and then the supernatant was decanted leaving any H^ O present i n the f l a s k . The neutral l i p i d s were separated from the phospho-l i p i d - f r e e f r a c t i o n by a procedure s i m i l a r to that of McCarthy and Duthie ( 1 9 . 6 2 ) . Four grams of activated 100 mesh s i l i c i c acid were weighted into a 100 ml. beaker. Eight ml. of the isopropanol-KOH solution and 24 ml. anhydrous d i e t h y l ether were then added to the s i l i c i c acid. The contents were mixed and then allowed to stand for 5 minutes. The mixture was then s l u r r i e d into a 2 cm, by 26 cm. glass column and washed with 1 0 0 ml. of anhydrous d i e t h y l ether. The 2 - 5 ml. of phospho-l i p i d - f r e e l i p i d was dissolved i n a small quantity of anhydrous d i e t h y l ether placed on the column and thoroughly washed into the packing by several small portions of anhydrous d i e t h y l ether. The phospholipid-free l i p i d f r a c t i o n was placed on the column under a nitrogen atmosphere. Cholesterol, cholesterol esters, mono-, d i - , and t r i g l y c e r i d e s were then eluted i n one f r a c t i o n -41-from the column with 200 ml. of anhydrous d i e t h y l e t h e r . The n e u t r a l l i p i d s e l u t e d from the column were c o l l e c t e d i n a 250 ml. Erlenmyer f l a s k . When the f r a c t i o n was c o l l e c t e d , the f l a s k was f l u s h e d with n i t r o g e n , stoppered, and p l a c e d i n a r e f r i g e r a t o r f o r subsequent weight d e t e r m i n a t i o n of the n e u t r a l l i p i d s . i ) Removal of Free F a t t y Acids The f r e e f a t t y a c i d s (FFA) r e t a i n e d on the column were removed by two 10 ml. a l i q u o t s of boron t r i f l u o r i d e s o l u t i o n , c o n t a i n i n g 125 gm. BF^ per 1000 ml. methanol (M e t c a l f e and Smith, 1961). j ) E s t e r i f i c a t i o n of Free F a t t y A c i d s The e l u t e d FFA were t r a n s - e s t e r i f i e d by m i l d l y r e f l u x i n g the e l u t e d s o l u t i o n f o r 15 minutes. The s o l u t i o n was then t r a n s f e r r e d to a separatory f u n n e l and the round bottom f l a s k r i n s e d with 5 ml. of n-pentane. Twenty ml. o f d i s t i l l e d water was added to the s e p a r a t o r y f u n n e l and the l a y e r s allowed to s e p arate. The aqueous l a y e r was then t r a n s -f e r r e d i n t o the o r i g i n a l round bottom f l a s k and the o r g a n i c l a y e r was t r a n s f e r r e d to a 50 ml. Erlenmyer f l a s k c o n t a i n i n g anhydrous sodium s u l p h a t e . F i v e m i l l i l e t e r s of s p e c t r a -s c o p i c a l l y analyzed n-pentane was added to the aqueous l a y e r and the s o l u t i o n r e - e x t r a c t e d as b e f o r e . The two o r g a n i c f r a c t i o n s were combined and w e l l d r i e d with anhydrous sodium - 4 2 -sulphate. The water-free organic f r a c t i o n was then transferred to a 15 ml. graduated test tube and the sodium sulphate rinsed with two portions of 3 nil. n-pentane. Using nitrogen gas, the volume i n the test tube was adjusted to 2 ml. and transferred to a 1 dram v i a l . The test tube was rinsed well with n-pentane. The a i r space i n the v i a l was flushed with nitrogen gas and the v i a l sealed, l a b e l l e d , and stored at - 3 0°C for subsequent gas chromatographic analysis. k) Determination of the Weight of the Neutral Lipids The neutral l i p i d content was determined by flas h evaporating the 200 ml. neutral l i p i d f r a c t i o n almost to dryness. Ten m i l l i l e t e r s of chloroform was then added to the round bottom f l a s k and the contents transferred to a pre-weighed aluminum weighing disk. The round bottom flask was rinsed well with chloroform. The weighing dishes were then placed on a hot plate, (the temperature set at low) i n a fume hood, i n order to evaporate most of the chloroform. The samples were dried to constant weight by placing them i n a vacuum oven at 70°C for 3 hours. 1) Gas Chromatographic Analysis of the Free Fatty  Acids 'The i s o l a t e d free fatty acid methyl esters were analyzed using a Micro-Tek Model 220 gas chromatograph equipped with a flame i o n i z a t i o n detector. The column used v/as of -43-stain l e s s s t e e l , 10 f t . i n length and 3/16" i n diameter, packed with 3% EGSP-Z (an ethylene g l y c o l - s u c c i n i c acid diphenyl-diethoxysilane polyester) on 100 - 120 mesh Gas-Chrom Q, Lot 4 3 0 3 , purchased from Applied Science Laboratories. Nitrogen at 5.7 ml/min was employed as the c a r r i e r gas. The i n l e t temperature was 250°C and the detector temperature was 240°C. A l l analyses were conducted isothermally .with a column temperature of 185°C. Methyl tricosanoate ( 2 3 : 0 ) purchased from Applied Science Laboratories was used as an i n t e r n a l standard. The fatty acid methyl esters were i d e n t i f i e d by comparison of t h e i r retention times with those of herring o i l and cod l i v e r o i l . The recorder was equipped with a Disc Chart Integrator and quantitation was based on the assumption that detector response was proportional to carbon content. The r e s u l t s were expressed both as a percentage of the t o t a l FFA analyzed and as mg. FFA per gm. neutral f a t . m) Determination of the Mineral Concentration i n the Flesh Sodium and potassium concentration i n the f i r s t half inch of muscle beneath the skin (outside muscle) and i n the next adjacent half inch of muscle (inside muscle) was determined on the frozen f l e s h using the method of Thompson ( 1 9 6 9 ) . The dry-ash method was used and the sodium and potassium concentrations i n the solutions were determined using an E.E.L. Flame Photometer. The concentrations were expressed as mg/gm f l e s h . - 4 4 -Chloride concentration i n the f i r s t h a l f inch of muscle beneath the skin (outside muscle) and i n the next adjacent hal f inch of muscle (inside muscle) was determined using Quantab Chloride T i t r a t o r s S031 No. 1 1 7 5 . The procedure used was s i m i l a r to that outlined i n the instructions for Quantab Chloride T i t r a t o r s SOkl No. 1176 except a t o t a l volume of 50 ml. rather than 100 ml. v/as used. C) S t a t i s t i c a l Analysis The sodium, potassium, and chloride concentration data as well as the color, thaw drip, and pH values were s t a t i s t i c a l l y analyzed using a randomized complete block s p l i t - p l o t design. The TBA values and the long chain free f a t t y acid data were s t a t i s t i c a l l y analyzed using a randomized complete block s p l i t - s p l i t plot design. Pairs of f i s h were the r e p l i c a t e s or blocks, methods of freezing were the whole p l o t s , locations (inside or outside muscle) were the s p l i t - p l o t s , and times of frozen storage were the s p l i t - s p l i t plots for the analyses of the TBA test and long chain free fatty acid data. Pairs of f i s h were the blocks, methods of freezing were the whole p l o t s , and locations were the s p l i t - p l o t s for the analyses of the sodium, potassium, and chloride concentration data, For the analyses of the color, thaw d r i p , and pH data pairs of f i s h were the blocks, methods of freezing were the whole plots and times of frozen storage were the s p l i t - p l o t s . -US-Pairs were considered random whereas a l l other effects were considered fixed i n a l l analyses. The thaw drip values and the free fatty acid data expressed as percentages were transformed,, using the arcsine transformation, before any analyses of variance or correlations were conducted. The ab-solute differences i n the pH, thaw dri p , color, TBA values, and the free f a t t y acids^between the brine-frozen samples and the plate-frozen samples were correlated with the taste panel scores (number of correct i d e n t i f i c a t i o n s ) . A l l f a t t y acids i n the text and i n a l l tables are described using the shorthand notation; chain length: number of double bonds, e.g. 18:2 where chain length = 18 carbons, number of double bonds = 2. RESULTS A • Analysis of Variance a). Flesh pH Analysis of variance of the pH data showed that ther were no s i g n i f i c a n t differences i n pH between brine-frozen and plate-frozen samples of P a c i f i c h a l i b u t , chinook salmon, and coho salmon (Table I ) . The analysis (Table I) also revealed s i g n i f i c a n t differences i n pH among d i f f e r e n t frozen storage times for P a c i f i c halibut (P 0 . 0 5 ) chinook salmon (P = 0 . 0 1 ) , and coho salmon (P - 0 . 0 0 1 ) . Also with chinook salmon there was a s i g n i f i c a n t difference (P - 0 . 0 5 ) among p a i r s . i ) P a c i f i c Halibut The pH of P a c i f i c halibut decreased steadily during frozen storage (Fig. - 3 ) . Duncan's new multiple range test showed that the pH at 81 weeks of frozen storage was not s i g n i f i c a n t l y d i f f e r e n t than the pH at 62 xveeks but was s i g n i f i c a n t l y lower (P - 0 . 0 5 ) than at 4 5 , 3 1 and 14 weeks (Table I I ) . i i ) Chinook Salmon The pH of chinook salmon did not change steadily during frozen storage. The pH increased from 9 weeks u n t i l 27 weeks, decreased u n t i l 58 weeks and f i n a l l y increased u n t i l 77 weeks of frozen storage ( F i g . 4 ) . The pH of chinook salmon at 58 weeks was s i g n i f i c a n t l y lower (P - 0 . 0 5 ) than the pH TABLE I Analysis of variance of pH values of P a c i f i c Halibut, Chinook Salmon, and Coho Salmon. P a c i f i c Halibut Chinook Salmon Coho Salmon Source • d.f. M.S. Prob 1 d.f. M.S. Prob d.f. M.S. Prob Pairs 2 0 . 0 1 2 6 0 . 1 5 4 2 2 0 . 0 3 3 7 0 . 0 2 9 9 * 2 0 . 0 0 0 9 0 . 8 1 5 4 Methods 1 0 . 0 0 5 9 0 . 5 1 6 8 1 0 . 0 0 3 4 0 . 3 2 3 6 1 0 . 0 0 8 9 0 . 3 1 2 8 Error a 2 0 . 0 0 9 6 2 0 . 0 0 2 0 2 0 . 0 0 4 9 Time 4 0 . 0 2 6 5 0 . 0 1 3 9 * 4 • 0 . 0 5 7 4 0 . 0 0 1 4 * * 2 0 . 1 5 6 1 0 . 0 0 0 1 * * * M x T 4 0 . 0 0 1 0 0 . 9 5 2 5 4 0 . 0 0 5 7 0 . 5 7 8 4 2 0 . 0 1 4 5 0 . 0 8 1 2 Error b 16 0 . 0 6 6 0 16 0 . 0 0 7 7 8 0 . 0 0 4 2 1 P r o b a b i l i t y of type 1 error 2 Error a was used to test Methods Error b was used to test a l l terms except Methods * S i g n i f i c a n t at the 5% l e v e l ** S i g n i f i c a n t at the 158 l e v e l *** S i g n i f i c a n t at the 0.1% l e v e l - 4 8 -TABLE II Duncan's new multiple range test on the s i g n i f i c a n t 'time effects of the analyses of variance on the pH values of P a c i f i c Halibut, Chinook Salmon, and Coho Salmon. ' P a c i f i c Halibut (2) 14 weeks^ ' 31 weeks 46 weeks 62 weeks 8 l weeks 6 . 2 0 3 ( 3 ) 6 . 1 5 6 6 . 1 3 8 6 . 1 1 8 6 . 0 2 3 Chinook Salmon 26 weeks^^ 9 weeks 40 weeks 77 weeks 58 weeks 6 . 0 8 2 ( 3 ) 6 . 0 6 7 6 . 0 1 8 5 . 9 7 2 5 . 8 3 8 Coho Salmon 27 weeks^ 2) 10 weeks 78 weeks 6 . 3 1 0 ( 3 ) 6 . 2 9 8 6 . 0 2 5 ( 1 ) Means not underlined by the same l i n e were s i g n i f i c a n t l y d i f f e r e n t (P ^ 0 . 0 5 ) from each other. ( 2 ) Weeks of frozen storage at - 3 0°C. ( 3 ) pH values. -H9-Pigure 3 Average f l e s h pH of P a c i f i c halibut stored at - 3 0°C. - 5 0 -Figure 4 Average f l e s h pH of chinook salmon stored at - 3 0°C. a t a l l o t h e r f r o z e n storage' t i m e s ( T a b l e I I ) . i i i ) Coho Salmon The pH o f t h e p l a t e - f r o z e n coho salmon d e c r e a s e d s t e a d i l y d u r i n g f r o z e n s t o r a g e w h i l e the pH o f t h e b r i n e -f r o z e n samples i n c r e a s e d from 10 weeks u n t i l 27 weeks t h e n d e c r e a s e d u n t i l 78 weeks o f f r o z e n s t o r a g e ( F i g . 5 ) . The pH o f coho salmon a t 78 weeks was s i g n i f i c a n t l y l o w e r (P - 0 . 0 5 ) t h a n t h e pH a t 10 and 27 weeks o f f r o z e n s t o r a g e ( T a b l e I I ) . b) Thaw D r i p i ) P a c i f i c H a l i b u t A n a l y s i s o f v a r i a n c e on t h e thaw d r i p o f f r o z e n s t o r e d P a c i f i c - h a l i b u t r e v e a l e d a h i g h l y s i g n i f i c a n t (P - 0.01) method x t i m e i n t e r a c t i o n ( T a b l e I I I ) , At 14 and 31 weeks o f f r o z e n s t o r a g e the thaw d r i p o f the b r i n e - f r o z e n samples was l e s s t h a n t h a t o f p l a t e - f r o z e n samples. However, a t 45, 6 2 , and 8 l weeks o f f r o z e n s t o r a g e the thaw d r i p o f t h e b r i n e - f r o z e n samples was g r e a t e r t h a n t h a t o f t h e p l a t e - f r o z e n samples ( F i g . 6 ) . The thaw d r i p o f the p l a t e - f r o z e n f i s h tended t o d e c r e a s e d u r i n g f r o z e n s t o r a g e w h i l e t h a t o f the b r i n e - f r o z e n -h a l i b u t tended t o i n c r e a s e ( F i g . 6 ) . s i i ) Chinook Salmon The mean thav; d r i p o f p l a t e - f r o z e n c h i n o o k salmon was not s i g n i f i c a n t l y d i f f e r e n t from t h a t o f b r i n e - f r o z e n samples ( T a b l e I I I ) . However, t h e r e was a ve r y h i g h l y -52-Pig u r e 5 Average f l e s h pH of coho salmon s t o r e d at -30°C. TABLE I I I A n a l y s i s H a l i b u t , o f V a r i a n c e> o f t h e Thaw D r i p Dai Chinook' Salmon, and Coho Salmon ^a o f . P a c i f i c P a c i f i c H a l i b u t Chinook Salmon Coho Salmon • Source . 2 ( 3 ) d . f . M.S. Prob d . f . M.S. Prob d . f . M.S. .. Prob P a i r s ,M 2 0 .3429 0 . 0 0 1 8 * * 2 0 . 0 0 5 1 0.6435 2 0.1509 0.0735 M e t h o d sK H } 1 0 .0439 0 . 7 7 8 8 1 0 . 0 0 7 4 0.5441 ' 1 0.6149 0.0341 E r r o r a 2 0 . 4 8 9 3 2 0.0141 2 0 . 0 1 5 9 • Time 4 0 . 0 4 8 5 0 . 2 8 6 3 4 0 . 1 1 3 8 0 . 0 0 0 3 2 . 0 . 2 2 0 1 0 . 0 3 3 5 M x T 4 0 . 1 3 5 8 0.0224* 4 0 . 0 3 2 3 0 . 0 5 5 1 2 0 . 0 9 3 7 . 0 . 1 6 4 2 E r r o r b 16 0 . 0 3 5 2 16 0.0114 8 0.0412 1 A n a l y s i s was conducted on t h e a r c s i n t r a n s f o r m e d p e r c e n t a g e s . 2 2 A l l mean sq u a r e s a re m u l t i p l i e d by 1 . 0 x 10 . (3) P r o b a b i l i t y o f t y p e 1 e r r o r ( 4 ) E r r o r a was used t o t e s t methods E r r o r b v/as used t o t e s t a l l terms except methods * S i g n i f i c a n t a t the 5% l e v e l ** S i g n i f i c a n t a t the 1% l e v e l I. 4.0 I 3.0 I 2.0 I I .0 0, _ l o.o o ^ 9.0 < X i- e.o « _. 6 b r i n e - f r o z e n « a p l o t e - f r o z e n 1 1 1 1 v 14 31 45 62 81 L E N G T H OF F R O Z E N S T O R A G E ( W E E K S ) Figure 6 Average thaw drip of P a c i f i c halibut stored at - 3 0°C. rr o 5; < x i-II. O 10.0 9. 0 8.0 7.0 6.0 5 .0 • • b r ine - f rozen • o p lo te - f rozen 9 26 40 58 77 L E N G T H OF F R O Z E N S T O R A G E ( W E E K S ] .Figure 7 Average thaw drip of chinook salmon stored at - 3 0°C. - 5 5 -s i g n i f i c a n t (P - 0 . 0 1 ) storage time e f f e c t . The thaw drip at 9 weeks was s i g n i f i c a n t l y lower (P - 0 . 0 5 ) at a l l other frozen storage times (Table IV, F i g . 7 ) . Mean thaw drip of brine-frozen chinook salmon was less than that of the plate-frozen samples at 9 weeks but approximately equal or greater than the plate-frozen samples at other storage times (Fig. 7 ) . i i i ) Coho Salmon Plate-frozen coho salmon had a s i g n i f i c a n t l y higher (P * 0 . 0 5 ) thaw drip than brine-frozen - (Table I I I , F i g . 8 ) . There was also a s i g n i f i c a n t difference (P - 0 , 0 5 ) i n thaw drip among frozen storage times (Table I I I ) . The thaw drip at 27 weeks was s i g n i f i c a n t l y higher (P = 0 . 0 5 ) than at either 10 or 78 weeks (Table IV), This pattern was p a r t i c u l a r l y pronounced i n plate-frozen samples (Fig. 8 ) , c) Color 1) Hunter Rd Values I) Chinook and Coho Salmon During frozen storage the Rd values of chinook salmon appeared f a i r l y i r r e g u l a r and variable (Fig. 9 ) . The Rd readings of coho salmon were r e l a t i v e l y constant during frozen storage (Fig. 1 1 ) . Analysis of variance on Hunter Rd readings of both chinook and coho salmon revealed no s i g n i f i c a n t e f f ects due to method or storage time (Table V. and VI). -56-TABLE IV Chinook Salmon 77 weeks 10. 0% (2) (3) <,(4) Duncan's new multiple range test on the s i g n i f i c a n t time means from the analyses of variance on thaw d r i p . ( l ) 40 weeks 9.1% 2 6 weeks 9.0% 58 weeks 7.8% 9 weeks 6/5% Coho Salmon 27 weeks 10.7% 78 weeks 10 weeks, 8.4% 6.9% (1) The test was conducted on the arcsine transformed data. (2) Means not underlined by the same l i n e are s i g n i f i c a n t l y d i f f e r e n t (P - 0.05) from each other. (3) Weeks of frozen storage (4) O r i g i n a l (not transformed) values expressed as % of the weight of the frozen sample. -57-Pigure 8 Average thaw drip of coho salmon stored at - 3 0 ° C-, -58 -2) Hunter 'a' Values i ) Chinook Salmon The mean Hunter 'a' values of Chinook salmon were not s i g n i f i c a n t l y affected by method of freezing. However, there was a highly s i g n i f i c a n t difference (P - 0.01) among f r o -zen storage times. Duncan's new multiple range test showed that the 'a' value at 9 weeks was s i g n i f i c a n t l y lower (P * 0.05) than at any other storage time (Table V). The change i n 'a' values during frozen storage was somewhat si m i l a r for both the plate-frozen and the brine-frozen chinook salmon (Fig. 1 0 ) . However, at 9 weeks of frozen storage the brine-frozen samples had s l i g h t l y higher 'a' reading while at a l l other frozen storage times the plate-frozen samples had the higher reading (Fig. 1 0 ) . i i ) Coho Salmon The mean Hunter 'a' value of plate-frozen coho salmon was not s i g n i f i c a n t l y d i f f e r e n t from that of brine-frozen. There was a s i g n i f i c a n t difference (P - 0.05) among the storage times means (Table VI). The 'a' reading at 78 weeks was s i g n i f i c a n t l y higher than at a l l other times (Table VII). The 'a' value at 10 weeks was not s i g n i f i c a n t l y d i f f e r e n t from the 'a' value at 27 weeks of frozen storage (Table VII). The general change i n 'a' values during frozen storage of coho salmon was s i m i l a r for both the brine-frozen and the plate-frozen samples (Fig. 1 2 ) . TABLE V ' Analysis of variance of color readings f o r chinook salmon Rd a b a/b Source d.f. M.S • P r o b ( 1 ) M.S. Prob M. s. Prob M.S. Prob Pairs . . Methods K d } 2 1 3 . 9 6 3 0 . 0 0 0 5 1 1 0 . 6 6 0 . 0 0 0 0 1 . 9 9 5 6 0 . 3 0 6 3 0.1422 0 . 0 0 0 0 1 2 . 9 4 5 0 . 7 5 9 6 3 6 . 8 1 0 . 1 1 0 9 1 . 9 0 5 1 0 . 5 8 1 1 0 . 0 3 7 5 0 .4014 Error b 2 2 6 . 604 4 . 6 3 4 . 4 7 9 5 0 . 0 3 3 4 Time 4 1 . 0 0 1 0 . 4 7 3 9 2 8 . 3 3 0 . 0 0 1 9 s * 1 . 4 3 6 9 0 . 4 7 8 3 0 . 0 7 0 8 0 . 0 0 0 0 M x T 4 1 . 0 2 3 0 . 4 7 3 6 5 . 6 5 0 . 2 7 9 8 1 . 4199 0 .4841 0 . 0 1 3 2 0 . 0 1 2 8 * Error b 16 1 . 0 8 0 4.05 1 . 5 6 3 7 0 . 0 0 2 9 V71. I P r o b a b i l i t y of Type 1 error * S i g n i f i c a n t at the 5$ l e v e l (P 0 . 0 5 ) 2 Error a was used to test methods. ** S i g n i f i c a n t at the 1% l e v e l (P - 0 . 0 1 ) Error b was used to test a l l terms except *** S i g n i f i c a n t at the 0 . 1 $ l e v e l (P 0 . 0 0 1 ) Methods TABLE VI' A n a l y s i s of. v a r i a n c e of... c o l o r r e a d i n g s f o r coho salmon Rd a b a/b Source d . f . M.S. P r o b ( 1 ) M.S. Prob M.S. Prob M.S. Prob P a i r s 2 0 . 1 5 1 8 . 0 . 9 1 8 5 2 . 0 3 6 0 . 4 9 6 3 • 0 . 8 0 6 5 0 . 7 6 8 7 0 . 0 0 7 8 0 . 1 6 4 9 M e t h o d s ^ . 1 0 . 3 7 8 5 ' 0.4181 0 . 0 0 9 0 . 9 2 1 4 1 . 8 2 4 1 0 . 2 8 4 8 0 . 0 0 2 9 0 . 4 0 5 4 E r r o r b 2 0 . 3 6 7 7 4 . 8 7 8 0 . 8 6 5 5 0 . 0 0 2 7 Time 2 0 . 0 6 8 2 0 . 9 5 4 9 12.848 0.0410* 1 . 2 3 7 7 0 . 6 7 4 0 0 . 0 1 3 9 0.0048** M x T 2 0 . 0 5 9 2 0 . 9 5 9 1 1 . 2 5 5 0 . 6 4 1 3 0 . 3 2 9 7 0 . 8 8 9 8 0.0045 0 . 3 2 0 7 E r r o r b 8 1 . 9 7 5 3 2 . 6 3 4 2 . 9 4 3 8 0 . 0 0 3 4 o I P r o b a b i l i t y of Type 1 e r r o r . E r r o r a was used to t e s t methods E r r o r b was used to t e s t a l l terms except Methods. . * S i g n i f i c a n t at the 5% l e v e l (P * 0 . 0 5 ) ** S i g n i f i c a n t at the 1% l e v e l (P 0 . 0 1 ) *** S i g n i f i c a n t at the 0 . 1 $ l e v e l (P ^ 0 . 0 0 1 ) -61--• brine — f rozen -o p l o t e - f r o z e n 13.75 13.25 12.75 O Z o 12.25 < U J rr rr o z D < rr 11.75 26 4 0 58 77 9 L E N G T H OF F R O Z E N S T O R A G E (w E E K s) Figure 9 Average Hunter Rd values of chinook salmon stored at -30°C. 30.0 29.0 28.0 27.0 26.0 . 2 5.0 24.0 4 23.0 ( . ( — i ; 1 fr-9 26 4 0 5 8 7 7 L E N G T H OF F R O Z E N S T O R A G E ( W E E K S ) Figure 10 Average Hunter 'a' values of chinook salmon stored at -30°C. - 6§-11.25 Figure 11 Average Hunter Rd values of coho salmon stored at - 3 0°C. o z 31.0 .. 3 0 0 .. 29. 0 . 2 8 . 0 . 2 7 . 0 .. 2 6 . 0 ° 25. 0 J < U J " 2 4 . 0 b r i n e — f r o z e n -* p la te — f r o zen 1 0 2 7 - L E N G T H O F F R O Z E N S T O R A G E ( W E E K S ) H 78 Figure 12 Average Hunter 'a' values of coho salmon stored at - 3 0°C. -63-3) Hunter 'b' Values 1) Chinook and Coho Salmon There was no s i g n i f i c a n t d i f f e r e n c e i n Hunter 'b' v a l u e s between p l a t e - f r o z e n and b r i n e - f r o z e n samples of e i t h e r chinook (Table ."V) or coho salmon (Table VI}, A l l o t h e r main e f f e c t s and i n t e r a c t i o n s were, a l s o n o n - s i g n i f i c a n t f o r both s p e c i e s (Table V and V I ) . Except f o r the 'b 1 values at 58 weeks of f r o z e n storage the values of the b r i n e - f r o z e n c h i n -ook salmon samples remained f a i r l y c o n s t a n t . With the p l a t e -f r o z e n samples, except f o r a sharp decrease between 26 weeks and 40 weeks the values were a l s o r e l a t i v e l y constant ( P i g . 13). From 9 weeks u n t i l 78 weeks of f r o z e n storage the 'b* values of p l a t e - f r o z e n coho salmon decreased about 0.90 u n i t s while the b r i n e - f r o z e n samples decreased about 0 . 8 5 u n i t s ( F i g . 14). 4) Hunter a/b R a t i o s i ) Chinook Salmon The method x time i n t e r a c t i o n of the a/b r a t i o s f o r •chinook salmon was s i g n i f i c a n t (P = 0.05) (Table V ). Except f o r the 9th week of f r o z e n storage the p l a t e - f r o z e n had h i g h e r a/b r a t i o s than the b r i n e - f r o z e n samples ( F i g . 1 6 ) . A l s o except f o r the p e r i o d between 26 and 40 weeks of f r o z e n storage the changes i n the a/b r a t i o of chinook salmon were s i m i l a r f o r both the p l a t e - and b r i n e - f r o z e n samples ( P i g . 1 6 ) , i i ) Coho Salmon There was no s i g n i f i c a n t d i f f e r e n c e i n the mean a/b r a t i o between b r i n e - f r o z e n and p l a t e - f r o z e n samples -64-j * . 1 1 a 4 — 9 26 40 5 6 77 L E N G T H OF F R O Z E N S T O R A G E ( w E E K s ) Figure 13 Average Hunter b values of chinook salmon stored at -30°C. - 6 5 -24.0 z o < UJ or 23.0 . 22.0 .. Figure 14 Average Hunter b values of coho salmon stored at - 3 0°C. o z 1.26 -1.24 I. 22 .. I.2<5> I.I 8 I.I 6 I 1.14 I. 12 L J I. 10 J-rr I. 08 Xi o s *- —»-brine — f r o z e n • » p l a t e - f r o z e n - » — 10 — » — 2 7 L E N G T H OF F R O Z E N S T O R A G E ( W E E K S ) Figure 15 Average Hunter a/b r a t i o s of coho salmon stored at - 3 0°C. 78 -66-< cc < a 1.3 0 j 1.26 1.22 4 1.18 I. 14 1.10 . I. 06 .. 1.0 2 ..' 0.98 .. 0 .94 0.90 1 • 9 L E N G T H O F -B br ine — f r o z e n -o p I o t e — f r o z e n — i , 1 j — 26 4 0 5 8 77 F R O Z E N S T O R A G E ( v / E E K s ) F i g u r e 16 Average Hunter a/b r a t i o s o f c h i n o o k salmon s t o r e d at - 3 0°C. / -67-(Table y i ) . However, there was a highly s i g n i f i c a n t difference (P - 0 . 0 1 ) among frozen storage times (Table v i ) . The a/b r a t i o at 78 weeks was s i g n i f i c a n t l y higher (P - 0 . 0 5 ) than at 10 and 27 weeks of frozen storage (Table v i i ) . From 10 u n t i l 78 weeks of frozen storage the a/b r a t i o of the plate-frozen samples increased 0 , 1 7 3 units while the brine-frozen samples increased 0 , 1 2 7 units (Fig. 1 5 ) . d) Flavor Differences The r e s u l t s of the t r i a n g l e tests on inside and outside muscle samples of P a c i f i c halibut, chinook salmon and coho salmon are shown i n Figure 1 7 . Levels of s i g n i f i c a n t difference were obtained from tables prepared by Larmond ( 1 9 6 7 ) . i ) P a c i f i c halibut There were no s i g n i f i c a n t differences i n f l a v o r be-tween freezing methods for either inside or outside muscle samples at 14 weeks of frozen storage. At 31 weeks, however, there was a very highly s i g n i f i c a n t difference (P - 0 , 0 0 1 ) between the outside muscle of the plate-frozen halibut and the outside muscle of the brine-frozen f i s h (Fig. 17 ). These "~ differences between freezing methods steadily declined a f t e r 3 1 weeks of frozen storage u n t i l they were just s i g n i f i c a n t at the 5% l e v e l a f t e r 8 l weeks (Fig. . 17) . The taste panel r e s u l t s for halibut inside muscle appeared a l i t t l e more e r r a t i c i n comparison with those for - 6 8 -TABLE VII Duncan's new multiple range test on the s i g n i f i c a n t time effects of the analyses of variance on the Rd, a, b, and a/b values of Chinook Salmon and Coho Salmon. ( 1 ) Chinook Salmon: a values 58 weeks 77 weeks 26 weeks 40 weeks 9 weeks 2 8 . 8 9 ( 3 ) 2 8 , 6 6 2 6 . 6 3 • . 2 6 . 4 5 Coho Salmon: a values 78 weeks^ 2) 10 weeks 27 weeks 2 8 . 6 0 ( 3 ) 2 6 . 1 1 2 6 . 0 2 2 3 . 5 0 Coho Salmon: a/b r a t i o 78 w e e k s 1 0 weeks 27 weeks 1 . 2 5 1 . 1 1 1 . 1 0 ( 1 ) Means not underlined by the same l i n e are s i g n i f i c a n t l y (P - 0 . 0 5 ) d i f f e r e n t from each other. ( 2 ) Weeks of frozen storage at - 3 0°C. ( 3 ) Hunter a values. (4) a/b r a t i o . Figure 17 Taste panel (triangle test) r e s u l t s of P a c i f i c halibut, chinook salmon, and coho salmon. x = s i g n i f i c a n t at the 5% l e v e l y = s i g n i f i c a n t at the 1% l e v e l z = s i g n i f i c a n t at the 0.1% l e v e l -69-o ro z o < »-z U J a H O U i rr cc o o u. o rr ui m z 19 17 15 13 II 9 PAC I FIC H A L I BUT + Z fy o outside muscle » ins ide muscle 14 31 45 62 81 19 i 17 15 13 I I 9 CHINOOK SALMON —• outside muscle * ins ide muse le Lx 26 40 58 77 19 4-17 15 ± 13 J . II + COHO S A L M O N - • o u t s i d e muscle •9 i n s i d e muscle y -x 10 27 78 LENGTH OF F R O Z E N S T O R A G E (WEEKS ) -70-outside muscle. There were highly s i g n i f i c a n t differences (P - 0 . 0 1 ) between freezing methods at 31 and 8 l weeks v;hereas the differences were non-significant at 14 and 45 weeks and s i g n i f i c a n t (P - 0 . 0 5 ) at 62 weeks of frozen storage (Fig. 1 7 ) . i i ) Chinook Salmon The taste panel r e s u l t s for chinook salmon were somewhat s i m i l a r to those for halibut. There were no s i g n i -f i c a n t differences between freezing methods at the f i r s t analysis ( 9 t h week of frozen storage) for either inside or outside muscle samples and with outside muscle there was a highly s i g n i f i c a n t difference (P - 0 . 0 1 ) between freezing methods at the second analysis ( 2 7 t h week of frozen storage) (Fig. 1 7 ) . This difference steadily declined u n t i l i t became non-significant at 58 and 77 weeks (Fig. 1 7 ) . The taste panel r e s u l t s for inside muscle samples were d i f f e r e n t from those for halibut as with chinook salmon the differences between freezing methods were non-significant at each sampling time (Fig. 1 7 ) . i i i ) Coho Salmon Taste panel results for coho salmon were very d i f f e r -ent from those for halibut and chinook salmon. At the f i r s t analysis ( 1 0 t h week of frozen storage) there was a n o n - s i g n i f i -cant difference between freezing methods for the outside muscle and a highly s i g n i f i c a n t difference (P - 0 . 0 1 ) for the inside muscle (Fig. 17). The number of correct i d e n t i f i c a t i o n s steadily increased with time and culminated with a. very highly TABLE VIII Acceptability Preferences for P a c i f i c Halibut, Chinook Salmon and Coho Salmon Preferences Species Location Length of Number of Plate- Brine-• No Frozen Correct _Frozen Frozen i Pre Storage I d e n t i f i c a t i o n 1 % % % P a c i f i c Outside 14 weeks 13 69 3 1 halibut muscle 31 i t 20 60 40 45 tt 18 39 50 1 1 62 tt 17 29 59 12 8 1 t i .15 47 53 Inside 14 weeks 9 22 78 muscle 31 tt 18 50 50 45 tt 14 43 50 7 62 tt 16 50 38 13 81 t t 18 44 28 28 Chinook Outside 9 weeks 12 67 33 Salmon muscle 26 tt 18 56 44 40 i t 17 59 35 • 6 58 tt 14 50 50 77 »t 9 33 56 11 Inside 9 weeks 14 57 43 muscle 26 t t 11 55 45 40 i t 12 50 50 58 t t 10 - 70 30 77 i t 14 50 21 29 Coho Outside . 10 weeks 12 83 .17 Salmon muscle 27 i t 14 57 43 78 t t 20 40 55 5 Inside 10 weeks 17 53 47' muscle 27 tt 13 54 46 78 i t 10 40 30 30 1 Number of correct i d e n t i f i c a t i o n s out of 30 obeervations. 2 Percentage of correct i d e n t i f i c a t i o n s . - 7 2 -s i g n i f i c a n t d i f f e r e n c e (P - 0.001) between f r e e z i n g methods at 78 weeks ( F i g . 1 7 ) . With coho salmon i n s i d e muscle there was a s i g n i f i c a n t d i f f e r e n c e between f r e e z i n g methods only at the 10th week. D i f f e r e n c e s at a l l other times were non-s i g n i f i c a n t ( F i g . 1 7 ) . Examination of the data f o r o u t s i d e muscle r e v e a l e d t h a t f o r a l l s p e c i e s p l a t e - f r o z e n samples were p r e f e r r e d to b r i n e - f r o z e n at the f i r s t sampling time. The p r e f e r e n c e d e c l i n e d as storage time i n c r e a s e d u n t i l at the l a s t sampling p e r i o d b r i n e - f r o z e n samples were s l i g h t l y p r e f e r r e d over the c o r r e s p o n d i n g p l a t e - f r o z e n ones. For both the i n s i d e and outside-muscle samples the percentage of "no p r e f e r e n c e s " tended to i n c r e a s e with i n c r e a s i n g time of f r o z e n storage (Table V I I I ) . e) M i n e r a l C o n c e n t r a t i o n i ) P a c i f i c H a l i b u t I n s o f a r as sodium and potassium c o n c e n t r a t i o n s i n the f l e s h were concerned, the d i f f e r e n c e s between the two methods of f r e e z i n g v/ere not s t a t i s t i c a l l y s i g n i f i c a n t and no c o n c l u s i o n s c o u l d be made about d i f f e r e n c e s i n c h l o r i d e c o n c e n t r a t i o n as there was a very highly, s i g n i f i c a n t (P - 0.01) method times l o c a t i o n i n t e r a c t i o n (Table I X ) , \ However, . g r a p h i c a l a n a l y s i s of the means i n d i c a t e d t h a t the sodium and c h l o r i d e c o n c e n t r a t i o n s of the p l a t e - f r o z e n i n s i d e muscle, p l a t e -f r o z e n o u t s i d e muscle and the b r i n e - f r o z e n i n s i d e muscle were q u i t e s i m i l a r ( F i g . 18). However, the b r i n e - f r o z e n o u t s i d e TABLE IX A n a l y s i s o f v a r i a n c e o f m i n e r a l c o n c e n t r a t i o n o f P a c i f i c H a l i b u t muscle Sodium P o t a s s i u m C h l o r i d e . Source d . f . M.S. P r o b ( l ) M.S. Prob M.S. Prob . P a i r s , 2 M e t h o d s ^ 1 E r r o r a 2 LOCATION 1 M x LOC. 1 E r r o r b 4 T o t a l 1 1 0.4532 1 . 9 7 6 4 0 . 4 7 1 0 2 . 3 4 9 7 1 . 6 5 0 2 0.4404 • 0 . 4 3 7 3 0 . 1 7 9 5 0 . 0 8 1 7 0.1241 0 . 4 7 6 0 0 . 0 6 7 5 0 . 3 9 5 4 2 . 2 1 8 8 0 . 1 6 3 3 0 . 0 5 0 1 0 . 0 3 2 1 0 . 7 1 3 7 0 . 0 0 3 7 * * 0.1445 0 . 0 0 7 0 . 8 7 4 9 1 0 . 6 6 0 0 . 0 1 3 1 0.047 1 2 . 6 6 9 0.0004 9 . 0 3 1 0 . 0 0 0 6 * * * 0 . 0 5 0 P r o b a b i l i t y o f Type 1 e r r o r E r r o r a was used t o t e s t methods. E r r o r b was used t o t e s t a l l terms except methods*** S i g n i f i c a n t S i g n i f i c a n t S i g n i f i c a n t a t t h e 5% l e v e l a t t h e 1% l e v e l a t t h e 0 . 1 $ l e v e l 4.60 -o brine — f r o z e n - o p i a t e f roz e n L I i n s i d e m u s c l e L 2 out s ide m u s c l e LI L2 Figure 18 Average sodium, potassium, and c h l o r i d e concentra-t i o n i n the f l e s h of P a c i f i c h a l i b u t . - 7 5 -muscle had 3 . 8 2 times as much sodium and 4 . 0 9 times as much chloride as the plate-frozen outside muscle (Fig. 18). The potassium concentration of the brine-frozen samples was about 1 . 1 1 times greater than the plate-frozen samples for the outside muscle and about 1 . 0 2 times greater for the inside muscle (Fig. 18). The sodium and chloride concentrations did not d i f f e r s i g n i f i c a n t l y among pairs (Table IX),, Sodium concentration did not d i f f e r s i g n i f i c a n t l y between the inside and outside muscle samples whereas potassium concentration was s i g n i f i c a n t l y (P - 0 . 0 1 ) greater i n the inside muscle (Table .IX, • F i g . 18). No conclusions about differences i n chloride concentration between the inside and outside muscle samples"could be made as there was a very highly s i g n i f i c a n t (P - 0 . 0 0 1 ) method times location i n t e r a c t i o n (Table IX), Chloride concentration i n the outside muscle was 6 , 0 3 times greater than that of the inside muscle with the brine-frozen samples and only 1 . 5 3 time's greater with the plate-frozen samples (Fig. 18), i i ) Chinook Salmon No simple assessment of the quantitative differences i n sodium concentration between methods and between locations could be made as there was a highly s i g n i f i c a n t (P - 0 . 0 1 ) method x lo c a t i o n i n t e r a c t i o n (Table X). Sodium concentration of inside muscle was e s s e n t i a l l y the same for the two methods of freezing but the concentration i n outside muscle was about 3 times higher i n brine-frozen compared to plate-frozen samples (Pig. 19). TABLE .X Analysis of variance of mineral concentration of Chinook Salmon muscle Sodium Potassium .... Chloride Source d.f. M .S. P r o b ^ M. S. • Prob M . s . Pr •ob Pairs Methods^ 7 2 0 . 0 2 8 1 0 . 2 4 5 3 0 . 0 6 5 2 0 . 2 1 7 3 0 . 0 8 9 3 0 . 5 9 0 4 1 0 . 4 4 8 5 0 . 0 3 7 1 1. 1844 0 . 2 3 7 2 1 . 3 2 6 7 0 . 0824 Error a 2 0 . 1 3 1 1 0 . 4 2 1 8 0 . 1 1 3 9 Location 1 1 . 0 2 0 8 0 . 0 0 1 8 2 . 0 7 5 0 0 . 0 0 1 9 * * 3 . 7 9 6 9 0 . 0 0 8 3 ' Method x LOC. 1 0 . 5 0 4 3 0 . 0 0 4 9 * * , 0 . 0014 0 . 8 1 5 8 0 . 9 2 4 1 0 . 0 6 6 1 Error b 4 0 . 0 1 3 7 0 . 0 2 8 4 0 . 1 4 6 9 T o t a l 11 P r o b a b i l i t y of Type 1 error Error a was used to test methods Error b v/as used to test a l l terms except methods * S i g n i f i c a n t at the 5% l e v e l ** S i g n i f i c a n t at the 1% l e v e l *** S i g n i f i c a n t at the 0 . 1 $ l e v e l Figure 19 Average sodium, potassium, and chloride concentra-t i o n i n the f l e s h of chinook salmon. - 7 8 -Potassium and chloride concentrations of chinook salmon did not d i f f e r s i g n i f i c a n t l y between the plate-frozen and brine-frozen samples (Table X ), The i n t e r a c t i o n between methods and locations for chloride approached si g n i f i c a n c e (P - 0 . 0 7 ) (Table ~x). The difference i n chloride concentra-t i o n between bririe-and plate-frozen samples was much greater for outside than for inside muscle (Fig..19). The potassium concentration was s i g n i f i c a n t l y •(P - 0 . 0 1 ) higher i n the inside muscle than i n the outside whereas chloride concentration was s i g n i f i c a n t l y (P - 0 . 0 1 ) lower i n the inside muscle (Table X, F i g . 1 9 ) . i i i ) Coho Salmon Assessment of differences i n sodium, potassium, and chloride concentration between methods and between locations v/as complicated by the presence of s i g n i f i c a n t (P ^ 0 . 0 5 ) method x location interactions (Table XI). Graphical analysis indicated that freezing method had l i t t l e e f fect on concentra-t i o n of sodium or chloride i n inside muscle. However, concen-t r a t i o n of these ions i n outside muscle were 2 . 6 5 times greater i n brine-frozen than i n corresponding plate-frozen samples (Fig. 20) . Potassium concentrations of samples of inside muscle was higher i n plate-frozen than i n brine-frozen f i s h but the reverse was true for outside muscle (Fig. 2 0 ) . TABLE-XI Analysis of variance of mineral concentration i n Coho Salmon muscle Sodium Potassium 1 Chloride Source d.f. M.S. -Prob ( 1 ) M .s. Prob M .s. Prob Pairs Methods 2 0 . 0 0 2 3 0 . 8 0 8 4 0 . 0 4 8 5 0 . 1 3 0 1 0 . 4 0 9 6 0 . 3 8 9 5 1 0 . 5 0 0 2 0 . 0 5 7 7 0 . 0 0 0 2 0 . 9 2 0 0 3 . 4 0 2 7 0 . 1 9 5 1 Error a 2 0 . 0 2 7 0 0 . 1 0 2 2 0 . 9 1 1 5 Locations 1 1 . 1 5 9 4 0 . 0 0 1 0 3 . 1 9 3 0 0 . 0 0 0 5 6 . 4 3 8 7 0 . 0 1 3 3 Method x LOC. 1 0 . 4 5 2 4 0 . 0 0 3 7 * * 0 . 4 4 4 7 0 . 0 0 5 9 s * 3 . 4 6 6 9 0 . 0 3 3 7 Error'b 4 0 . 0 1 0 1 0 .0.137 ' 0 . 3 3 8 9 P r o b a b i l i t y of Type 1 error Error a was used to test methods Error b was used to test a l l terms except methods * S i g n i f i c a n t at the 5$ l e v e l ** S i g n i f i c a n t at the 1% l e v e l * s * S i g n i f i c a n t at the 0 . 1 $ l e v e l - 8 a -• i Figure 20 Average sodium, potassium, and chloride concentra-t i o n i n the f l e s h of echo salmon. -81-f ) TBA Values 1) P a c i f i c H a l i b u t The TBA values of the b r i n e - f r o z e n samples were s i g n i f i c a n t l y g r e a t e r (P - 0 . 0 5 ) than those o f the p l a t e f r o z e n samples (Table X I I ) . A s i g n i f i c a n t (P. ^  0 . 0 5 ) l o c a t i o n x time i n t e r a c t i o n p r e c l u d e d t e s t i n g of l o c a t i o n or time e f f e c t s (Table X I I ) . TBA values of o u t s i d e muscle were g e n e r a l l y g r e a t e r than those of i n s i d e muscle with the g r e a t e s t d i f f e r e n c e s at 45 and 62. weeks "of storage ( F i g . 21). i i ) Chinook Salmon The s i g n i f i c a n t (P ^ 0 . 0 5 ) method x l o c a t i o n i n t e r -a c t i o n e f f e c t r e v e a l e d by a n a l y s i s of v a r i a n c e of TBA values of chinook salmon (Table XII) i s g r a p h i c a l l y r e p r e s e n t e d i n F i g u r e 22. I t i s obvious that the extremely high TBA values f o r o u t s i d e muscle of b r i n e - f r o z e n f i s h at 26 and 40. weeks of storage were l a r g e l y r e s p o n s i b l e f o r the i n t e r a c t i o n . There were h i g h l y s i g n i f i c a n t d i f f e r e n c e s (P - 0.01) i n the TBA values among the d i f f e r e n t times of f r o z e n storage ~ . XTable X I I ) . Mean TBA values at the f i r s t and l a s t two sampling p e r i o d s were s i m i l a r (Table X I I I ) . The h i g h e s t mean TBA value o c c u r r e d at 26 weeks of storage and was s i g n i f i c a n t l y h i g h e r (P ^ 0 . 0 5 ) than those at 9, 77, and 58 weeks but was not s i g n i f i c a n t l y d i f f e r e n t from that at 40 weeks of f r o z e n storage (Table X I I I ) . TABLE X I I A n a l y s e s o f v a r i a n c e o f TBA v a l u e s o f P a c i f i c h a l i b u t , c hinook salmon and coho salmon P a c i f i c H a l i b u t Chinook Salmon <- Coho Salmon Source d . f . M.S.' d . f . M.S. d . f . M.S. P a i r s 2 2 Method 1 M x P = E r r o r a 2 L o c a t i o n 1 M X L . 1 E r r o r b 4 Time 4 M x T 4 L x T 4 M x L x. T 4 E r r o r c 32 11.234 2 0 . 7 9 5 s 0.333 80 . 8 8 7 14.260 2 . 8 2 2 7 . 5 6 1 4 . 5 1 0 12.004 3 . 9 6 9 4 . 4 1 8 .2 . 1 2 1 1 4 4 4 4 4 32 0.455 87.554 1 1 . 4 9 8 2 4 9 . 3 6 0 „ 74.202 4.360. 41.541 20.500 22.356 14.553 9.032 2 1 2 1 1 4 2 2 2 2 16 0.278 14.161 .. 19.664* 212,210 12.469 6.415 64.677 3.826 47.325"" 3.535 5.388 1 A l l mean square v a l u e s are m u l t i p l i e d by 1 x 10 2 Methods were t e s t e d by e r r o r a L o c a t i o n and M X L were t e s t e d u s i n g e r r o r b A l l o t h e r terms were t e s t e d by e r r o r c * S i g n i f i c a n t a t the 5% l e v e l , " " S i g n i f i c a n t a t the 1% l e v e l • - 8 3 -TABLE XIII Duncan's new m u l t i p l e range t e s t on s i g n i f i c a n t time means from the a n a l y s i s of v a r i a n c e of the TBA values of chinook salmon. 26 weeks 40 weeks 9 weeks 77 weeks 58 weeks 0.2138 2. ' 0.1695 0.0929 0.0902 0.0820 1 Weeks of f r o z e n storage at - 3 0°C 2 The average TBA value i n absorbance u n i t s . Each mean i s the average of 3 o b s e r v a t i o n s . Means not u n d e r l i n e d by the same l i n e are s i g n i f i c a n t l y (P = 0 . 0 5 ) d i f f e r e n t from each other. -8.4-0.30 0.28 0.26 0.24 4% 0.22 .. 0.20 1 0.18 U ) 0 0.16 z < 0.14 oo C 0.12 O CO co 0.10 < 0.08 ui •3 0.06 _i > 0.04 w O.0-2 0 © — © b r i n e - f r o z e n o u t s i d e musc le p l a t e - f r o z e n o u t s i d e muscle brine - f r o z e n i n s i d e muscle p l a t e - f r o z e n i n s i d e muscle 14 —I— 4 5 31 5 62 81 L E N G T H OF F R O Z E N S T O R A G E ( W E E K S ) Figure 21 Average TBA values of P a c i f i c halibut stored at -30°C. - 8 5 -ui o z < 0.60 0.5 6 0.5 2 4* 0.4 8 0.44 0.4 0 0.36 4-0 T32 0.28 m cc o 0-24 in m 0. 20 < 0.1 6 U I 3 0. I 2 .. > 0.08 1 < CD 0.04 <§) © brine-frozen o u t s i d e muscle p lote- frozen o u t s i d e musc le br ine-frozen i n s i d e muscle p la te - f rozen i n s i d e muscle •4-L E N G T H 26 40 O F F R O Z E N 58 S T O R A G E 77 (w E E K S ) Figure 22 Average TBA values of chinook salmon stored at - 3 0°C. -86-0.44 0.40 0.36 .. 0.32 0.28 ^ 0 . 2 4 ui o \ 0-20 C D C C O 0.16 C O C D < ' — 0.12 UJ p < > C O 0.08 0. 04 © © brine-frozen outside muscle 0 *0 plote-frozen outside muscle « o brine-frozen inside muscle o o plate-frozen inside muscle _—_^  10 L E N G T H 0 F 2 7 F R O Z E N 78 S T O R A G E ( W E E K S ) Figure 23 Average TBA values of coho salmon stored at -30°C. -87-i i i ) Coho Salmon No simple evaluation of differences between inside and outside muscle or among d i f f e r e n t times of frozen storage was possible as there was a highly s i g n i f i c a n t (P - 0.01) l o c a t i o n x time i n t e r a c t i o n (Table X I I ) . Graphical analysis of the means indicated that exceptionally high values for out-side muscle .at the f i r s t two sampling periods with a peak at 27 weeks were responsible for the i n t e r a c t i o n e f f e c t (Fig. 23) and that by 78 weeks, differences between inside and outside muscle had v i r t u a l l y disappeared, g) Long Chain Free Fatty Acids P a c i f i c Halibut D T.Fee H^ -^ y ^£ids Expressed as Percent of TotaJ Free" Fatty Acids Analyzed There was a s i g n i f i c a n t difference (P = 0.05) between freezing methods only for f a t t y acid 15:0 (Table I, Appendix) which was greater i n the brine-frozen than i n the plate-frozen samples. A s i g n i f i c a n t (P -• 0.0 5). method x time i n t e r -action existed for f a t t y acid 17:0 (Table I , Appendix). . V i s -ual analysis indicated that the changes in f a t t y acid 17:0 over time were q u a l i t a t i v e l y d i f f e r e n t for the two methods and no obvious trends could be observed (Table I I , Appendix). The percentage of f a t t y acid 18:0 was s i g n i f i c a n t l y greater 1 (P - 0.05) for inside muscle while the percentage of f a t t y acids 16:1, 17:0, 18:2, 18 : 4 , 20:1 and -88-and 2 2 : 1 was s i g n i f i c a n t l y (P ^ 0 . 0 5 ) greater f o r the outside muscle (Tables I and I I , Appendix). A s i g n i f i c a n t (P - 0 . 0 5 ) time x l o c a t i o n i n t e r a c t i o n e x i s t e d f o r both 14:0 and 2 0 : 5 and s i g n i f i c a n t (P ^ 0 . 0 5 ) method x l o c a t i o n , and method x l o c a t i o n x'time i n t e r a c t i o n s f o r 18:3 (Table 1, Appendix). V i s u a l a n a l y s i s o f the means f o r these three f a t t y acids d i d -not r e v e a l any meaningful trends (Table I I , Appendix). There were s i g n i f i c a n t d i f f e r e n c e s (P - 0 , 0 5 ) between frozen storage times f o r f a t t y acids 1 6 : 0 , and 1 6:1, 1 6 : 2 , 18:2,;.18:4, 2 0 : 1 , 2 0 : 2 , 2 2:1, 2 2 : 5 , and 22:6 (Table 1 , Appendix). No conclusions could be made about d i f f e r e n c e s between frozen storage times f o r f a t t y acids 1 4 : 0 , 1 7 : 0 , 18:3> and 2 0 : 5 as there were s i g n i f i c a n t (P - 0 . 0 5 ) l o c a t i o n x time I n t e r a c t i o n s f o r 14:0 and 2 0 : 5 , a s i g n i f i c a n t (P ^ 0 . 0 5 ) method x time, i n t e r a c t i o n f o r 17 : 0 and a s i g n i f i c a n t (P - 0 . 0 5 ) method x l o c a t i o n x time i n t e r a c t i o n f o r 18:3 (Table I , Appendix). The changes i n the percentages of fr e e f a t t y acids 1 6 : 0 , 1 6:1, and 16 : 2 during frozen storage, although s i g n i -f i c a n t (P - 0 . 0 5 ) , were random i n nature and no meaningful trends could be observed (Table XIV). Free f a t t y acids 18:2, 18:4, 2 0:1, 2 0 : 2 , 2 2:1, and 2 2 : 5 were lowest at 14 weeks of frozen storage and increased e r r a t i c a l l y t h e r e a f t e r (Table XIV). In c o n t r a s t the percentage of free f a t t y a c i d 2 2 : 6 d e c l i n e d s t e a d i l y from 14 to 81 weeks of fr o z e n storage, 2 ) Free F a t t y Acids Expressed as ug F a t t y Acid per Gram of N e u t r a l L i p i d , -89-TABLE XIV Method, l o c a t i o n , and time means from the analyses of variance of free f a t t y acids (expressed as percent of t o t a l free f a t t y acids analyzed) of P a c i f i c halibut-*-. 14:0 15 :0 16 : 0 16 :1 16 :2 17 :0 M l 2 2.18 : ns 0. 35^  * 15. 93 ns 5.33 ns 0.29 ns 1.21 M2 2.35 0. 29 15. 26 6.29 0 . 38 1.30 L I 3 1.945 0. 32 nsl6. 29 ns 5.13* . 0.29 ns 1.15* L2 2.60 • 0. 33 14. 88 6.49 0.37 1.36 T l 4 1.98 0. 35 6 a 15. 99 ab 5.69 ab 0.27 ac 0.96 T2 2.13 0. 35 a 17. 62 a 4.91 b 0.39 ab 1.26 T3 2.33 0. 37 a 13. 69 b 6.40 ab 0.49 b 1.42 T4 1.68 0. 30 a 14. 67 b 4.95 b 0.21 c 1.13 T5 3.22 0. 23 a 15. 79 ab 7.08 a 0.31 ac 1.51 18:0 18 :1 18 : 2 18:3 18:4 20 :1 Ml 7.23 ns 17. 58 ns . 1. 33 ns 0.41 0.33 ns 5.28 ns M2 4.90 15. 36 1. 30 0.46 0.72 6.83 LI 6.84: ^ . 16. 24 ns . 1. 19* 0.40 0. 34** 5. 74s' L2 5.32 16. 63 1. 43 0.48 0.71 6.36 T l 6. 35 a 16. 21 a 1. 06 a 0.41 0.34 a 4.90 a T2 6. 21 a 15. 85 a 1. 45 b 0 . 31 0.39 ab 5 .30 a T3 6.12 a 16. 35 a 1. 30 ab 0.53 0.62 be 6.55 be T4 6.62 a 16. 99 a 1. 44 b 0.52 0.5 3 abc : 6.39 b T5 4.17 a 17. 00 a 1. 32 ab 0.42 0.74 c 7.65 c -90-TABLE XIV (Continued) 20:2 20:4 20:5 22:1 22:5 22 :6 Ml 0 . 22 ns 2.61 nsll.80 ns 1.58 ns 1.85 ns 22 .56 ns M2 0.19 1.38 14.07 3.54 1.61 22 .00 LI 0.20 ns 2.19 nsl2.99 1.9 6* 1.67 ns 23 .18 ns L2 0.20 1.80 12.89 3.16 1.80 21 . 36 T l 0.10 a 1.61 a 13.48 1.90 a 1.44 a 24 .76 a T2 0.28 b 2.76 a 11.88 2. 39 a 1.52 ab 22 .93 ab T3 0. 25 be 1.94 a 12.95 2.64 a 1. 82 be 22 .48 ab T4 0. 20 c 2.08 a 14.00 2.27 a 2.02 c 22 .13 b T5 0.18 c 1.59 a 12 .38 3.62 b 1. 86 be 18 . 85 c 1 The analyses were conducted on the arsine transformed percentages but the actual percentages (not transformed) are recorded i n TABLE XIV. 2 Ml = brine-frozen and M2 = plate-frozen and each mean i s the average of 30 observations. 3 LI = inside muscle and L2 = outside muscle and each mean i s the average of 30 observations. 4 T l = 14 weeks, T2 = 31 weeks, T3 = 45 weeks, T4 = 62 weeks and T5 = 81 weeks of frozen storage. Each mean i s the average of 12 observations. ns = no s i g n i f i c a n t difference at the 5% l e v e l . j. j . j. S i g n i f i c a n t at the 5% l e v e l . S i g n i f i c a n t at the 1% l e v e l . S i g n i f i c a n t at the 0.1% l e v e l . I f a column of methods, locati o n s , or storage times i s completely blank ( i . e . no *, l e t t e r or ns) then the methods, lo c a t i o n s , or storage times f o r that f a t t y acid were not tested for s i g n i f i c a n c e . Time means sharing the same l e t t e r are not s i g n i f i c a n t l y (P - 0.05) d i f f e r e n t from each other. -91-Free f a t t y a c i d s 14:0, 17:0, 18:1, 18:3, and 20:2 were s i g n i f i c a n t l y g r e a t e r (P - 0 , 0 5 ) i n the b r i n e - f r o z e n than i n the p l a t e - f r o z e n h a l i b u t . There were no s i g n i f i c a n t d i f -f e r e n c e s between f r e e z i n g methods f o r f a t t y a c i d s 15:0, 16:0,' 16:1, 16:2, 18:2, 18:4, 20:1, 20:5, 22:1 and 22:6 (Table I I I , Appendix). D i f f e r e n c e s between b r i n e - f r o z e n and p l a t e -f r o z e n samples f o r f a t t y a c i d s 18:0, 20:4 and 22:5 were dependent upon l o c a t i o n as i n d i c a t e d by a s i g n i f i c a n t (P - 0.05) method x l o c a t i o n i n t e r a c t i o n (Table I I I , Appendix), V i s u a l a n a l y s i s of the means i n d i c a t e d that i n s i d e muscle contained more 20:4 and 22:5 than o u t s i d e muscle with both the b r i n e -and p l a t e - f r o z e n samples but that the d i f f e r e n c e s between . f r e e z i n g methods was f a r g r e a t e r f o r the i n s i d e muscle than f o r the o u t s i d e muscle samples, the b r i n e - f r o z e n samples having the g r e a t e r content of both f r e e f a t t y a c i d s (Table IV, Appendix), In c o n t r a s t the i n s i d e muscle contained more 18:0 than the o u t s i d e muscle with the b r i n e - f r o z e n samples but the r e v e r s e was t r u e f o r p l a t e - f r o z e n samples. Consequently, the magnitude by which the f a t t y a c i d 18:0 content of the b r i n e -f r o z e n samples exceeded that of the p l a t e - f r o z e n samples was very much g r e a t e r with the i n s i d e than with the o u t s i d e muscle (Table IV, Appendix). The i n s i d e muscle samples contained s i g n i f i c a n t l y (P ^ 0.05) more 14:0, 15:0, 16:0, 16:1, 17:0, 18:1, 18:2, 18:3, 18:4, 20:1, 20:2, 20:5, 22:1, and 22:6 f r e e f a t t y a c i d s than the o u t s i d e muscle samples (Table XV and Appendix, Table I I I ) . -92-TABLE XV Method, l o c a t i o n , and time means from the analyses of variance of free f a t t y acids (expressed as jug free f a t t y acid per gram of neutral l i p i d ) of P a c i f i c h a l i b u t . 14:0 15 : C 16:0 16 :1 16:2 17 :0 M l 1 2386. 8* - 4 2 3.4 ns 22737. 8 ns 6365 . 9 ns 448. 2 ns 15 34.7* M2 1257. 9 229 .8 9183. 0 3272 . 6 208 . 7 710 . 6 L I 2 2889 . 9 « '* 551.5- 27299. 1* 7871. 3* 549 . 4 ns 1860.4-L2 754. 7 101.6 4621. 7 1767 . 2 107. 5 384 , 9 T l 3 571. ?H a 119.9 a 5247. 5 a 1385 . 7 a 93. 3 a 285.4 a T2 2140. 3 ab369.4 a 25363. 4 a 6726 . 5 a 681. 9 a 1658 .0 a T3 2662. 8 b 636.5 a 18753. 4 a 6795 . 9 a 544. 4 a 1636,6 a T4 1087. 6 ab2 36.9 a 10780. 7 a 2803. 7 a 117. 4 a 752 .1 a T5 2649 . 7 b 270.2 a 19157. 1 a 6384. 4 a 205. 1 a 12 0 8.2 a 18:0 18:1 18 :2 18 : 3 18:4 20:1 Ml 9858, i 5 ' 22245.2* 1513. 8 ns 502 . 5« 260 . 9 ns 6255.4 ns M2 3427 . 7 8636.5 696. 7 252 . 5 264. 0 3706.4 LI 1146 3. 7 25 8 33.0-* 1783. 6* sit 617. 83' 366. 4- A A 8156.0* L2 1822 . 1 504 8.7 426. 9 137. 1 158. 5 1805.8 T l 2214. 6 a 4861.2 a 360, 9 a 116. 0 a 42. 4 a 1206 .4 a T2 8340 . 7 al95 80.9 a 1331. 5 b 403 . 4 a 2 34 . 6 b 5222.8 b T3 9550. 8 a21004.5 a 1524 . 8 b 60 7 . 0 a 384. 7 be 7665.1 b T4 5507 . 5 al2753.2 a 956 . 7 ab 359. 1 a 222 . 9 b 4067.2 ab T5 7601. 0 al9004.4 a 1352 . 3 b 401. 8 a 427. 6 c 6742 .9 b -93-TABLE XV (Continued,) 20: 2 20 :4 20:5 22:1 22:5 22:6 Ml 304. 4* 3239.7 1626.89 ns 1466 .1 ns 2352.8 31428 . 6 ns M2 125 . 1 .893.5 8268.2 1441 .5 928.2 13889 .0 LI 368. 3* 3559.5 20743.0* 2101 .1* * 2747.8 38694 .7* L2 61. 2 573. 8 3794.1 806 .5 533.2 6622 .8 T l 38. 4 a 546.9 a 3747.3 a 321 .7 a 467.6a 7894 .4 a T2 376. 8 a 3246. 8 a2+607 .1 a 1649 .1 b 1786.6a30118 .8 a T3 330. 6 a 2708.6 al6817 .5 a 1891 .9 b 2530.8a33630 . 3 a T4 144. 9 a 1599.7 a 9834.5 a 12 85 .1 ab 1374.4al6815 .5 a T5 183. 0 a 2 2 31.0 a!6336 .4 a 2121 .3 b 2039.la24834 .8 a 1 Ml = brine-frozen and M2 = plate-frozen and each mean i s the average of 30 observations. 2 LI = inside muscle and L2 = outside muscle and each mean i s the.average of 30 observations. 3 T l = 14 weeks, T2 = 31 weeks, T3 = 45 weeks, T4 = 62 weeks, and T5 = 81 weeks of frozen storage. ns = no s i g n i f i c a n t difference at the 5% l e v e l * S i g n i f i c a n t at the 5% l e v e l ** S i g n i f i c a n t at the 1% l e v e l *** S i g n i f i c a n t at the 0.1% l e v e l . -4 Time means sharing the same l e t t e r are not s i g n i f i c a n t l y (P - 0.05) d i f f e r e n t from each other. 5 I f a column of methods, locations, or storage times i s completely blank ( i . e . no *, l e t t e r , or ns) then the methods, locations, or storage times f o r that f a t t y acid were not tested for s i g n i f i c a n c e . -94-F a t t y a c i d 1 6 : 2 c o n c e n t r a t i o n v;as not s i g n i f i c a n t l y d i f f e r e n t between the i n s i d e and o u t s i d e muscle samples. There was a s i g n i f i c a n t d i f f e r e n c e (P - 0 . 0 5 ) among f r o z e n s t o r a g e t i m e s f o r f r e e f a t t y a c i d s 14:0, 1 8:2, 18:4, 20:1, and 22 : 1 ( T a b l e I V , A p p e n d i x ) . I n g e n e r a l , the concen-t r a t i o n o f f r e e f a t t y a c i d s 14:0, 18:2, 20:1 and 22:1 was s i g n i f i c a n t l y d i f f e r e n t from the c o n c e n t r a t i o n at 62 weeks o f f r o z e n s t o r a g e ( T a b l e XV). With f r e e f a t t y a c i d 18:4 the c o n c e n t r a t i o n a t 14 weeks was s i g n i f i c a n t l y l ower t h a n t h a t a t any o t h e r f r o z e n s t o r a g e time w h i l e the c o n c e n t r a t i o n at 81 weeks was s i g n i f i c a n t l y g r e a t e r t h a n the c o n c e n t r a t i o n a t 1 3 , 3 1 , and 62 weeks ( T a b l e XV). i i ) Chinook Salmon 1) F r e e F a t t y ' A c i d s E x p r e s s e d as P e r c e n t o f T o t a l ' F r e e F a t t y A c i d s A n a l y z e d . The p e r c e n t a g e o f f r e e f a t t y a c i d 17:0 was s i g n i f i -c a n t l y (P - 0 , 0 5 ) g r e a t e r i n b r i n e - f r o z e n samples than i n p l a t e - f r c z e n samples ( T a b l e XVI and Appendix, T a b l e V ) . The p e r c e n t a g e o f f a t t y a c i d 18 : 3 was s i g n i f i c a n t l y g r e a t e r (P - 0 . 0 5 ) i n t h e o u t s i d e t h a n i n the i n s i d e muscle o f c h i n o o k salmon ( T a b l e XVI and Appendix, T a b l e V ) . The i n s i d e and o u t s i d e muscle samples d i d not d i f f e r s i g n i f i c a n t l y i n the p e r c e n t a g e s o f f a t t y a c i d s 14:0, 15:0, 16:1, 17:0, 18:0, 18:1, 18:2, 18:4, 20:1, 20:2, 20:4, 20:5, 22:5, and 22:6 ( T a b l e V, A p p e n d i x ) . C o n c l u s i o n s about d i f f e r e n c e s between i n s i d e and o u t s i d e muscle c o u l d not be e a s i l y drawn -95-TABLE XVI Method, location and time means from the analyses of variance of free f a t t y acids (expressed as percent of t o t a l free f a t t y acids analyzed) of Chinook salmon1 14:0 15: 0 • 16 :0 16 :1 16:2 17 :0 M l 2 .5.07 ns 0.26 ns 16.75 6 9.13 ns 0.45 1.17' ,». M2 4.58 0.28 16.16 8.23 0.49 1.04 L I 3 4.89 ns 0.27 ns 16. 56 8.98 ns 0.43 1.02 ns L2 4. 75 0.27 16. 34 8. 39 0.50 1.19 T l 4 4.43 5a 0.33 a 20 .40 7.89 a 0. 32 0.78 a T2 4.59 a 0.19 b .18 .93 8.52 a 0.67 1.15 be T3 5.20 a 0. 35 b 13.49 9.20 a 0.65 1.37 c T4 4.82 a 0. 27 b 14.87 8. 80 a 0. 31 0.97 ab T5 5.07 a 0.18 a 14.40 9.02 a 0. 39 1.24 be 18: 0 18:1 18:2 18 :3 18 :4 20 :1 Ml 3.26 ns 24.23 ns 2.19 ns 1.18 ns 1.72 ns 4.32 ns M2 3.85 23.51 2.10 1.22 1.64 4.57 LI 3.49 ns 23.72 ns 2.03 ns 1.16' 1.61 ns 4.40 ns L2 3.63 24.02 2.26 1.24 1.75 4.49 T l 3. 80 a 23. 46 ab 1. 35 a 1.01 a 1.07 a 3.69 a T2 2.99 b 21.83 b 3.12 b 1.2 3 ab 1.62 b 3.68 a T3 3. 89 b 24 .80 a 1.97 c 1.29 b 1. 84 b 4. 89 be T4 3.71 b 25.10 a 1.88 c 1.41 b 1.97 b 5.26 be T5 3.39 ab 24.11 a 2.41 d 1.05 a 1.89 b 4.68 b -96-TABLE XVI (Continued.) 20: 2 20 :4 20 :5 22:1 22:5 22:6 Ml 0.28 ns 0.50 ns 13.90 ns 3.05 ns 2.14 ns 8.62 ns M2 0.25 0.45 13.10 2.86 2.62 11.24 LI 0. 30 ns 0.44 ns 13.56 ns 2.86 2.30 ns 10.14 ns L2 0. 24 0. 51 13.44 3.04 2.46 9.73 T l 0. 20 a 0.64 a 13. 70 a 2.66 2.28 9.87 ab T2 0. 64 b 0.53 a 12.19 a 3.44 2.06 10.84 a T3 0.17 a 0.38 a 13. 71 a 2.92 2.42 9.62 ab T4 0.18 a 0.42 a 13.63 a 3.16 2.54 8.70 b T5 0.14 a 0.41 a 14. 30 a 2.57 2.60 10.62 a 1 The analyses were conducted on the arcsine transformed percentages but the actual percentages (not transformed) are recorded i n TABLE XVI. 2 Ml = brine-frozen and M2 = plate-frozen and each mean i s the average of 30 observations. 3 LI = inside muscle and L2 = outside muscle and each mean i s the average of 30 observations. 4 Tl" = 9 weeks, T2 = 26 weeks, T3 = 40 weeks, T4 = 58 weeks, and T5 = 77 weeks of frozen storage. Each mean i s the average of 12 observations. ns = no s i g n i f i c a n t difference at the 5% l e v e l * S i g n i f i c a n t at the 5% l e v e l . ** S i g n i f i c a n t at the 1% l e v e l . *** S i g n i f i c a n t at the 0.1% l e v e l . 5 Time means sharing the same l e t t e r are not s i g n i f i c a n t l y (P - 0.05) d i f f e r e n t from each other. 6 If a column of methods, locati o n s , or storage times i s completely blank ( i . e . no *, l e t t e r , or ns) then the methods, lo c a t i o n s , or storage times f o r that f a t t y acid were not tested f o r s i g n i f i c a n c e . -97-for free fatty acids 1 6 : 0 , 1 6 : 2 , and 2 2 : 1 as there were s i g n i f i -cant (P - 0 . 0 5 ) method x location x time Interactions for 1 6 : 0 and.16:2 and a s i g n i f i c a n t (P - 0 . 0 5 ) location x time in t e r a c t i o n for fat t y acid 2 2 : 1 (Table V, Appendix). Visual analysis of the means indicated that although the percentage of 2 2 : 1 of both inside and outside muscle increased to a maximum and then rapidly decreased, the outside muscle reached a maximum at 26 weeks while that of the inside muscle did not reach a maximum u n t i l 58 weeks of frozen storage (Table VI, Appendix). The percentages of free fatty acids 1 5 : 0 , 1 7 : 0 , 1 8 : 0 , 1 8 : 1 , 1 8 : 2 , 1 8 : 3 , 18:4, 2 0 : 1 , 2 0 : 2 , and 2 2 : 6 d i f f e r e d s i g n i f i -cantly (P - 0 . 0 5 ) among storage times (Table V, Appendix), There were no s i g n i f i c a n t differences between frozen storage times for fatty acids 14 : 0 , 1 6 : 1 , 20V4, 2 0 : 5 , and 2 2 : 5 . D i f f e r -ences between frozen storage times for fatty acids 1 6 : 0 , 1 6 : 2 , and 2 2 : 1 could not be simply evaluated as there were s i g n i f i -cant (P - 0 , 0 5 ) method x location x time interactions for 1 6 : 0 and 1 6 : 2 and a s i g n i f i c a n t (P - 0 , 0 5 ) location x time i n t e r a c t i o n for 2 2 : 1 (Table V, Appendix). The percentages of fatty acids 1 5 : 0 , 1 7 : 0 , and 1 8 : 0 reached a maximum at 40 weeks while 1 8 : 1 , 1 8 : 3 , 18:4, and 2 0 : 1 reached a maximum at 58 weeks and fatty acids 1 8 : 2 , 2 0 : 2 and 2 2 : 6 reached a maximum at 26 weeks of frozen storage (Table XVI). In a l l cases the maximum concentration was s i g n i f i c a n t l y (P - 0 . 0 5 ) greater than the concentration at one or more other frozen storage times (Table XVI). -98-2 ) Free F a t t y A c i d s E x p r e s s e d as yg_ F a t t y A c i d per Gram o f N e u t r a l L i p i d ~\ . The b r i n e - f r o z e n c h i n o o k salmon c o n t a i n e d s l g n i f i - . c a n t l y (P ^ 0 . 0 5 ) l e s s 1 6 : 0 , 1 8 : 2 , and 22.: 6 f r e e f a t t y " a c i d s t h a n p l a t e - f r o z e n samples ( T a b l e X V I I and Appendix, T a b l e V I I ) . There were no s i g n i f i c a n t d i f f e r e n c e s between f r e e z i n g methods f o r f a t t y a c i d s 1 5 : 0 , 1 6 : 2 , 1 8 : 0 , 1 .8:1, 18:3, 2 0 : 1 , 2 0 : 2 , 2 0 : 4 , 2 0 : 5 , and 2 2 : 5 (Table X I I , A p p e n d i x ) . No c o n c l u -s i o n s c o u l d be made about d i f f e r e n c e s between f r e e z i n g methods f o r f a t t y a c i d s 14: 0 , 1 6 : 1 , 1 7 : 0 , 1 8:4, and 2 2 : 1 as t h e r e were s i g n i f i c a n t (P - 0 . 0 5 ) method x l o c a t i o n x time i n t e r a c t i o n s ( T a b l e V I I , A p p e n d i x ) . There were s i g n i f i c a n t d i f f e r e n c e s (P - 0 . 0 5 ) between i n s i d e and o u t s i d e muscle f o r f r e e f a t t y a c i d s 1 5 : 0 , 1 8 : 0 , 1 8 : 1 , 1 8 : 2 , 1 8 : 3 , 2 0 : 1 , 2 0 : 4 , 2 0 : 5 , and 2 2 : 5 ( T a b l e V I I , A p p e n d i x ) . The c o n c e n t r a t i o n o f a l l the above mentioned f a t t y a c i d s was g r e a t e r i n the i n s i d e muscle than I n the o u t s i d e muscle ( T a b l e X V I I ) , No c o n c l u s i o n s c o u l d be made about d i f -f e r e n c e s between i n s i d e and o u t s i d e muscle f o r f r e e f a t t y a c i d s 1 4 : 0 , 1 6 : 0 , 1 6:1, 1 6 : 2 , 1 7 : 0 , 1 8:4, 2 0 : 2 , 2 2 : 1 , and 2 2 : 6 as t h e r e were s i g n i f i c a n t (P - 0 . 0 5 ) method >: l o c a t i o n x time i n t e r a c t i o n s f o r f a t t y a c i d s 1 4 : 0 , 1 6 : 1 , 1 7 : 0 , 1 8 : 4 , and 2 2 : 1 and s i g n i f i c a n t (P - 0 , 0 5 ) l o c a t i o n x time i n t e r a c t i o n s f o r f a t t y a c i d s 1 6 : 0 , 1 6 : 2 , 2 0 : 2 , and 2 2 : 6 ( T a b l e V I I , A p p e n d i x ) . V i s u a l a n a l y s i s o f the means o f f a t t y a c i d s 1 6 : 0 , 1 6 : 2 , 2 0 : 2 , and 2 2 : 6 i n d i c a t e d t h a t i n each case t h e r e were no o b v i o u s m e a n i n g f u l t r e n d s ( T a b l e V I I I , A p p e n d i x ) . -99-TABLE XVII Method, l o c a t i o n , and time means from the analyses of variance of free f a t t y acids (expressed jog free f a t t y acid per gram of neutral l i p i d ) of Chinook salmon. Ml" M2 14:0 15:0 16:0 16:1 16:2 17:0 622.7 r 30.5ns 2005.6* 1143.2 61.1 157.6 695.2 38.8 2350.7 1260.1 71.4 157.8 895. 7 47.1** 2987.0 .1651. 8 88. 0 203. 4 L2 422 . 2 22.2 1369 . 3 751.5 44 . 5 112. 0 J. x 272. 5 21.1 5a 1227.0 502. 3 19. 8 48. i T2 558. 1 24.0 a 2630.5 1073.5 95. 4 162. 3 T3 873. 0 56.6 b 2362.1 15 75.0 107. 8 218. 3 T4 652. 8 38. 3 a 2036.6 12 01.1 42 . 0 130 . 8 T5 938. 3 3 3.4 a 2634.6 1656.2 66. 3 228. 3 18 : 0 18 :1 18 :2 18:3 18 : 4 20 : 1 Ml 390 . 0 ns3061.9 ns 2 7 8.4- 150. 3 ns22 5 . n 1 536. 6 ns M2 546. 2 3676.7 312. 8 174.0 248. r D 696 . 0 LI 623. i}« 4 511.9** 395 .9 . 213.2 = 312. 8 820. 1* L2 • 313. 6 2225.7 19 6. 2 111.1 161. 5 412. 5 T l 236. 7 a 1502.4 a 87.0 a 61. 0 a 69. 2 241. u a T2 371. 3 ab2945.1 b 391. 8 b 164. 5 b 206. L 441. 3 b T3 654 . 6 c 4415.0 c 3 21.5 be ! 213.0 b 301. U 832 . 1 c T4 497. 0 bc3531.3 be 251.5 c 185,6 b 255 . 7 697. 9 c T5 583. 0 c 445 2.6 c 426 .1 b 186, 6 b 353. 2 868 . 7 c -100-TABLE XVII (Continued) 20:2 20 :4 20:5 22 : 1 22:5 22:6 Ml 30.8 ns 55.4 ns 1682.4 ns 368. 0 254.6 ns 1051.2 M2 37. 8 63.0 1964.7 420 . 9 387. 2 1715.5 LI 51.7 78.5* 2477 . 8s' .5 31. 5 430.7** 1920. 8 L2 16.9 40.0 1169.4 257. 4 211.2 845.9 T l 7.0 34.1 a 870.9 a 164. 2 145.0 a 681. 5 T2 79 . 2 64.2 a 1535.8 b 39 2. 6 265.8 b 1407.6 T3 33.5 69.0 a 2327.3 cd 483. 3 419.7 c 1721.5 T4 24.5 53.9 a 1822.3 be 432. 5 307.2 b 1116.1 T5 27.5 75.0 a 2561.4 d 49 9 . 7 466.8 c 1990.0 Ml Brine-frozen and M2 Plate-frozen and each mean i s the average of 3 0 observations. 2 LI = inside muscle and L2 = outside muscle and each mean i s the average of 30 observations. 3 T l = 9 weeks, T2 = 26 weeks, T3 = 40 weeks, T4 = 58 weeks, and T5 = 77 weeks of frozen storage. ns = no s i g n i f i c a n t difference at the 5% l e v e l . * S i g n i f i c a n t at the 5% l e v e l . ** S i g n i f i c a n t at the 1% l e v e l . *** S i g n i f i c a n t at the 0.1% l e v e l . 4 If a column of methods, locations , or storage times i s completely blank ( i . e . no *, l e t t e r , or ns) then the methods, locations, or storage times for that f a t t y acid were not tested f o r si g n i f i c a n c e . 5 Time means sharing the same l e t t e r are not s i g n i f i c a n t l y (P - 0.05) d i f f e r e n t from each other. -101-There were s i g n i f i c a n t d i f f e r e n c e s (P - 0 . 0 5 ) among f r o z e n s t o r a g e t i m e s f o r f r e e f a t t y a c i d s 1 5 : 0 , 1 8 : 0 , 1 8 : 1 , 1 8 : 2 , 1 8 : 3 , 2 0 : 1 , 2 0 : 5 , and 2 2 : 5 ( T a b l e V I I , A p p e n d i x ) . Free f a t t y a c i d 2 0 : 4 d i d not d i f f e r s i g n i f i c a n t l y between f r o z e n s t o r a g e t i m e s . No c o n c l u s i o n s about d i f f e r e n c e s between f r o z e n s t o r a g e t i m e s c o u l d be made f o r f r e e f a t t y a c i d s 1 4 : 0 , 1 6 : 0 , 1 6 : 1 , 1 6 : 2 , 1 7 : 0 , 1 8 : 4 , 2 0 : 2 , 2 2 : 1 , and 2 2 : 6 as t h e r e were s i g n i f i c a n t (P - 0 . 0 5 ) method x . l o c a t i o n x time i n t e r a c t i o n s f o r 1 4 : 0 , 1 6 : 1 , 1 7 : 0 , 1 8 : 4 , and 2 2 : 1 and s i g n i f i c a n t (P - 0 . 0 5 ) l o c a t i o n x time i n t e r a c t i o n s f o r 1 6 : 0 , - 1 6 : 2 , 2 0 : 2 and 2 2 : 6 ( T a b l e V I I , A p p e n d i x ) . The c o n c e n t r a t i o n o f f r e e f a t t y a c i d 1 5 : 0 appeared t o i n c r e a s e t o a maximum at 45 weeks and t h e n d e c r e a s e u n t i l t h e 7 7 t n week o f f r o z e n s t o r a g e ( T a b l e X V I I ) . I n g e n e r a l , w i t h f r e e f a t t y a c i d s 1 8 : 1 , 1 8 : 2 , 1 8 : 3 , 2 0 : 1 , 2 0 : 5 , ' and 2 2 : 5 t h e r e was a s i g n i f i c a n t i n c r e a s e i n c o n c e n t r a t i o n between 9 and 26 weeks but t h e d i f f e r e n c e s among the o t h e r f r o z e n s t o r a g e t i m e s ( i . e . 4 5 , 5 8 , and 77 weeks) were v a r i a b l e ( T a b l e X V I I ) . i i i ) Coho Salmon 1) F r e e F a t t y A c i d s E x p r e s s e d as P e r c e n t o f T o t a l  F ree F a t t y A c i d s A n a l y z e d None o f the f r e e f a t t y a c i d s a n a l y z e d d i f f e r e d s i g n i f i c a n t l y between f r e e z i n g methods ( T a b l e I X , A p p e n d i x ) . The p e r c e n t a g e o f f a t t y a c i d 18:4 was s i g n i f i c a n t l y (P - 0 . 0 5 ) g r e a t e r I n o u t s i d e muscle t h a n i n i n s i d e muscle -102-(Table XVIII and Appendix, Table IX). Free f a t t y acids 1 4 : 0 , 1 5 : 0 , 1 6 : 0 , 1 6 : 1 , 1 6 : 2 , 1 7 : 0 , 1 8 : 0 , 1 8 : 1 , 1 8 : 2 , 1 8 : 3 , 2 0 : 1 , 2 0 : 2 , 2 0 : 4 , 2 2 : 1 , 2 2 : 5 and 2 2 : 6 did not d i f f e r s i g n i -f i c a n t l y with location (Table IX, Appendix). No conclusions about differences between inside and outside muscle or . • between fret-zing methods. could be made for free fatty acid 2 0 : 5 as there was a s i g n i f i c a n t (P - 0 , 0 5 ) method x location x time i n t e r a c t i o n (Table IX, Appendix), There was a ' s i g n i f i c a n t (P ^ 0 . 0 5 ) difference among the d i f f e r e n t frozen storage times for free fatty acids 1 5 : 0 , 1 6 : 1 , 1 7 : 0 , 1 8 : 1 , 1 8 : 2 , 1 8 : 4 , 2 0 : 2 , 2 0 : 4 and 2 2 : 1 (Table IX, Appendix), No conclusions about differences among frozen storage times could be made for free f a t t y acid 2 0 : 5 as there was a s i g n i f i c a n t (P - 0 . 0 5 ) method x location x time i n t e r a c t i o n (Table IX, Appendix), The concentrations of free fatty acids 1 7 : 0 , 1 8 : 0 , and 1 8 : 4 were s i g n i f i c a n t l y greater (P - 0 . 0 5 ) at 78 .weeks than at 27 and/or 10 weeks of frozen storage (Table XVIII). The concentrations of free fatty acids 1 5 : 0 , 1 6 : 1 , 1 8 : 1 , 1 8 : 2 , 2 0 : 2 , 2 0 : 4 and 2 2 : 1 were s i g n i f i c a n t l y greater (P £ 0 . 0 5 ) at the 1 0 t h or 2 7 t h week than at the 7 8 t h week (Table XVIII). 2 ) Yvce. Fatty Acids Expressed as pg_ Free Fattj/ Acid per Gram of Neutral L i p i d There were not any s i g n i f i c a n t differences due to freezing method, location of or storage time for any of the 18 free fatty acids analyzed (Table XIX, and Appendix Table XI), -103-TABLE XVIII Method, lo c a t i o n , and time means from the analyses of variance of free f a t t y acids (expressed as percent of t o t a l free f a t t y acids analyzed) of Coho salmon. 14:0. 15:0 16:0 16:1 16:2 17:0 Ml 1. 3.37 ns 0.23 ns 15.58 ns 8.44 ns 0.43 ns 1.05 ns M2 3.49 0.18 14.49 9.07 0.50 1.19 L I 2 3.24* 0.20 ns 15.40 ns 8.78 ns 0.35 ns 0.95 ns L2 3.62 0.21 14.67 8.34 0.57 1.29 T l 3 3.66 4a 0.30 a 15.20 a 8.37 a 0.43 a 0.90 a T2 3.19 a 0.15 b 14.44 a 10.06 b 0.55 a 1.15 ab T3 3.44 a 0.17 b 15.47 a 7.26 a 0.41 a 1.31 b 18:0 18:1 18:2 18:3 18:4 20:1 Ml 3.26 ns 21.60 ns 2.01 ns 1.10 ns 1.55 ns 4.24 ns M2 3.09 20.81 2.01 1.00 1.51 4.56 LI 3.12 ns 21.05 ns 1.87 ns 0.99 ns 1.41* 4.30 ns L2 3.23 21.37 2.15 1.10 1.65 4.49 T l 3.19 a 20.94 a 1.49. a 1.11 a 1.57 a 4.37 a T2 2.20 b 22.81 b 2.47 b 0.93 a 1.19 b 4.21 a T3 4.15 c 19.84 a 2.07 c 1.11 a 1.83 c 4.61 a -104-TABLE XVIII (Continued) 20:2 20:4 20:5 22:1 22:5 22:6 Ml 0.68 ns 0.72 ns 15.II 5 3.07 ns 2.87 ns 13.04 ns M2 0.59 0.76 15.16 4.09 2.96 13.02 LI • 0.70 ns 0.77 ns 15.16 3.07 ns 2.66 ns 13.71 ns L2 0.57 0.77 15.02 3.46 3.17 12.34 T l 0.39 a 1.06 a 15.23 3.69 a 2.62 a 13.78 a T2 1.22 b 0.56 b 13.28 4.17 a 2.80 a 12.54 a T3 0.29 a 0.60 b 16.88 2.89 b 3.33 a 12.78 a Ml Brine-frozen and M2 Plate-frozen and each mean i s the average of 30 observations. 2 LI = inside muscle and L2 = outside muscle and each mean i s the average of 30 observations. 3 T l = 9 weeks, T2 = 26 weeks, T3 = 40 weeks of frozen storage. ns = no s i g n i f i c a n t difference at the 5% l e v e l . * S i g n i f i c a n t at the 5% l e v e l . ** S i g n i f i c a n t at the 1% l e v e l . is** S i g n i f i c a n t at the 0.1% l e v e l . 4 I f a column of methods, l o c a t i o n s , or storage times i s completely blank ( i . e . no *, l e t t e r , or ns) then the methods, lo c a t i o n s , or storage times for that f a t t y acid were not tested for s i g n i f i c a n c e . 5 Time means sharing the same l e t t e r are not s i g n i f i c a n t l y (P - 0.05) d i f f e r e n t from each other. -105-TABLE XIX Method, lo c a t i o n , and time means from the analyses of variance of free f a t t y acids (expressed as free f a t t y acid per gram of neutral l i p i d ) of Coho salmon. 14:0 15:0 16:0 16:1 16:2 17:0 M l 1 4817.8 ns 201.7 ns 17661.9 ns 9743.2 ns 632.9 ns 1876.0 ns M2 804.1 41.0 3692.8 2120.8 100.3 308.4 L I 2 5288.5 ns 224.7 ns 20002.2 nsil064.2 ns 671.9 ns 2053.6 ns L2 333.4 18.0 1352.5 799.8 61.4 130.8 T l 3 372. 2 ^ 31.1 a 1640. 7 a 872 . 0 a 43.4 a 93.8 a T2 371.4 a 18.6 a 1800.5 a 1270.1 a 59.3 a 122.5 a T3 7690.3 a 314.3 a 28590.8 a 15653.9 a 997.2 a 3060.3 a 18:0 18:1 18:2 18:3 18:4 20:1 Ml 3999.3 ns24819.3 ns2138.4 ns 1441.0 ns 2799.6 ns 7468.6 ns M2 904.2 5148.3 520.7 288.5 421.0 1106.1 LI 4533.1 ns27932.5 ns2464.0 ns 1621.4 ns 3056.0 ns 8162.9 ns L2 370,4 2035.1 195.0 108.2 164.7 411.8 T l 344.8 a 2233.6 a 151.4 a 114.6 a 166.0 a 442.0 a T2 275.2 a 2836.4 a 289.4 a 112.7 a 133.9 a 483.7 a T3 6735.1 a 39881. 5 a 3547 .8 a 2367,1 a 453.1 .1 a 11936, 3 a -106-TABLE XIX (Continued) 20:2 20:4 20;5 22:1 22:5 22:6 Ml 804.5 ns 569.3 ns 19905.2 ns 4600.8 ns 2691.0 ns 14666.1 ns M2 106.9 154.4 3998.9 ns 849.0 779.3 3725.1 LI 873.1 ns 664.6 ns 22377.3 ns 5165.5 ns 3152.8 ns 17163.9 ns L2 38.3 59.2 1526.8 284.2 317.5 1227.3 T l 37.9 a 104.5 a 1622.2 a 364.4 a 253.3 a 1541.7 a T2 159.3 a 70.4 a 1636.2 a 507.3 a 348.9 a 1637.5 a T3 1169.9 a 910.8 a 32597.8 a 7302.9 a 4603.1 a 24407.6 a Ml Brine-frozen and M2 Plate-frozen and each mean i s the average of 30 observations. 2 LI = inside muscle and L2 = outside muscle and each mean i s the average of 30 observations. T l 3 = 9 weeks, T2 = 26 weeks, T3 = 40 weeks of frozen storage. ns - no s i g n i f i c a n t difference at the 5% l e v e l . * S i g n i f i c a n t at the 5% l e v e l . ** S i g n i f i c a n t at the 1% l e v e l . *** S i g n i f i c a n t at the 0.1% l e v e l . 4 Time means sharing the same l e t t e r are not s i g n i f i c a n t l y (P - 0.05) d i f f e r e n t from each other. -107-h ) yQ't.al Free Fatt y Acids (Expressed as JJJ^  Free Fatty Acid  per Gram of Neutral LlpldT' i ) P a c i f i c Halibut There was no s i g n i f i c a n t difference between method, of freezing or among storage times (Table XIII, Appendix), The concentration of t o t a l free fatty acids v/as s i g n i f i c a n t l y (P - 0 , 0 1 ) greater i n the inside than i n the outside muscle (Table XX and Appendix, Table XIII). Chinook Salmon The concentration of t o t a l free fatty acids was s i g n i f i c a n t l y greater (P = 0 , 0 5 ) i n the plate-frozen than i n the brine-frozen samples (Table XX and. Appendix, Table XIII), Inside muscle contained s i g n i f i c a n t l y (P - 0 . 0 1 ) greater free fa t t y acids than the outside muscle (Table XX and Appendix Table XIII). There v/as also a very highly s i g n i f i c a n t d i f f e r -ence (P - 0 , 0 0 1 ) among storage times (Table XIII, Appendix). The samples at 9 weeks were s i g n i f i c a n t l y (P - 0 . 0 5 ) lower in t o t a l free fatty acid, concentration than samples at any other storage time (Table XX), The samples at. 77-.weeks v/ere s i g n i f i c a n t l y (P - 0 , 0 5 ) greater than samples at any other storage time except those at 58 weeks (Table XX), i i i ) Coho Salmon There v/ere no s i g n i f i c a n t differences between method of freezing, location of sampling, or among storage -108-TABLE XX Method, lo c a t i o n , and time means from the analyses of variance of t o t a l free f a t t y acids (expressed as ug free f a t t y acid per gram of neutral l i p i d ) of P a c i f i c h a l i b u t , Chinook salmon and Coho salmon. P a c i f i c halibut Chinook salmon Coho salmon Ml" M2 12959.3 ns 5739.2 1210.7* 1481.7 12083.6 ns 2506.9 LI' L2 15745.6** 2952.8 1824.0** 868.4 13647.2 ns 943.4 14 weeks' 29 5 2 .2.Ha PACIFIC HALIBUT 31 weeks 45 weeks 62 weeks 12433.9 a 12967.6 a 7070.3 a 81 weeks 11322.2 a 9 weeks 619.2 a 26 weeks 12 80.9 b CHINOOK SALMON 40 weeks 5 8 weeks 1698.5 cd 1327.7 be 77 weeks 1804.8 d 10 weeks 1042.9 a COHO SALMON 27 weeks 1213.3 a 78 weeks 19629.6 a 1 2 3 ns ft Ml = brine-frozen and M2 = plate-frozen. LI - inside muscle and L2 = outside muscle. Weeks of frozen storage. = no s i g n i f i c a n t difference at the 5% l e v e l . S i g n i f i c a n t at the 5% l e v e l . S i g n i f i c a n t at the 1% l e v e l . Time means sharing the same l e t t e r are not s i g n i f i c a n t l y (P = 0.05) d i f f e r e n t from each other. -109-times (Table XIII, Appendix). Correlations a ^ Correlations of p_H, Thaw Drip and Color- (Hunter Rd, 'a' , k» a n d a/B jfaJ-J^es) with Each Other 1 ^ £-G.? 1 / H a l i b u t There was a s i g n i f i c a n t (P - 0.05) negative c o r r e l a t i o n (r = -0.358) between thaw drip and pH (Table XV, Appendix). i-* - ^ Chinook Salmon There were s i g n i f i c a n t (P - 0.05) correlations among the three color parameters (Table XV, Appendix), i i i ) Coho Salmon The negative, correlations between pK and Hunter 'a' values (r = -0.596) and between pH and Hunter a/b values (r = -0.721) were both highly s i g n i f i c a n t (P = 0.01). There were also s i g n i f i c a n t (P - 0,05) correlations among the three color parameters (Table XV, Appendix). Correlations of pJH, Thaw Drip, Color, TBA values and Free Fatty Acids with Flavor i ^ P a c i f i c Halibut Flavor was not s i g n i f i c a n t l y correlated with any of the other parameters measured (Table XV, Appendix). -110-i i ) Chinook Salmon . Only the Hunter b values (r = -0.527), the TBA values (r = +0.460), and free f a t t y acid 20:1 (expressed as percent of t o t a l free f a t t y acids analyzed (r = -0.410) correlated s i g n i f i c a n t l y (P - 0.05) with flavor 1 (Table XVI, Appendix). i i i ) Coho Salmon Only the Hunter 'a' values (r = +0.653) and free f a t t y acid 18:0 (expressed as percent of t o t a l free f a t t y acids analyzed (r = +0.515) correlated s i g n i f i c a n t l y (P ^ 0.05) with f l a v o r (Table XVI, Appendix). - 1 1 1 -D I S C U S S I O N a) p_H The presence of a small and non-significant differenc i n pH between brine-frozen and plate-frozen halibut indicates that brine-freezing l i k e l y would not Increase the incidence of the undesirable chalky condition i n halibut as i t has been found that chalkiness i s related to the pK of the muscle (Tom-linson et a l . , 1 9 6 5 ) . The decline i n pH during frozen storage of P a c i f i c halibut agrees v/ith the findings of Tomlinson et a l . ( 1 9 6 9 ) . However, i n both cases sampling was confounded with the p o s i t i o n along the length of the halibut and Tomlinson et_ a l . ( 1 9 6 6 ) observed that the pH of halibut tends to be higher near the head of the f i s h and lower near the center of the body. Thus the observed decline i n pH may be related to the position of sampling, or i t may have resulted from a combination of frozen storage time and po s i t i o n of sampling. No data could be found i n the l i t e r a t u r e regarding f l e s h pH at d i f f e r e n t points along the length; of chinook and coho salmon, Nevertheless this does not preclude a. p o s i t i o n a l e f f e c t on f l e s h pH, p a r t i c u l a r l y i n view of the findings for P a c i f i c halibut (Tomlinson e_t a l , 1 9 6 6 ) . Therefore the re s u l t s are inconclusive. -112-b) Thaw Drip The mean thaw drip of P a c i f i c halibut and Chinook salmon, at the f i r s t and/or second sampling time, was less for the brine-frozen than for the plate-frozen samples. At a l l other frozen storage times the brine-frozen samples had approximately equal or greater thaw drip than the plate-frozen samples (Fig. 6 and F i g . 7 ) . Fish frozen slowly tends to form more drip than those frozen quickly and drip increases with increased frozen stor-age time (Miyauchi, 1 9 6 3 ) . Also Tarr ( 1 9 ^ 2 ) showed that NaCl can markedly reduce free drip from f i s h muscle. The brine-frozen halibut were frozen over a 47 hour period and the chinook salmon were brine-frozen over a 9 . 5 hour period whereas both the halibut and chinook salmon were plate-frozen i n approximately 3 . 5 hours. Thus, the fact that the brine-frozen halibut and chinook salmon had less thaw drip than the plate-frozen samples, •during the early part of frozen storage, may be related to the e f f e c t NaCl has on reducing d r i p . Whereas brine-frozen halibut and chinook had approximately the same ( p a r t i c u l a r l y the chinook salmon) or more ( p a r t i c u l a r l y the halibut) thaw drip than the plate-frozen samples during the l a t e r part of frozen storage suggests that freezing rate may have been a determinant. However, this explanation does not apply to the thaw drip from coho salmon as the plate-frozen samples -113-consistently had greater thaw drip than the brine-frozen samples. The decline i n thaw drip during frozen storage of plate-frozen halibut has been observed i n other work with t h i s species (Roach et_ a l . , 1 9 6 6 ) . The thaw drip values of P a c i f i c halibut were higher than those reported by Tomlinson e_t a l . ( 1 9 6 9 ) but t h i s may be due to differences i n the r a t i o of cut surface area to t o t a l weight of the f i s h f l e s h as the amount of drip formed i s related d i r e c t l y to the above mentioned r a t i o (Miyauchi, 1 9 6 3 ) . Differences i n pH may also be involved as Tomlinson et j Q . ( 1 9 6 6 ) found that thaw drip of halibut increased continuously with decreasing pH i n the range pH 6 . 8 - 5 . 7 . The f l e s h pH of the halibut i n the present work varied between pH 6 . 2 5 and pH 5 . 8 5 whereas the f l e s h pH of the halibut used by Tomlinson e_t a l . ( 1 9 6 9 ) varied between pH 6 . 6 6 and pH 6 . 2 5 . c) Color The Hunter Rd, a, and b color readings on raw salmon have, by themselves, l i t t l e p r a c t i c a l meaning. However, Schmidt and Idler ( 1 9 5 8 ) found that the 'a' reading alone was a suitable measure of the color of processed salmon and that i t could best be predicted from the a/b r a t i o of the raw flesh.. The higher the a/b r a t i o of the raw f l e s h , the higher the 'a' value of the r e s u l t i n g processed product and the higher the v i s u a l redness. Analysis of variance indicated that i n comparison -114-wlth plate-freezing, brine-freezing did not s i g n i f i c a n t l y decrease the a/b r a t i o of coho salmon. For chinook salmon, between 26 and 77 weeks of frozen storage, the brine-frozen samples always had a s l i g h t l y lower a/b r a t i o than the plate-frozen (Fig. i(p.). Oxidative r a n c i d i t y of frozen red salmon (chinook and coho salmon) i s accompanied by a fading of the red pi g -ments of the fle s h (Tarr, 1 9 ^ 7 ; 1 9 5 5 ; Boyd et a l . , 1 9 5 7 ) . Thus, i f oxidative r a n c i d i t y was occuring during the frozen storage of chinook and coho salmon one would expect a decrease i n the a/b r a t i o . However, with both species the a/b r a t i o increased during frozen storage. With the coho salmon the a/b r a t i o at 78 weeks v/as s i g n i f i c a n t l y higher (P - 0 . 0 5 ) than the a/b r a t i o at 10 and 27 weeks of frozen storage (Table VI). With brine-frozen chinook salmon the a/b r a t i o increased from a low of 1 . 0 0 3 at 9 weeks to a high of 1 . 2 0 3 at 58 weeks of frozen storage (Fig. 16.1)* Thus either oxidative r a n c i d i t y was not taking place to a s u f f i c i e n t degree to reduce the a/b r a t i o or oxidative r a n c i d i t y does not s i g n i f i c a n t l y affect the red pigments of salmon. d) Flavor Differences For halibut and chinook salmon the taste panel found that the differences i n f l a v o r between outside muscle samples from brine-frozen and plate-frozen f i s h reached a maximum at the second period ( i . e . , at the end of approximately 29 weeks -115-of frozen storage at - 3 0°C) and then steadily decreased. This r e l a t i o n s h i p may be related to the higher s a l t content i n the brine-frozen outside muscle samples. The higher s a l t content may have accelerated the induction period of l i p i d oxidation as i n the chinook salmon the greatest difference i n TBA values between the brine-frozen and plate-frozen outside muscle sample occurred at the second sampling period and i n halibut at the t h i r d sampling period. After approximately 30 weeks of frozen storage the plate-frozen samples may begin to 'catch-up' with the brine-frozen samples i n development of ra n c i d i t y and o f f - f l a v o r s . The fact that, with a l l three species, the plate-frozen outside muscle samples were preferred at the f i r s t two or three sampling periods while at the l a s t sampling period the brine-frozen outside muscle samples were s l i g h t l y pre-ferred suggests that s a l t possibly masked increasing deterior-ation i n f l a v o r . It i s unknown to what degree the higher s a l t content of the brine-frozen outside muscle samples influenced the panel members a b i l i t y to i d e n t i f y the odd sample and no explanation i s apparent for the difference between coho and the other two species. The taste panel also detected l i t t l e difference i n fl a v o r between the brine-frozen and plate-frozen inside muscle samples from the two salmon species. One possible reason for -116-the difference detected between inside halibut muscle samples i s that halibut were brine-frozen during a 47 hour period while the salmon were brine-frozen i n 9 . 5 hours. The fact that the present r e s u l t s d i f f e r e d from those of Peters e_t a l . ( 1 9 6 8 ) may be related to the fact that A t l a n t i c cod i s a much leaner f i s h than salmon and to a lesser degree halibut. e) Mineral Concentration The sodium concentration i n the brine-frozen inside muscle, plate-frozen inside muscle and plate-frozen outside muscle of P a c i f i c halibut, chinook salmon and coho salmon was si m i l a r to that reported by McBride and MacLeod ( 1 9 5 6 ) but potassium concentration was s l i g h t l y lower. The fact that the percentage change i n potassium i n the outside muscle was much less than the percentage change i n sodium and chloride concentrations may be related to the length of time the f i s h were i n the brine. Tomlinson et a l . ( I 9 6 5 a; 1 9 6 5 b) reported that the decrease i n potassium con-centration of coho and sockeye salmon and rainbow trout was much slower, than the increase i n sodium'concentration that occurs during storage of f i s h i n refrigerated sea water (RSW) or f o r t i f i e d r e f r i g e r a t e d sea water (FRSW). Even though there was an increase i n sodium and chloride content of the brine-frozen outside muscle of a l l three species, the s a l t concentration was s t i l l below that -117-generally accepted for p a l a t a b i l i t y , which i s usually taken to be 1%, Similar results v/ere found by Harrison and Roach ( 1 9 5 3 ) for chinook salmon, chum salmon and gray cod and by Holston and Pottinger (195*0 for haddock. f) TBA Values The TBA values for the inside muscle of a l l three species were r e l a t i v e l y s i m i l a r for both brine- and p l a t e -frozen samples suggesting l i t t l e difference i n degree of oxidation i n inside muscle frozen by the two d i f f e r e n t methods. In outside muscle, by contrast, between 26 and 45 weeks of frozen storage (depending on the species) there occurred a r e l a t i v e l y large maximum difference i n TBA values between the brine-frozen and plate-frozen samples. The differences then decreased as storage progressed u n t i l at approximately 80 weeks the differences i n TBA values between the inside and outside muscle samples were r e l a t i v e l y small. The fact that, except at the f i r s t or l a s t analyses, (depending on the species) average TBA values of the brine-frozen outside muscle v/ere greater than those of the corresponding plate-frozen samples indicated that oxidative r a n c i d i t y , p a r t i c u l a r l y at approximately 29 weeks of frozen storage, was greater i n the brine-frozen outside muscle than i n the plate-frozen outside muscle. The observation that the brine-frozen outside and the plate-frozen outside muscle of chinook and coho salmon -118-as we'll as the brine-frozen outside muscle of halibut reached a maximum and then steadily decreased are not i n agreement with the r e s u l t s of Awad et a l , ( 1 9 6 9 ) . They observed that the TBA values of fresh-water whitefish, stored at - 1 0°C, steadily increased with time of frozen storage. In contrast, C a s t e l l et^ al_. ( 1 9 6 6 ) observed that the l i p i d s of cod muscle stored at - 18°C and at ~25°C did not undergo oxidation as measured by the TBA method. C a s t e l l et a l . ( 1 9 6 6 b) also found that during frozen storage the l i p i d s of cod became markedly more resistant to metal - (Cu or or to hemoglobin-catalyzed oxidation. They suggested that the free fatty acids may have reacted with proteins or some other component of the muscle i n a manner that pro-tected t h e i r double bonds against oxidation. An analogous reaction may explain the decrease In the TBA values that occurred during the present study. The TBA test i s based on the reaction of malonalde-hyde with 2 Thiobarbituric acid to form a pink to red colored product i n solution. .Consequently, a possible explanation for the observed decrease i n TBA values of the outside muscle samples i s that malonaldehyde may have reacted with some other components of the muscle and thus would have been unavailable for the TBA reaction. Buttkus ( 1 9 6 7 ) showed that malonaldehyde reacted with the E-amino groups of trout myosin. The rate of reaction at 0°C was less than that at 20°C while the reaction rate at - 2 0°C was almost equal to that at +20°C. Buttkus ( 1 9 6 9 ) also showed that malonaldehyde -119-reacted with cysteine and methionine, Kwon e_t a l . ( 1 9 6 5 ) reported that t h i o b a r b i t u r i c acid reactive substances react with protein and i n tuna stored at - l 8 ° C became p a r t i a l l y unrecoverable for the TBA t e s t . An additional factor probably contributing to the decrease i n TBA values observed i n the present study could be the fact that the presence of oxygen i s necessary for oxidative r a n c i d i t y to occur (Lundberg, 1 9 6 1 ) . P r i o r to freezing, the f i s h f l e s h was exposed to oxygen whereas after the f i s h were frozen and heavily glazed access of oxygen to the f l e s h would have been very greatly impeded. During each sampling, the f i s h were reglazed immediately aft e r a sample was taken. Thus even at t h i s point very l i t t l e oxygen would penetrate the f l e s h . Consequently, oxidative r a n c i d i t y could only proceed while oxygen was s t i l l present i n the f l e s h . Once the o r i g i n a l oxygen was used up randidity could no longer continue as no 'new' oxygen (or very l i t t l e ) should have been entering the f l e s h . Awad e_t a l , ( 1 9 6 9 ) "observed increasing oxidative r a n c i d i t y (TBA values) with increasing storage time. Although the f i s h used i n t h e i r study were wrapped i n saran f i l m p r i o r to freezing, the f i s h were not frozen u n t i l approximately 5 days post-mortem. g) Free Fatty Acids The percentage of free fatty acid 17:0 was s i g n i f i -cantly (P - 0.05) greater i n the brine-frozen than i n the -120-plate-frozen chinook salmon (Table XVI and Appendix, Table V. The concentrations ( J X g free f a t t y acid per gram of neutral l i p i d ) of fat t y acids' 1 6 : 0 , 1 8 : 2 , and 2 2 : 6 as well as the concentrations of t o t a l free f a t t y acids analyzed were s i g n i -f i c a n t l y smaller (P = 0 . 0 5 ) in'the brine-frozen than i n the plate-frozen chinook salmon.' (Table XVII and Appendix, Table VII), With halibut the percentage of fatty acid 1 5 : 0 and the concentrations of free fatty acids 14 : 0 , 1 7 : 0 , 1 8 : 1 , 1 8 : 3 , and 2 0 : 2 were al].' s i g n i f i c a n t l y greater (P" - 0 , 0 5 ) i n brine-frozen than in plate-frozen f i s h (Tables XIV and XV and Appendix, Tables I and I I I ) . There were no s i g n i f i c a n t differences i n the percentages or concentrations of any i n d i v i d u a l free fatty acids between freezing methods for cohc salmon (Tables IX and XI, Appendix), Also there were no s i g n i f i c a n t differences between freezing methods i n the concentration of t o t a l free f a t t y acids analyzed for either coho salmon or halibut (Table 1 XIII, Appendix), Thus brine-freezing appeared to affe c t the formation of some Individual free fatty acid i n both chinook salmon and halibut but brine-freezing did not s i g n i f i c a n t l y a f f e c t the t o t a l free f a t t y acid formation i n either coho salmon' or halibut while it. did s i g n i f i c a n t l y decrease the concentration of t o t a l free fatty acid analyzed i n chinook salmon. The fact that there were many more s i g n i f i c a n t (P - 0 . differences between methods when free fatty acids were ex-pressed as JUL g per gram of neutral l i p i d rather than as per-cent of free fatty acids analyzed indicated that brine-freezing -121-a l t e r s the composition of the f r e e f a t t y acids formed by a very s l i g h t degree. The f a c t that b r i n e - f r e e z i n g s i g n i f i c a n t l y a f f e c t e d the t o t a l f r e e f a t t y a c i d c o n c e n t r a t i o n ( t o t a l of those analyzed) of chinook salmon but not of coho salmon or of h a l i b u t may be r e l a t e d to the higher l i p i d content i n chinook salmon. In g e n e r a l , where there was a s i g n i f i c a n t d i f f e r e n c e , the i n s i d e muscle contained more free f a t t y acids per gram of n e u t r a l l i p i d than the outside muscle. This may be the r e s u l t of the higher n e u t r a l l i p i d content of the outside muscle. The con c e n t r a t i o n of the t o t a l f r e e f a t t y acids of chinook salmon and the I n d i v i d u a l free f a t t y acids of chinook salmon and h a l i b u t that d i f f e r e d s i g n i f i c a n t l y (P - 0.05) among frozen storage times tended to increase r a t h e r r a p i d l y during the f i r s t 26 or 45 weeks of frozen storage and then increase ( i f at a l l ) at a much slower r a t e . Even wi t h the c o n c e n t r a t i o n of t o t a l f r e e f a t t y acids of h a l i b u t , although they d i d not d i f f e r s i g n i f i c a n t l y among frozen storage times, the greatest Increase occurred between 14 and 31 weeks. This trend ( i f present) was not very evident with the coho salmon. This r a p i d increase i n free f a t t y acids followed by a much slower increase i s somewhat s i m i l a r to the p a t t e r n observed i n P a c i f i c gray cod and A t l a n t i c cod (Wood and Haqq, 1962) except in. the present study the slower r a t e of Increase ( i f any) was not as uniform as that observed by Wood and Haqq (1962). -122-The r e s u l t s of the present study d i f f e r from those of Varesmea et a l . ( 1 9 6 9 ) who observed that with rainbow t r o u t s t o r e d f o r 8 months at - l 8 ° C or at - 3 2°C the percentage of f a t t y a c i d .18:1 decreased from 2 3 $ to approximately 0% while the percentage of f a t t y a c i d 1 8 : 0 i n c r e a s e d from 6% to approx-imate l y 24$. O l l e y et a l . ( 1 9 6 9 ) showed t h a t with haddock and lemon s o l e there was a p r e f e r e n t i a l h y d r o l y s i s of 1 6 : 0 , 1 8 : 1 , and 2 0 : 5 p h o s p h o l i p i d s . In the present study the con-c e n t r a t i o n of f a t t y a c i d s 1 6 : 0 , 1 8 : 1 , and 2 0 : 5 of P a c i f i c h a l i b u t , chinook salmon, and coho salmon d i d not s i g n i f i c a n t l y i n c r e a s e as time of f r o z e n storage progressed (Tables XIV, XVI, and XVIII and Appendix, Tables I, V, and I X ) . The s i g n i f i c a n t d i f f e r e n c e s i n t o t a l f r e e f a t t y a c i d c o n c e n t r a t i o n between b r i n e - f r o z e n and p l a t e - f r o z e n chinook salmon d i f f e r s from the f i n d i n g s of Peters et a l . ( 1 9 6 8 ) who observed no s i g n i f i c a n t d i f f e r e n c e i n t o t a l t i t r a t a b l e f r e e f a t t y a c i d s between b r i n e - f r o z e n and p l a t e - f r o z e n A t l a n t i c cod. The d i s c r e p a n c y between the two o b s e r v a t i o n s may be r e l a t e d to the much hi g h e r l i p i d content of chinook salmon (Stansby and O l c o t t , 1 9 6 3 ) . . The r e s u l t s suggest that l i p i d h y d r o l y s i s may occur at a g r e a t e r r a t e i n P a c i f i c h a l i b u t than i n A t l a n t i c h a l i b u t . In the- present study, although there were no s i g n i f i c a n t d i f f e r e n c e s among storage times and the c o n c e n t r a t i o n at 31 weeks was not s i g n i f i c a n t l y g r e a t e r than that at 14 weeks the c o n c e n t r a t i o n of t h e ' t o t a l long c h a i n f r e e f a t t y a c i d s i n -creased from 2 , 9 5 2 . 2 u g per gram of n e u t r a l l i p i d at 14 -123-weeks to 1 2 , 4 3 3 , 9 yx, g at 31 weeks of frozen storage at - 3 0°C whereas Dyer et_ a l . ( 1 9 5 8 ) observed no l i p i d hydrolysis i n A t l a n t i c halibut stored at - 1 0 ° ? for approximately 6 months. It should be emphasized that when the free fatty acids were expressed a u g free fatty acid per gram of neutral l i p i d i t was assumed that the increase i n free fatty acids arises from hydrolysis of phospholipids and not from hydroly-s i s of t r i g l y c e r i d e s . During the course of t h i s study E i l i n s k i and Lau ( 1 9 ^ 9 ) showed that rainbow trout possessed l i p o l y t i c a c t i v i t y towards long-chain t r i g l y c e r i d e s . Also Bosund and Ganrot ( '1969) showed that hydrolysis of long-chain t r i g l y c e r -ides occurs during the frozen storage of.herring, However, with other species, notably A t l a n t i c cod and related species ( l i n g , saithe and hake) several workers have shown that almost a l l the free fatty acids arise from hydrolysis of phospholipids (Olley et a l . , 1 9 6 2 ; Bligh, I 9 6 I ; and Bligh and Scott, 1 9 6 6 ) , It should also be emphasized that the free fatty acid c a l l e d 2 2 : 6 probably also contains (depending on the species) 5 to 10$ fatty acid 24:1 (Ackman, 1 9 6 6 ; and Gruger et a l . , 1 9 6 4 ) . -124-SUMMARY AND CONCLUSIONS Experiments' were conducted to determine the eff e c t of brine- and. plate-freezing at sea and the length of subsequent frozen storage upon f l e s h pH, thaw - d r i p , color, f l a v o r , TBA values, and various long chain free fatty acids of P a c i f i c halibut, chinook salmon, and coho salmon. The effect of the two freezing methods upon mineral (sodium, potassium, and chloride) concentration was also determined. Evaluations were conducted on outside and inside muscle, where appropriate. Method, of freezing had no s i g n i f i c a n t e f f e c t upon, the f l e s h pH of any of the 3 species. Flesh pK s i g n i f i c a n t l y (P ~ 0 . 0 5 ) decreased (the pattern of decrease varied with the species) as length of frozen storage increased but this decrease i n pH may have been the resu l t of the method of sampling. The mean thaw drip from P a c i f i c halibut and chinook salmon was less for the brine-frozen than for the plate-frozen samples after storage for 9 to 31 weeks whereas subsequently the brine-frozen samples had. approximately equal or greater thaw drip than the plate-frozen samples, The plate-frozen coho salmon had a s i g n i f i c a n t l y (P - 0 , 0 5 ) higher average thaw drip than the brine-frozen. During frozen storage the thaw drip of the plate-frozen halibut tended to decrease while that of the brine-frozen halibut tended to increase. In general, with both chinook and coho salmon, the thaw drip of both the brine-arid plate-frozen samples tended to s l i g h t l y increase. -125-In general, redness ('a'-value) and the a/b' r a t i o tended to Increase with length of frozen storage of both brine- and plate-frozen chinook and coho salmon. The differences i n flavor between brine- and plate-frozen samples of outside muscle of both halibut and chinook salmon reached a maximum, which depending upon the species was either highly (P ^  0 . 0 1 ) or very highly (P ^  0 . 0 0 1 ) s i g n i -f i c a n t at 26 or 31 weeks of frozen storage. The differences then steadily decreased. In contrast, the differences i n fla v o r between brine- and plate-frozen coho salmon outside muscle steadily increased. With the exception of the samples of coho salmon stored for 10 weeks there appeared to be l i t t l e difference i n fl a v o r of inside muscle of the salmon species frozen by the two d i f f e r e n t methods. Conversely, s i g n i f i c a n t (P ^ 0 . 0 5 ) differences i n flavor between brine- and plate-frozen samples of halibut inside muscle were detected at 3 1 , 6 2 , and 8 1 weeks of storage. In general, with a l l three species, during frozen storage the TBA values of both brine- and plate-frozen outside muscle increased to a maximum then decreased. With a l l three species, the differences i n TBA values between brine- and plate-frozen outside muscle samples rapidly increased and reached a maximum at 4 5 , 26 or 27 weeks then decreased u n t i l there was approximately no difference at 8 1 , 7 7 , and 78 weeks of frozen storage for h a l i b u t , chinook, and coho salmon, respe c t i v e l y . -126-Th e TBA values f o r the i n s i d e muscle of a l l three species were r e l a t i v e l y constant during frozen storage and there appeared to be l i t t l e d i f f e r e n c e between samples, frozen by the two d i f f e r e n t methods. When expressed as percent of t o t a l free f a t t y acids analyzed there was a s i g n i f i c a n t d i f f e r e n c e between f r e e z i n g methods f o r only free f a t t y acids 1 5 : 0 of h a l i b u t and 1 7 : 0 of chinook salmon. In both cases the percentage was greatest i n the b r i n e - f r o z e n samples. Although f o r each species there were a f a i r number of free f a t t y acids that d i f f e r e d s i g n i f i -c a n t l y (P ~ 0 . 0 5 ) among frozen storage times the changes were e r r a t i c and no meaningful trends could be discerned. When expressed a jig per gram of n e u t r a l l i p i d b r i n e - f r o z e n h a l i b u t contained s i g n i f i c a n t l y (P - 0 . 0 5 ) more fr e e f a t t y acids 14:0, 17:0, 1 8 : 1 , 1 8 : 3 , and 2 0 : 2 , and br i n e f r o z e n chinock salmon contained s i g n i f i c a n t l y (P - 0.05) l e s s 1 6 : 0 , 1 8 : 2 , and 2 2 : 6 than the corresponding p l a t e - f r o z e n samples. In g e n e r a l , w i t h a l l three s p e c i e s , although not always s i g n i f i c a n t , the concent r a t i o n of the i n d i v i d u a l f r e e f a t t y acids was greater i n the i n s i d e than i n the outside muscle. Also f o r h a l i b u t and chinook salmon, p a r t i c u l a r l y where there was a s i g n i f i c a n t d i f f e r e n c e among storage times, the c o n c e n t r a t i o n of the free f a t t y acids appeared to r a p i d l y increase during the f i r s t 26 to 31 weeks of frozen storage. -127-T o t a l f r e e f a t t y a c i d s a n a l y z e d per ,ug o f n e u t r a l l i p i d was s i g n i f i c a n t l y (P - 0 . 0 5 ) lower i n the b r i n e - f r o z e n t h a n i n the p l a t e - f r o z e n c h i n o o k salmon whereas t h e r e v/ere no s i g n i f i c a n t d i f f e r e n c e s between f r e e z i n g methods f o r h a l i b u t and coho salmon. The c o n c e n t r a t i o n o f t o t a l f r e e f a t t y a c i d s was h i g h l y s i g n i f i c a n t l y (P - 0 . 0 1 ) g r e a t e r i n the i n s i d e t h a n i n t h e o u t s i d e muscle f o r h a l i b u t and c h i n o o k salmon. W i t h coho salmon the . c o n c e n t r a t i o n o f t o t a l f r e e f a t t y a c i d s did. not d i f f e r s i g n i f i c a n t l y between l o c a t i o n s . The c o n c e n t r a t i o n o f t o t a l f r e e f a t t y a c i d s d i f f e r e d s i g n i f i c a n t l y (P - 0 . 0 5 ) among s t o r a g e t i m e s f o r o n l y c h i n o o k salmon. There was a l a r g e s i g n i f i c a n t (P ^ 0 . 0 5 ) i n c r e a s e between 9 and 26 weeks of s t o r a g e . A l t h o u g h not s i g n i f i c a n t t h e r e was a f a i r l y l a r g e i n c r e a s e i n the c o n c e n t r a t i o n o f t o t a l f r e e f a t t y a c i d s between 14 and 31 weeks of s t o r a g e f o r h a l i b u t . V/ith a l l t h r e e s p e c i e s the sodium and c h l o r i d e con-c e n t r a t i o n s o f the b r i n e - f r o z e n o u t s i d e muscle was two t o t h r e e -t i m e s g r e a t e r t h a n t h a t o f t h e b r i n e - f r o z e n i n s i d e , p l a t e -f r o z e n i n s i d e and p l a t e - f r o z e n o u t s i d e muscle samples. Method o f f r e e z i n g had a s n a i l and v a r i a b l e e f f e c t upon p o t a s s i u m c o n c e n t r a t i o n o f t h e f l e s h . There was a s i g n i f i c a n t (P - 0 , 0 5 ) n e g a t i v e c o r r e l a -t i o n ( r = - 0 . 3 5 8 ) between pH and thaw d r i p o f h a l i b u t , There -128-were a l s o h i g h l y s i g n i f i c a n t (P - 0 . 0 1 ) negative c o r r e l a t i o n s between pH and Hunter 'a' values (r = - 0 , 5 9 6 ) and pH and Hunter a/b r a t i o s ( r + -0.721) f o r coho salmon. With h a l i b u t f l a v o r was not s i g n i f i c a n t l y correlated, w i t h any of the other parameters measured. Only the Hunter b values ( r = - 0 . 5 2 ) , the TBA values ( r = +0.460) and free f a t t y a c i d 20:1 (expressed as percent of t o t a l f r e e f a t t y acids analyzed ( r = -0.410) c o r r e l a t e d s i g n i f i c a n t l y (P = 0.01) with f l a v o r of chinook salmon. V/ith coho salmon only the Hunter 'a' (r = + .0 .653) values and f r e e f a t t y a c i d 1 8 : 1 (expressed as percent of t o t a l free f a t t y acids analyzed) (r = + 0 . 5 1 5 ) c o r r e l a t e d s i g n i f i c a n t l y with f l a v o r . The e f f e c t of b r i n e - f r e e z i n g upon most v a r i a b l e s measured was e i t h e r small and/or complex. For a l l three species the sodium and c h l o r i d e c o n c e n t r a t i o n was two to three times greater i n the b r i n e - f r o z e n outside muscle than i n a l l other samples. When the concentration (jj.g per gram of n e u t r a l l i p i d ) of free f a t t y acids d i f f e r e d s i g n i f i c a n t l y (P ^ 0 . 0 5 ) among frozen storage times the f r e e f a t t y acids increased, most r a p i d l y during the f i r s t 26 to 31 weeks of storage. The t a s t e panel r e s u l t s and the TBA values i n d i c a t e that b r i n e - f r e e z i n g does Impair the q u a l i t y of the outside muscle of h a l i b u t and chinook salmon during the e a r l y stages of frozen storage i n comparison to p l a t e - f r e e z i n g . -129-LITERATURE CITED Ackman, R.G. ( 1 9 6 6 ) , Simplified Gas-Liquid Chromatography of Marine O i l Fatty Acid Esters on EGSP-Z Organosilicone Polyester. . J . Gas Chromatog. 4 : 2 5 6 - 2 5 9 . Anderson, M.L., M.A. Steinberg, and F.J. King. ( 1 9 6 5 ) . Some Physical Effects of Freezing Fish Muscle and t h e i r Relation to Protein-Fatty Acid Interaction i n Kreuzer, R. (ed.) 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Canada 2 2(4): 9 5 5 - 9 6 8 . Tomlinson, N., S.E. Geiger, and E. Ddllinger. ( 1 9 6 6 ) . Free Drip, Flesh pH, and Chalkiness i n Halibut. J . F i s h . Res. Bd. Canada 2 3 : 6 7 3 - 6 8 0 . Tomlinson, N,, S.E. Geiger, and S.W. Roach, ( 1 9 6 9) . Quality of Sea Frozen Fish from the North-eastern P a c i f i c Ocean, i n Kreuzer, R. (ed.), Freezing and I r r a d i a t i o n of F i s h , Fishing News (Books) Ltd., London, I T D T 7 ~ P P • 6 8 - 7 5 . Varesmaa, E,, J . J . Lane, and E.P. Niinivaara, ( 1 9 6 9 ) . Rainbow Trout (Salmo irideus) Produced i n Finland. VII. Changes i n the Organoleptic Quality and. Fatty Acid Composition During Frozen Storage. Maataloustieteelinen Aikakauskirja 4 l ( 3 ) : 1 6 0 - 1 6 4 . White Fish Authority ( 1 9 5 7 ) . Report on an Experiment into the Freezing of Fish at Sea, Tilbury House, Petty France, London, June, 1 9 5 7 , 82 pp. Yonker, W.V. ( 1 9 6 3 ) . The Salmon F i s h e r i e s , i n M.E. Stansby (ed.), I n d u s t r i a l Fishery Technology, Reinhold Publishing Co., New York, 1 9 6 3 . Young, O.C. ( 1 9 4 1 ) . Thawing of Fish I I . Progress Reports P a c i f i c Biolog. Station P a c i f i c Fisheries Experimental Station F i s h . Res. Bd. Canada 5 0 : 1 6 - 2 0 . Yurkowski, M. and H. Brockerhoff. ( 1 9 6 5 ) . Lysolecithinase of Cod Muscle. J, F i s h . Res. Bd. Canada 23:643-652. APPENDIX TABLE I Analyses of variance of free f a t t y acids (expressed as percent of the t o t a l free fatty acid analyzed) of P a c i f i c h a l i b u t 1 14:0 15:0 16:0 16:1 17:0 16:2 Source d.f. M.S. 3 M.S. M.S. M.S. M.S. M.S. Pairs 2 7.80 0.50 77. 07 4.55 2.19 0 . 38 Method 2 . 1 4.09 0.49* 77.41 145.03 1.27 1..2 7 E r r o r a 2 3.64 0.02 6.21 25 .47 2.11 0.53 Location 1 65.32 0.01 335.06 281.93* 6.53* 0.90 M x L 1 1. 39 0.05 41.66 0.58 3.97 1.21 Error b 4 0.37 0.20 231.86 33.28 0.57 0.25 Time 4 40.94 0.40 310.04* 10 8.84* 5.90 • 1.4 3* M x T 4 0.66 0.07 62.89 12.87 3.3 3* 0. 44 L x T 4 9.50* 0. 37 63.40 29 .24 2 .44 0.67 M x L x T 4 2.53 0.14 45 .44 24 .56 0.90 0 . 35 Error c 32 2.97 0.23 95.72 28.62 1.22 0 .29 18:0 18 :1 18:2 18:3 18:4 20 :1 Pairs , 2 Method 6 1 Error a 2 Location 1 M x L 1 Err o r b 4 Time 4 M x T 4 L x T 4 M x L x T 4 Err o r c 32 2.93 184.12 847.16 865.61 62 .13 396.77 358.40* 52.72 0 .68 30.40 40.77 13.49 30.51 36.15 20.41 9.07 14.52 21.91 6.45 62.51 519.89 37.97 1.40 0.03 0.15 0.40 0.95 0.48 8.79* 1.09 0.05. . 0.54* 0.86 0.05 3.10* 0.98 0. 81 0 .18 1.40 0.17 1.00 0. 39* 1.0 2 0.13 1.98 58.55 23. 64 372 .47 1.69 54.21 2 0.64** 60.69 4.11 0.50 0.56 1.14 3.14** 418.08 0.80 1.86 0.51 16. 77 0.69 3.00 0.75 6.45 TABLE I (Continued) 20:2 20:4 20:5 22:1 22:5 22:6 Pairs 2 0.24 36.20 218.05 9 . 52 4.98 192. 54 Metho.d 1 0.19 225.39 843.65 582.37 8.89 61.52 Err o r a 2 0.3.2 96.67 127.79 68.65 265.62 865.71 Location 1 0.00027 23.01 • 1.46 216.54** 2.36 676.40 M'x L 1 0.00027 37.25 15. 03 28.96 1.23 67. 85 Err o r b 4 0.17 32.95 57 .60 5.42 1.21 96.62 Time 4 ' 0.59*** 27.22 94. 81 5 0.54** 7 .17** 724 .28 T x M 4 0.069 28.99 8.40 18.13 0.80 43.63 T x L 4 0.088 24.02 161.01** 19.16 1.64 213 .04 T x M x L . 4 0.027 31.37 21.48 0.43 2 .24 236.95 Error c 32 0. 070 27. 74 29.43 11.04 1.80 96.76 Analyses of variance was conducted on arcsin transformed percentages. 2 Methods were test by error a. . Location and M x L were tested using error b. A l l other terms were tested by error c. 3 +5 Mean square values are a l l multiplied by 1.0 x 10 * S i g n i f i c a n t at the 5% l e v e l (P < 0.05) ** S i g n i f i c a n t at the 1% l e v e l (P £ 0.01) *** Si g n i f i c a n t ' at the 0.1% l e v e l (P * 0.001) TABLE I I . Method x location x time i n t e r a c t i o n means from the analyses of variance of the free f a t t y acids (expressed as percent of t o t a l free f a t t y acids analyzed) of P a c i f i c halibut . Free Length of Inside Muscle Outstide Muscle Fatty Frozen Brine Plate Brine Plate Acid Storage Frozen Frozen Frozen Froze] 1 4 : 0 14 weeks 1 . 9 8 2 1 .99 1 . 9 7 1 . 9 9 31 n 1 . 5 4 1 . 7 8 2 .66 2 . 5 2 45 II 1 . 9 1 2 . 2 7 2 . 4 1 2 .73 62 n 1 . 5 4 1 . 39 1 . 7 1 2 . 0 6 81 it 2 . 0 6 2 . 9 0 4 . 0 6 3 . 8 5 1 5 : 0 14 weeks 0 . 4 1 0. 34 0 .38 0 . 2 7 31 ti 0 . 3 1 0 . 2 1 0 . 4 6 0 . 4 3 45 it 0 .36 0 . 5 0 0 . 38 0 . 2 6 62 II 0 . 3 8 • 0 . 2 3 0. 34 0 . 2 6 81 ti 0 . 2 2 0 . 2 1 0 . 2 6 0 . 2 2 16 :0 14 weeks 1 8 . 2 7 1 5 . 1 9 1 6 . 0 9 1 4 . 4 3 31 ii 1 8 . 3 9 2 0 . 3 7 1 6 . 1 9 1 5 . 76 45 II 1 4 . 1 2 1 4 . 4 3 1 3 . 3 7 1 2 . 8 8 62 II 1 5 . 56 1 5 . 1 4 1 3 . 9 2 14 . 47 81 ti 1 5 . 5 4 1 5 . 75 1 7 . 8 2 1 4 . 0 2 1 6 : 1 14 weeks 4 . 6 7 5 . 9 0 5 . 4 7 6 . 7 0 31 ti 4 . 7 3 5 . 0 3 3 . 5 2 6. 37 45 II 5 . 4 7 5 . 9 4 7 . 3 0 6 . 9 1 62 ti 3 . 6 1 3 . 6 7 . 5 . 7 0 6. 81 81 tt 4 . 88 7 . 3 9 7 . 8 3 8 . 2 2 16 :2 14 weeks 0. 29 0 . 2 8 0 . 3 3 0 . 1 7 31 II 0 . 3 7 0 . 5 3 0 . 2 2 • 0 . 4 5 45 II 0 . 5 1 0. 34 0 .44 0 . 6 7 62 II 0 . 1 6 0 . 1 3 0 .16 0 . 4 0 81 » 0 .14 0 . 2 0 , 0 . 2 6 0 . 6 2 1 7 : 0 14 weeks 0 . 9 4 0 . 8 3 1 . 0 5 1 . 0 1 31 tt 1 . 29 1 . 3 8 0 . 9 9 1 . 3 7 45 it 1 . 6 0 1 . 1 5 1 . 5 7 1 . 35 62 ti 1 . 1 3 0 . 75 1 . 0 5 1 . 5 9 81 tt 0 . 9 7 1 . 4 7 1 . 5 0 2 . 1 1 1 8 : 0 14 weeks 8 . 9 1 5 .62 6 . 5 1 4 . 2 5 31 it 8 . 0 1 5 . 5 8 6 . 6 2 4 . 5 8 45 tt 6. 64 6 . 0 6 6 . 5 9 5 . 1 9 62 it 8 . 6 5 6 . 5 2 6 . 9 0 3 . 9 2 81 it 7 .90 4 . 39 5 . 6 6 2 . 9 4 TABLE II (Continued) Free Length of Inside Muscle Outside Muscle Fatty Frozen Brine Plate• Brine Plate Acid Storage Frozen Frozen Frozen Frozen 18:1 14 weeks 17.23 14. 71 17.75 15.16 31 ti 17.11 14 .99 16. 31 15.31 45 II 15.78 16.01 19.14 14. 38 62 n 18.68 15.56 17.49 16.20 81 IT 17.07 15.23 19.54 16.10 18:2 14 weeks 1.19 1. 02 1.09 0.92 31 it 1.46 1.19 1.42 1.75 45 it 1.03 1.33 1.42 1.40 62 II 1.26 1.26 1.54 1.70 81 it 1.14 1.03 1.73 1. 30 18: 3 14 weeks . 0.36 0.44 . 0. 34 0.52 31 it 0.33 0.22 0. 35 0. 35 45 ti 0.46 . 0.55 0.59 0.52 62 it 0.51 0.32 0.49 0.78 81 it 0. 34 0.43 0. 37 0.53 18:4 14 weeks 0. 09 0. 37 0.23 0.66 31 tt 0.18 0.22 0.26 0.92 4 5 II 0.41 0. 54 0.62 0.90 62 ti 0.23 0.28 0.43 1.19 81 tt 0.21 0.85 0.60 1.2 8 20:1 14 weeks 4.17 6. 01 3.96 5.45 31 4.23 5.56 4.94 6.47 45 ti 5.91 6.49 6.24 7.55 62 ti 5.04 7.41 5.85 7.27 81 ti 7.41 7.27 7.08 8.82 2 0:2 14 weeks 0.16 0.11 0.06 0.07 31 ti 0.29 0.31 0.25 0.28 45 ti 0.27 0.20 0.29 0.23 62 tt 0.21 0.19 0.20 0.21 81 ti 0.17 0.12 0.29 0.14 20:4 14 weeks 1.72 1.21 2.01 1.49 31 it 6. 32 1.11 2.11 1.47 45 ti 2.29 1.68 2.23 1.54 62 ti 2.50 1.54 2.73 1.54 81 tt 2.41 1.10 1.73 1.13 TABLE II (Continued) Free Length of Inside Muscle Outside Muscle Fatty Frozen Brine Plate Brine Plate Acid Storage Frozen Frozen Frozen Frozen 2 0 : 5 m weeks 1 1 . 1 1 1 4 . 0 6 1 3 . 1 7 1 5 . 7 4 31 ti 9 . 7 5 1 2 . 3 7 1 1 . 7 9 1 3 . 5 9 45 it 1 2 . 2 3 1 4 . 03 1 1 . 1 2 1 4 . 4 0 62 n 1 2 . 7 0 1 5 . 1 1 1 3 . 1 5 1 5 . 0 1 81 II 1 4 . 2 0 1 4 . 1 7 8 .94 1 2 . 0 5 2 2 : 1 14 weeks 0 . 9 6 2 . 33 1 . 0 0 3 . 3 0 31 it 0 . 9 8 2 . 5 2 1 . 7 5 4 . 3 3 45 .it 1 . 72 2 . 4 0 2 . 34 4 . 1 1 62 II 1 . 4 6 2 . 5 3 1 . 5 9 3 . 5 1 81 ti 0 . 8 8 3 . 8 7 3 . 1 2 6 . 5 8 22: 5 14 weeks 1 . 3 3 1 . 4 9 1 . 8 5 1 . 0 7 31 II 1. 36 1 . 37 1 . 8 7 1 . 5 0 45 II 2 . 0 3 1 . 8 4 1 . 9 5 1 . 4 7 62 n 2 . 14 1 . 5 6 2 . 3 1 2 . 0 8 81 II 1 . 8 8 1 . 7 1 1 . 8 3 2 . 0 2 22 :6 14 weeks 2 3 . 9 4 2 5 . 8 4 2 4 . 6 2 2 4 . 6 4 31 II 2 1 . 8 3 2 3 . 0 9 2 5 . 89 2 0 . 89 45 II 2 5 . 1 4 2 2 . 4 8 20 . 35 2 1 . 9 6 62 II 2 2 . 2 3 2 4 . 2 5 2 2 . 5 9 19 .45 81 n 2 2 . 6 4 2 0 . 2 3 1 5 . 7 5 1 6 . 7 7 The analyses were conducted on the arcsine transformed percentages but the actual percentages (not transformed) are recorded i n TABLE I I . Each value i s the average of 3 observations. TABLE I I I Analyses of variance of free f a t t y acids (expressed as tt,g free f a t t y acid per gram of neutral l i p i d ) of P a c i f i c halibut 14:0 15:0 16:0 16:1 16:2 17:0 Source d.f. M.S.1 M.S. M.S. M.S. M.S. M.S. Pairs 2 2 12.54 0.98 2203.00* 16 2 .5* 1.37 7.31* Method 1 19.12* 0.56 2756.00 14 3.52 0. 86 10.19* Error a 2 0.61 0.04 299 . 28 17.56 0.13 0.15 Location 2_ 63.38** 3. 04 7 714.0 0* 5 58.88* 2.92 32.6 6* M x L 1 5.65 0,20 1663.20 71.75 0.87 6.52 Err o r b 4 1.96 0.23 514.28 30.74 0.40 1.06 Time 4 10.79* 0.46 773.06 77.54 0.86 4.23 M x T 4 3.02 0.25 409 .26 33.15 0.33 1.05 L x T 4 5.13 0.30 689.17 53.00 0 .70 3 .11 M x L x T 4 2.33 0.23 404.04 38 .90 0 .36 1. 35 Error c 32 3.85 0.26 628.72 45 .13 0.50 2.16 18:0 18 :1 18 : 2 18:3 18 :4 20:1 Pairs 2 203.07* 1247.60* 5.4 3** 0.8 8* 0 .12949 79.4 3* Method X 620.26 2777.30* 10.02 0.94* 0.00014 97.46 Error a 2 27.83 121.54 0.90 . 0.0 4 0.02179 30.06 Location 1 1394.40 6479.80** 2 7.61** 3.47** 0.64 8 3 3*** 604.90* M x L 1 35 5.31* 1319.50 3.51 0.39 0.00006 46.30 Error b 4 37.76 186.11 1.08 0.12 0.00280 21.45 Time 4 99.47 539.84 2. 59* 0. 37 0.27891*** 76.35* M x T 4 5 2.66 294.60 1.55 0.21 0.05578 15. 34 L x T 4 74. 01 384.27 1.75 0 . 24 0.06924 35.09 M x L x T 4 47.98 30 6.6 3 1.33 0.25 0.08484 11.19 Error c 32 62.05 303.20 0.93 0.20 0 .04440 20.31 TABLE III (Continued) 2 0:2 20:4 2 0:5 22 :1 22:5 22 :6 Pairs 2 0.411 16.65 1126.60** 8.964** 11.48 2396.10 Method 1 0 .48 3* 82.57 960.18 0 .009 30 .44 4614.60 Err o r a 2 0. 007 1.16 160 .09' 3.601 0.21 481.04 Location 1 1.111* 133.72 4309.00* 25.141** 73.57 15429.00** M x L 1 0. 264 48.28* 581.08 0.048 15.8 5* 2788.40 Err o r b 4 • 0.104 3.09 223.02 0.726 1.11 500.91 Time 4 0.230 13.08 363.73 5.957* 7.25 1298.70 M x T 4 0.113 10.37 162.53 1.561 1.87 397.36 L x T 4 0.163 12.29 288.89 1.734 . 5.22 1035.00 M x L x T 4 0 .116 10.96 138.36 1.778 1.83 394.46 Error c 32 0.178 5.05 206 .95 1.530 4.53 817.26 1 A l l mean square values are mul t i p l i e d by 1.0 x 10~ 6 • Methods were tested by error a. Location and M x L were tested using error b. A l l other terms were tested by error c. S i g n i f i c a n t at the 5% l e v e l (P * 0.05) S i g n i f i c a n t at the 1% l e v e l (P ^ 0.01) S i g n i f i c a n t at the 0.1% l e v e l (P 0.001) TABLE IV Method x location x time i n t e r a c t i o n means from the analyses of variance of the free f a t t y acids (expressed as iig free f a t t y acid per gram of neutral l i p i d ) of P a c i f i c halibut Free Length of Inside Muscle Outside Muscle' Fatty Frozen Brine Plate Brine Plate Acid Storage Frozen Frozen Frozen Frozen 14:0 14 weeks 1128.4 1 368 .2 6 8 8.8 3 9.4 31 it 5903.7 14 74.8 786. 9 396 . 8 45 it 4215.7 4671.0 874.5 89 0 .1 62 tt 2732.6 760. 7 549.9 307. 4 81 »t 4326.0 2819,2 2161 .4 792,2 15:0 14 weeks 237.4 66. 2 162.4 13.6 . 31 II 1132.2 166.6 129.9 48.9 45 it 817.6 1377,8 18 3. 7 161.8 62 1! 666.5 129.4 112.3 39.4 81 it 677.8 24 3. 8 109.3 50.1 15:0 14 weeks 10515.6 3221.1 6513.8 739 .3 31 ti 79519 .9 1656 3.6 4901.5 2368.5 45 tt 33961.1 27829.4 6189.7 7033.2 62 it 28666.6 8285.5 4018.0 215 2.5 81 it 43944.3 20383.9 9 04 7.5 3252 . 7 16 :1 14 weeks 2661.7 991,2 1560. 0 330.1 31 ti 20494.9 4272.3 1058 ,1 1080,5 45 tt 9872.9 12048.4 2927.6 2 344.7 62 it 6555.7 1945.6 1662 .7 1050.8 81 tt 12971.6 6898.1 390 3.4 176 4.6 16 :2 14 weeks 196. 3 53.7 114.5 8.7 31 t! 2122.3 437.6 66.8 101.1 45 II 928.8 822 .6 175.1 251.2 62 1! 294.3 66. 3 52.8 56.3 81 II 404.9 167.3 , 126.1 122.1 17:0 14 weeks 572.4 168 .2 353 .5 51.6 31 ti 4841.9 1269 . 5 274.8 245.7 45 it 3025.8 2361.3 600 .9 558,5 62 tt 2026.7 426.1 329 .1 226 .4 81 tt 25 4 3.7 1368.9 778 .6 429 .7 18 :0 14 weeks 4705.8 1231.0 2705.1 216.6 31 tt 26261.2 4353.4 2001.7 746.6 45 tt 17485.6 144 38.8 3193.5 3085.1 62 tt 15887.5 3728.7 1854.4 559 .4 81 tt 21239.3 5306.0 3247. 3 611.2 TABLE IV (Continued) Free Length of Inside Muscle Outside Muscle Fatty Frozen Brine Plate Brine Plate Acid Storage Frozen Frozen Frozen Frozen 18:1 . 14 weeks 9917.2 2589.4 6181.. 8 756. 2 31 it 60580.9 10543.3 4913.0 2286 . 3 45 II 34699.3 34752.9 8820.8 5744.7 62 II 34814.0 8471.5 5319.3 - 2408.0 81 ti 46622.3 15338.8 10582.8 34 7 3.7 18:2 14 weeks 682 .9 2 09 .9 504.4 46.3 31 ti 3708.4 923.1 4 3 3.4 261.4 45 II 22 3 3.0 2877.6 567.3 421.3 62 II ^ 2347 .9 685.6 553 .9 239 .4 81 II 3152. 7 1015.2 954. 2 287.1 18: 3 14 weeks 200.3 73.1 164. 8 25. 7 31 it 1275.9 175.4 100.6 61.6 45 II 846.5 1162.2 256.1 164.1 62 ti 946.5 189.2 188.4 112.1 81 it 846. 6 462 .6 199. 0 98.8 18 :4 14 weeks 40. 6 44.2 52.4 32.6 31 tt 527 .3 193.6 77.9 139.6 45 it 390.4 762 .0 213.2 173. 3 62 ti 405.2 152.4 162.9 171.1 81 ti ' 455.9 692 .4 283.5 278.6 20:1 14 weeks 2237.5 1079.1 1236.6 272 .5 31 tt 14121.2 4320.5 1505.9 943.8 45 it 12216.5 12826.8 2670.7 2946.2 62 II 9278.1 4198.2 1757.8 1034.8 81 tt 13691.3 7590.9 3838.4 1851.1 20:2 14 weeks 108. 6 2 3.0 18.9 3. 3 31 tt 1191.0 209.0 70.9 36.1 45 tt 549 .2 543.6 137.6 92.0 62 it 375 .1 122.5 53.4 28.7 81 ti 397.5 163.0 142.0 29. 3 20:4 14 weeks 1089.2 219.5 804.3 74. 7 31 II 11207.2 944.4 612.8 222 .9 45 tt 5224.5 3890.8 983.6 735 .5 62 ti 4390.5 830 .2 961. 8 216.7 81 tr 6236.5 1562.2 886 .7 238.5 TABLE IV (Continued) Free Length of Inside Muscle Outside Muscle Fatty Frozen Brine Plate Brine Plate Acid Storage Frozen Frozen Frozen Frozen 20 :5 14 weeks 6735.7 2534.2 4926.6 793. 0 31 IT 42409.6 10155.9 3604 .7 2258.2 45 II 26434. 3 29085.8 5017.2 6734.7 62 II 23352.8 8771.1 • 5028.9 2185.0 81 40346.8 17606.1 4834 .9 2557.9 22:1 14 weeks 477.4 355 . 3 283 .9 170.4 31 II 3455.8 2000.9 540.3 599 .5 45 i i 1829 .9 3659 . 7 943.1 1134.6 62 i i 2680.9 1381.-6 553.8 524.0 81 it 1981.6 3188.3 1914.6 1400.8 22:5 14 weeks 851.7 269 .1 694.1 55.4 31 II 5252.6 1113.6 543.5 236.7 45 II 4913.2 3832.8 726.4 6 51.0 6 2 II 3718.9 824.1 663.0 307.5 81 II 5133.8 1567.9 1030.0 423.9 22:6 14 weeks 14342.0 5844.1 10092.0 1299 .6 31 it .88118.4 20767.9 8051. 7 3536.9 45 II 65744.5 48089 .6 8737.8 11949.2 62 II 41765. 0 14384. 6 8140.7 2971.7 81 II 614.37. 8 26452.8 7855.4 3593.1 Each mean i s the average of 3 observations. TABLE V Analyses o f v a r i a n c e of f r e e f a t t y a c i d s (expressed as p e r c e n t o f the t o t a l f r e e f a t t y a c i d s analyzed) of Chinook Salmon-*-. 14:0 15:0 16:0 16:1 16:2 17:0 Source d.f. 2 M.S. M.S. M.S. . M.S. M.S. M.S. P a i r s 3 2 2.39 0.26865*. 7 3.91 23.58 0.46 2.67 Method 1 36.13 0.04507 ' 64.36 128.30 0.28 2.2 8* E r r o r a 2 74.31 0.00522 166.03 14 .54 0.13 0.03 L o c a t i o n 1 3.16 0.00007 9.95 54.81 " 0.72 4.17 M x L 1 10.24 0.00427 9.12 5.86 1.06 0.38 E r r o r b 4 0.74 0.01277 90.32 18.96 0. 54 3.00 Time 4 12.31 0.69 9 65*** 1332.90 33. 30 3.65 6. 40* M x T 4 3.68 0.06573 26. 30 16. 86 0.17 0.65 L x T 4 4.10 0.07073 143.45 2.21 0. 01 0.28 M x L x T 4 0.71 0.03168 179.5 0* "2.9.8 0.78* 2.91' E r r o r c 32 6.49 0.07954 58. 34 12.92 0.27 1.27 18:0 18 :1 18 :2 18:3 18:4 20:1 P a i r s 2 11.20 292.34- 0.55 0 .069 5. 24 85 . 230 Method 1 52. 58 70 .45 1.18 0 .232 0.87 9.463 E r r o r a 2 ' 11.61 • 543.81 2.92 2.695 18 .06 75.094 L o c a t i o n 1 2. 87 12.17 8.46 ' 1.110* 3.04 1.147 M x L 1 4.60 9.02 0.12 0.003 1.94 0.006 E r r o r b 4 1.21 47.50 5.25 . 0.070 1.18 12. 52 3 Time 4 16.55* 275.47** 52.29*** 3.352** 15.8 7*** 6 3.17 5* M x T 4 9.64 37.62 5.23 0.623 1.49 3.324 L x T 4 6.15 19 .33 1.70 0.904 0.63 • 2. 019 M x L x T 4 3.08 67.47 1.15 0.190 1.43 4.149 E r r o r c 32 5.70 69.60 2.53 0.736 2 . 38 4.667 TABLE V (Continued) 20:2 20 :4 20:5 22:1 22:5 22:6 Pairs 2 2.3 3* 4.1924** 109 .54 55.92 2.86 12 3.9 4* Method 1 0.11 0.3174 106.20 5.23 25.46 1090.90 Error a 2 0. 05 0.5202 192.63 71.55 29.25 128.92 Location 1 0 .60 0.6017 2 .49 5.08 . 4.24 25.48 M x L 1 0. 04 0.0006 0.09 0.21 3.03 1.38 Error b 4 0.29 0.3079 13.16 3.35 5.94 9.77 Time 4 5.27*** 1.3597 80.99 15.51 5.67 9 0.21* M x T 4 0.26 0.7080 8.95 8.47 4.27 19 .96 L x T 4 0.17 0.4411 24.67 26.97** 4.66 65.52 M x L x T 4 0.48 0.4775 7.42 7.88 2.53 2.82 Error c 32 0..50 0.5207 38.44 5.85 4.04 33.55 Analyses of variance was conducted on the arcsin transformed percentages. 2 +5 A l l mean square values are multiplied by 1.0 x 10 . 3 Methods were tested by error a. Location and M x L were tested using error b. A l l other terms were tested using error c. * S i g n i f i c a n t at the 5% l e v e l (P ^ 0.05). ** S i g n i f i c a n t at the 1% l e v e l (P ^ 0.01). *** S i g n i f i c a n t at the 0.1% l e v e l (P ^ 0.001). TABLE VI Method x loc a t i o n x time i n t e r a c t i o n means from the analyses of variance of the free f a t t y acids (expressed as percent of t o t a l free f a t t y acids analyzed) of chinook salmon^ Free . Length of Inside Muscle Outside Muscle Fatty Frozen Brine Plate Brine Plate Acid Storage Frozen Frozen Frozen Frozen 14 :0 9 weeks 4 . 7 0 2 4 . 5 8 4 . 8 1 3 .64 26 II 4 . 6 3 4 . 1 8 5 . 2 0 4 . 3 5 40 II 5 . 4 2 5 . 06 5 . 4 6 4 . 85 58 II 5 . 39 4 . 8 3 5 . 0 2 4 . 0 5 77 II 4 . 8 9 5 . 2 6 5 . 1 3 4 . 9 9 1 5 : 0 9 weeks 0. 31 0. 38 0 . 2 7 0 . 37 26 n 0 . 1 7 0 . 1 9 0 . 2 3 0 . 1 8 40 II 0 . 3 0 0 . 3 3 0 . 3 8 0 . 38 58 II 0. 30 0. 30 0 . 2 8 0 . 2 1 77 ii 0 . 1 9 0 . 1 9 0 . 1 4 0 . 2 2 16 :0 9 weeks 2 1 . 39 1 7 . 1 4 19 . 62 2 3 . 35 26 ii 2 0 . 4 7 2 0 . 1 4 1 8 . 4 5 1 6 . 2 8 40 ii 1 3 . 9 3 1 4 . 8 0 1 3 . 27 12 . 87 58 II 1 4 . 6 6 1 4 . 2 3 1 5 . 4 9 1 4 . 1 6 77 ti 1 4 . 0 9 1 4 . 38 1 5 . 6 9 1 3 , 6 4 1 6 : 1 9 weeks 8 . 3 3 8 .33 7 . 8 7 7 . 0 3 26 II 9 . 1 0 8 . 2 6 8 . 6 7 8 . 0 3 40 II 9 . 9 3 8 . 7 8 9 . 49 8 . 6 1 58 ti 9 . 9 6 8 . 4 3 9 . 7 2 7 . 0 7 77 ii 9 . 34 8 . 7 0 8 . 9 3 8 . 4 7 16 :2 9 weeks 0. 30 0. 31 0 . 3 8 --• 0 . 3 2 26 II 0 . 7 3 0 . 5 1 0 . 4 6 0 . 9 6 40 n 0 .62 0 . 6 1 0 . 6 0 0 . 7 5 58 II 0 . 2 3 .0 .33 0 .32 0 . 38 77 II 0 . 38 0 . 3 0 0 . 4 3 0 . 4 3 17 :0 9 weeks 0 . 5 9 0 .81 1 .02 0 . 7 1 26 II 1 . 4 5 0 . 8 1 0 . 9 9 1 . 35 40 ii 1 . 4 9 1 . 0 1 1 .52 1 . 4 5 58 it 1 .04 0 . 7 9 1 . 1 5 0 . 9 0 77 II 0 . 9 7 1 . 2 4 1 . 4 4 1 . 3 3 18 :0 9 weeks 3 . 2 6 4 . 2 1 3 . 2 8 4 . 4 5 26 II 2 . 4 5 3 . 2 3 2 . 6 5 3 . 6 1 40 it 3 . 2 9 3 . 8 9 3 . 7 7 4 . 6 2 58 ii 3 . 60 4 . 4 4 - 3 . 1 4 3 . 6 7 77 it 3 . 8 0 2 . 7 0 3 . 37 3 . 69 TABLE VI (Continued) Free Length of Inside Muscle Outside Muscle Fatty Frozen Brine Plate Brine Plate Acid Storage Frozen Frozen Frozen Frozen 18 :1 9 weeks 23.73 23.89 24.25 21.98 26 II 20.87 22.40 23.09 20.98 40 ii 24.52 24 .88 24.66 25.30 58 ti 25.27 23.83 25.35 25.95 77 tt 25.28 22 .50 25.28 23.38 18 :2 9 weeks 1.17 1.46 1.45 1.33 26 tt 3. 38 2. 32 3. 65 3.10 40 tt 1.76 1.84 2. 09 2.18 58 tt 1.90 1.94 1.93 1.76 77 n 2.22 2.2 8 2.35 2. 80 18 : 3 9 weeks 0.98 0.99 . 1.06 1.02 26 it 1.21 1.45 0.98 1.29 40 it 1.26 1.08 1.42 1.41 58 tt 1. 36 1. 32 1.50 1.48 77 ti 0.87 1.06 1.17 1.11 18 :4 9 weeks 1.08 1.01 1. 22 0.98 26 ti 1.61 1.61 1. 70 1. 57 40 tt 1.89 1.4 6 2.11 1. 88 58 it 1. 87 1. 79 2.19 2 .04 77 ti 1.49 2. 26 2.01 1.80 20 :1 9 weeks 3.42 4.00 3.76 3.56 26 tt 3.42 3.55 3.83 3.92 40 it 4. 69 4.98 5.16 4. 74 58 ti 5.48 5.32 4.83 5.42 77 it 4/32 4.78 4.21 5. 37 20 : 2 9 weeks 0.47 0.07 0.10 0.14 26 tt 0.67 0.73 0.63 0 .53 40 tt 0.24 0.18 0.13 0.14 58 tt 0 .11 0.23 0.23 0.16 77 tt 0.11 0.18 0.10 .0.18 20 :4 9 weeks 0.77 0.52 0.78 0.47 26 tt 0.48 0.37 0.53 0.75 40 it 0.46 0. 31 0. 37 0. 39 58 tt 0. 29 0.37 0.61 0.39 77 it 0.33 0.53 0. 36 0.41 TABLE VI (Continued) Free Fatty Acid Length of Frozen Storage Inside Muscle Outside Muscle Brine Frozen Plate Frozen Brine Frozen Plate Frozen 20 :5 9 weeks 14.14 14.02 1398 12.69 26 it 12.09 11. 37 12.57 12. 73 40 ii 14. 30 13.07 14.29 13.19 58 II 14.14 14.40 13.42 12.56 77 ti 15 .03 .13.21 14.99 13.98 22 :1 9 weeks 2. 51 2.51 3. 34 2.27 26 it 3.29 2.08 4.16 4 .24 . 40 ii 3.21 2.48 3.17 2.84 58 II 3. 89 3.60 2. 39 2.77 77 it 1.96 3.06 2.53 2.73 22 :5 9 weeks 1.83 2.55 2.37 2.36 26 II 1.99 2.17 1.76 2. 32 40 it 2.56 2. 32 2.20 2.60 58 ti 1.75 2.49 2.24 3. 69 77 ti 2.49 2.80 2.18 2.92 22 :6 9 weeks 8.71 11.42 8.28 11.08 26 it 9.83 12.65 8.58 12 . 30 40 ii 9. 51 10.97 8.05 9.94 58 ti 5.76 9.60 8.19 11.28 77 ii 10.44 12.47 8.74 "10.83 The analyses were conducted on the arcsine transformed percentages but the actual percentages (not transformed) are recorded i n TABLE VI. Each value i s the average of 3 observations. TABLE VII Analyses of variance of free f a t t y acids (expressed as jxg free f a t t y acid per gram of neutral l i p i d ) of Chinook salmon. 14:0 15:0 16:0 16:1 16:2 17:0 Source d.f. M. S.x M.S. M. S. M. S. M. ,S. M. S. Pairs 0 2 33. 86* 0 .045 408. 40** 110. 50** 0. ,8 3* 3. 8 3* Method T Jt. 7. 87 0 .105 178. 72 20. 49 0. .16 0. 0008 Error a 2 ' 14. 32 0 .021 4. 00 • 1. 99 0. ,14 0. 32 Location 1 336. IS 0 ,9 30** 392 5. 20 1215. 80 2. . 85 12 . 54 M x L I 31. 3 3* 0 .107 283. 44 90 . 73 0. , 006 . 0. 33 Error b 4 X . 94 0 .014 106 . 86 13. 19 0. , 54 1. 44 Time 4 85. 01 0 .237*** 411. 48 255. 43 1, .60 6. 38 M x T 4 12. 08 0 .041 73. 31 38. 40 0. .40 1. 08 L x T U 7. 33 • 0 .009 273. 12* 32 . 70 0. .52 1. 10 M x L x T Li 20. 10* 0 .011 171. 04 61. 2 2* 0. .30 2. 84* Error c 32 6. 72 0 . 037 69. 00 20. 09 0. ,18 0. 98 18 :0 18:1 18 :2 18 :3 18:4 20 :1 Pairs 2 12 .92 809.55* 3.21* 1.69 8 * 61 * * 66.26* Method "i 36.21 557.07 1.7 8** 0.8 8 0.79 38.09 Error a 2 8.32 126.58 0.01 • 0.44 1.96 26.37 Location 1 14 3.9 7* * 78 33.20** 5 9.2 2** 15. 5 3* 34. 32 249.11* M x L 1 15.56 315.31 1.35 1.26 4.21 22.93 Error b 4 3.48 197.07 2.09 0.76 1. 35 3.53 Time 4 33.52* **1787.50*** 21.7 3*** 4.19*** 14 .1-3 86.43* M x T • 4 3.01 136.20 3.06 0.33 2.45 10.07 L x T 4 2.49 259 .96 5.63 1.09 1.62 5.22 M x L x T 4 2.02 237.21 5.45 1.00 6.2 2** 12.27 Error c 32 4.99 164.01 2.27 0.53 1.48 5 .76 TABLE VII (Continued) 20:2 20:4 20:5 22:1 22:5 22:5 Pairs 2 0.11 • 0. 86** 130.41* 11.0 2*** 2 7 6.10* Method 1 0.07 0.09 119.55 1 4.20 26.38 661.9 2* Error a 2 0.12 0 .17 41.95 17. 81 4.65 25. 54 Location 1 1.82 2.2 2* 2568.00** 112.67 7 2.2 6** 1733.20 M x L 1 0.10 0.21 200.37 2.71 7.04 • 283. 04 Error b 4 0.06 0.18 52.69 4. 81 . 1.91 37. 94 Time 4 0.87 0. 31 536. 58**' * 2 2.03 19.56*** 314.11 M x T 4 0. 02 0.15 26.04 8.02 1.12 19.01 L x T 4 0. 54** 0.18 44.52 2.33 3. 32 91.46* M . x L x T 4 0.02 0.22 96.87 13.19** 2.91 33.64 Error a 32 0.11 0.13 38.00 2.49 1.48 28. 75 A l l mean square values are mul t i p l i e d by 1.0 x 10 Methods were tested by error a. Location and M x L were tested using error b. A l l other terms were tested by error c. Si g n i f i c a n t at the 5% l e v e l . S i g n i f i c a n t at the 1% l e v e l . S i g n i f i c a n t at the 0.1% l e v e l . TABLE VIII Method x location x time i n t e r a c t i o n means from the analyses of variance of the free f a t t y acids (expressed as jug free f a t t y acid per gram of neutral l i p i d ) of chinook salmon. Free Length of Inside Muscle Outside Muscle Fatty Frozen Brine Plate Brine Plate Acid Storage Frozen Frozen Frozen Frozen 14:0 9 weeks 275.0 1 5-31.6"' 146.0 137.5 26 n 920 .9 781. 7 2 2 7,1 302.7 4 0 ?! 110 2.4 12 70.4 588 .8 53C.4 5 8 II 906.0 758.6 560.9 385.5 77 tl 728.5 1681.3 771.4 -571.9 15:0 9 weeks 18.3 44.7 7.9 13.6 26 it 37.2 37. 8 9.1 1 1 . 8 40 II 59. 6 87. 2 38.7 40.8 58 ti 50.9 48.2 34. 7 19 .4 77 it 27.6 59.8 20,9 25.2 16:0 9 weeks 1200.6 2135.0 613. 6 958.7 26 tt 4380.9 40 71.8 800. 2 1269.2 40 it 2659.7 38 81.9 1454.2 1452.6 58 ti 2690.0 2333.6 1779.7 1343.1 77 ti 2053.9 4462.3 2422.8 1599 . 3 16 :1 9 weeks .• 465.9 1010.2 2 33 ,7 299 .4 26 1! 1820.7 1561,7 348. 2 563 .5 40 t! 1977.4 2347.7 1019.7 955.0 58 tt 1713.4 1343.8 1090.2 656,9 77 II 1374.3 2902,4 1388.1 959 ,8 16:2 9 weeks 14.3 40.3 1 1 . 6 12.9 26 ti 175.2 117,2 20.5 68,5 40 n 124.9 164. 5 6 3.5 78. 3 58 it 38.6 53.1 41.2 35.3 77 t! 56.5 95.6 65.0 47,9 1 7 :0 9 weeks 36.9 9 6.7 3 1 . 2 30.2 26 ti 324.9 180.2 43.2 100.9 40 it 300.9 256.9 159. 3 156.1 58 it 176.9 124, 3 137,4 84.6 77 !l 139.8 396 .6 225 .4 151.5 18 :0 9 weeks 159.7 505.4 96.7 185.0 26 ii 502 . 6 629 .4 104,6 248. 6 40 tt 627.8 1085. 3 387 . 3 517.9 58 tt 608.7 703.1 *3 3 3 9 5 342 .9 77 it 575 .3 837.1 512.4 407 .4 TABLE VIII (Continued) Free Length of Inside Muscle Outside Muscle Fatty Frozen Brine Plate Brine Plate Acid Storage Frozen Frozen Frozen Frozen 18:1 9 weeks 1235.6 3081.7 755. 0 937 . 3 26 JI 4445.7 4785.6 986.4 1562.7 40 it 5012.3 6958.1 2757.7 2932.0 58 tt 4524.0 4012 .9 3041.4 2546.9 77 tt 3961.5 7101.5 3898.9 2848.4 18:2 9 weeks 66.3 185 . 3 43.4 52.9 26 ti 747.6 466, 6 139 .7 213.1 40 tt 346. 0 486.3 216.4 237 .4 58 it 317. 5 307.9 211. 3 169.1 77 it 335.7 690.0 359. 5 319.3 18 :3 9 weeks 46.7 123.2 32.0 42.1 26 it 272 .6 254.4 38.6 92.4 40 it 253 .6 295 .4 152.5 150.4 5 8 tt 229 .7 208.9 167.1 136. 5 77 ti 131. 6 315.4 178. 3 120.9 18 :4 9 weeks 69.8 129. 3 37.3 40.4 26 it 369 .6 257.4 74.1 123. 8 40 tt 397.9 378.4 233 .3 196.1 58 tt 311.5 286 .9 234.2 189.9 77 ti 225.2 701.5 304 .0 182.1 20:1 9 weeks 201.9 496. 5 114.6 152.8 26 it 668.1 639 . 3 156. 3 301.4 40 ti 964. 3 1281.8 540. 4 541.7 58 ti 913.7 857. 8 520 .1 499.9 77 it 644. 6 1532 .0 641.9 656.1 20:2 9 weeks 9.8 9 . 0 3.2 6.2 26 ti 122.8 142.0 26. 8 25 . 3 40 it 52.6 ,54.5 12.9 13.9 58 it 18.9 36.2 28.6 14. 3 77 it 17.0 54.8 16.0 22.0 20:4 9 weeks 38.5 53.9 23.7 20.4 26 tt 103. 8 84.6 25.5 42 .7 40 it 101.3 93.9 37.9 42. 8 58 tt 50.7 60. 3 68.9 35.6 77 tt 49.7 148.2 54.3 47. 8 TABLE VIII (Continued) Free Length of Inside Muscle Outside Muscle Fatty Frozen Brine Plate Brine Plate Acid Storage Frozen Frozen Frozen Frozen 20 : 5 9 weeks 737 .4 1778.1 421.5 546. 6 26 2409.3 2344 .1 530.9 859 .1 40 i t 2848.2 3494.8 1505.1 1461.1 58 II 2503.6 2330.2 1274.4 1181.1 77 II 2270.9 4061. 2 2322.7 1590.9 22:1 9 weeks 145.9 305 .1 102.5 103. 3 26 II 680.3 410.4 179.9 299 . 8 40 II 674. 2 600.6 341.1 317.1 58 t i 626.8 588.8 255.9 258 .7 77 Tl 291.6 991.2 381. 8 334 .1 22 :5 9 weeks 83.3 325 .8 69.2 101. 8 26 II 377 .9 460. 2 74.6 150. 7 4 0 i t 515.7 640 .9 225. 2 296. 9 58 i t 306. 7 402 . 8 195.0 324.5 77 i t 366. 8 826.2 331. 5 342.5 22 :6 9 weeks 459.2 15 34.2 248.6 483.7 26 tt 1803.4 2543.9 365.4 917.6 40 i t 2000.6 2907.3 869 .5 1108.4 58 t t 1009.5 1557.7 843.7 1053.3 77 i t . 1584.4 3807.5 1327.3 1240.9 Each value i s the average of 3 observations. TABLE IX Analyses of variance of free f a t t y acids (expressed as percent of the t o t a l free f a t t y acids analyzed) of Coho salmon x. 14 :0 15:0 16 :0 16:1 16 :2 17:0 Source d.f. M.S.2 M.S. M.S. M.S. M.S. M.S. Pairs ? 2 ' 22.00* 0.147** 227.31 115.81* 0.61 3.46 Method j . 1.28 0 .187 120.15 36. 27 0. 39 1.65 Error a 2 38. 36 0.206 21.24 5 .94 0.09 4. 31 Location JL 12 .48 0.009 54.69 0.10 4.35 10.47 M x L 1 0.66 0.009 0 .18 0. 01 0. 35 0.43 Error b 4 1.61 0.024 37. 34 17.82 0. 84 3. 29* Time 2 6.73 0.787*** 39.48 335.62*** 0.68 5.13* M x T 2 1.33 0.004 20.50 78.46 0 .25 0.44 L x T 2 3.05 0.003 51. 28 6.28 0.20 0.65 M x L x T 2 0.40 0.002 248.00 6.96 0.55 0.03 Error c 16 3. 05 0. 019 102.80 . 29.33 0. 35 0. 84 18 :0 18:1 18 :2 18 : 3 18 :4 20:1 Pairs 2 4.62 131.72 0.2707 1.93 6.3806 3.50 Method J. 2.60 73.23 0.0001 0. 84 0.1691 9. 36 Error a 2 57. 64 935.48 0.0751 6.17 14.9650 107.93 Location 1 1.14 11.4 3 7.2296 1.06 5.1376* 3.14 M x L 1 11.09* 35.25 0.4142 0.44 0.0001 2.78 Error b 4 0. 88 83.52 0.5400 0..19 0 . 5353 4.24 Time 2 114.54*** 357 .48** 29.1910*' ** 1.3 2 1].36 70*** 4. 79 M x T 2 2.67 12.56 1.4854 0. 38 0.9405 0.02 L x T 2 0 .40 97.98 2.7911 0.14 0.0568 4.24 M x L x T 2 16.73 173.87 7.0184 0.10 0.5089 5.18 Error c 16 8.00 59.89 2.0885 0.55 0.6689 3.46 I TABLE I X (Continued) 20:2 20:4 20:5 22:1 22:5 22:6 Pairs 2 2.43 . 2.13* 151.82 . 4.85 2.2 9 282.89 Method I 0.71 0.12 291.67 94.10 0 . 75 0.05 Error a 2 0.07 0.50 139 .56* 2 0 7.9 5*** ... 7.90 95.90 Location 1 1.50 0.37 0. 22 5.46 22.72 187.03 M x L 1 0.62 0.01 33.47 0.35 14.57 2.67 Error b 4 2.21 0.42 34.97 1.20 3. 82 82. 31 Time 2 30.79*** 9.11*** 449.19 49.75* 16.77 56.91 T x M 2 0.15 . 0.65 35.16 2.60 ' 8 .38 2.97 T x L 2 1.80 0.14 7.59 11.76 8.65 8.16 T x M x L 2 1.10 0. 27 115.60* 2.69 6.65 56.39. Error c 32 0.88 0.53 25. 76 6.18 4.99 152.23 Analyses of variance was conducted on the arcsin transformed percentages. 2 5 A l l mean square values are multiplied by 1.0 x 10 \ 3 Methods were tested by error a. Location and M x L were tested using error b. A l l other terms were tested by error c. * S i g n i f i c a n t at the 5% l e v e l . ** S i g n i f i c a n t at the 1% l e v e l . *** S i g n i f i c a n t at the 0.1% l e v e l . TABLE X Method x loc a t i o n x time i n t e r a c t i o n means from the analyses of variance of the free f a t t y acids (expressed as percent of t o t a l free f a t t y acids analyzed) of coho salmon . Free Length of Inside Muscle Outside Muscle Fatty Frozen Brine Plate Brine Plate Acid Storage Frozen Frozen Frozen Frozen 1-4:0 10 weeks 2 3.51* 3.63 3.60 3.93 27 ii 3.14 3.05 3. 36 3.20 78 it 3.03 3.10 3.58 4.04 15:0 10 weeks 0. 32 0.27 0. 33 0.27 27 ii 0.16 0.12 0.18 0.14 78 it 0.17 0.15 0.20 0.14 16 :0 10 weeks 18.48 13.98 13. 29 15.02 27 ti 13. 71 14.90' 15.21 13. 77 78 it 15.63 15. 64 16.92 13. 65 16 :1 10 weeks 8.78 6.90 9. 39 7.92 27 II 9.74 11.77 10.42 11.02 78 ti 6.87 7.53 6.42 8.20 16:2 10 weeks 0.27 0. 36 0.58 0.49 27 tt 0.52 0.44 0.55 0.69 78 it 0.26 0.26 0.40 0.73 17 :0 10 weeks 0.73 0.90 •1.00 0.97 27 II 0.79 1.10 1.24 1.48 78 it 1.03 1.16 1.54 1.53 18:0 10 weeks 3.2 2 3.06 3.49 2.98 27 II 2.26 1.90 2.48 2.15 78 tt 4.66 3.63 3.46 4.83 18 :1 10 weeks 21.46 19.58 20.57 22 .09 27 ti 21.44 22.61 25. 39 21.74 78 ti 21.48 20.57 20.00 18.20 18 :2 10 weeks 1.45 1.48 1.77 1.26 27 tt 2. 37 1.94 2.38 3.18 78 it 1.88 2.08 2.20 2 .11 18 : 3 10 weeks 1.20 0.95 1.25 1. 04 27 it 0.98 0. 80 0.92 1.00 78 ti 1.05 0.97 1.18 1.22 TABLE X (Continued) Free Length of Inside Muscle Outside Muscle Fatty Frozen Brine Plate Brine Plate Acid Storage Frozen Frozen Frozen Frozen 18 :4 10 weeks 1 . 4 9 1 . 4 5 1. 80 1 .52 27 ii 1 . 0 6 1 . 0 8 1 . 1 6 1 . 4 6 78 n 1 . 7 4 1 . 6 4 2 . 0 4 1 . 8 9 2 0 : 1 10 weeks 4 . 0 1 4 . 7 6 4 . 40 4 . 2 7 27 it 3 . 54 4 . 2 4 4. 56 4 . 4 7 78 it 4 . 58 4 . 6 3 4. 26 4 . 9 2 20 : 2 10 weeks 0 . 5 6 0. 24 0 . 2 6 0 . 5 0 27 ii 1 . 4 5 1 . 3 9 1 . 1 5 0 . 8 7 78 ii 0. 34 0 . 2 1 0. 31 0 . 3 2 20 :4 10 weeks 0 . 99 1 . 1 4 1 . 0 6 1 . 0 4 27 ii 0 . 5 3 0. 74 0 . 4 3 0 . 5 5 78 ii 0 . 7 2 0 . 5 4 0 . 6 0 0 . 5 7 2 0 : 5 10 weeks 1 4 . 9 9 1 6 . 53 1 5 . 2 3 1 5 . 0 6 27 II 1 5 . 1 1 1 1 . 8 5 1 2 . 2 9 1 3 . 80 78 ii 1 7 . 0 3 1 6 . 26 1 6 . 8 4 1 7 . 3 8 22 :1 10 weeks 2 . 7 5 4 . 3 3 3 . 2 7 4 . 3 7 27 II 4 . 34 4 . 9 1 3. 05 4 . 3 3 78 n 2 . 5 7 3 . 2 9 2 . 4 3 3 . 2 8 22 : 5 10 weeks 1. 30 2 . 9 4 3 . 2 4 2 .99 27 ti 2 . 9 1 2 . 7 5 2 . 9 5 2 . 5 8 78 ii 3 . 05 3 . 0 4 3 . 7 8 3 . 4 7 22 :6 10 weeks 1 3 . 55 1 4 . 9 3 1 3 . 9 8 1 2 . 5 9 27 ti 1 4 . 25 12 .72 1 1 . 1 2 1 2 . 0 0 78 II 1 3 . 1 2 1 3 . 6 7 1 2 . 1 8 1 2 . 1 5 The analyses were conducted on the arcsine transformed percentages but the actual percentages (not transformed) are recorded i n TABLE X. Each value i s the average of 3 observations. TABLE XI Analyses of variance of free f a t t y acids (expressed as jag free f a t t y acid per gram of neutral l i p i d ) of Coho salmon. 14:0 15:0 16:0 16:1 16:2 17:0 Source d.f. M.S.1 M.S. M.S. M.S. M.S. M.S. Pairs ? 2 16.11 0.026 215.82 54.44 0.28 24.90 Method 1 14. 50 0.02 3 175.62 52.23 0.2 6 22.12 Error a 2 18.05 0.026 203.99 66.90 0.30 28.67 Location 1 22.10 0.038 313.03 94. 82 0. 34 33.28 M x L •y j_ 14.83 0.023 176.00 54.10 0.28 22. 59 Error b 4 17. 67 0.028 220.22 69 .35 0. 31 27. 84 Time 2 21.42 0.034 2 8 8.81 85.11 0.36 34.86 M x T 2 14.8 3 0.023 178.28 54.96 0.26 22.85 L x T 2 18 .90 0. 030 251.99 75.38 0. 30 30. 24 M x L x T 2 15.46 0.024 181.89 58, 05 0.29 23. 59 Error c 16 17.38 0.027 216.07 68.53 0. 31 27 .47 18:0 18 :1 18 :2 18 : 3 18 :4 20:1 Pairs 2 11.19 422.40 3.09 1.4 5 5.88 42.82 Method 1 8.52 348.25 2.36 1.20 5.09 36.64 Error a 2 10.27 405.71 3.09 1.59 6.36 44.20 Location 1 15.50 60 3.61 4.63 2. 0 5 7.52 54. 07 M x L 1 9.51 352.09 2.44 1.26 5. 21 37.52 Error b 4 11.20' 437.03 3.22 1.55 6,25 44. 30 Time 2 16.51 558.01 4,43 2.03 7.67 , 52.65 M x T 2 • 8. 81 257.71 2.41 1.22 5.23 37.78 L x T 2 13.27 489.40 3.80 1.75 6. 79 47. 74 M x L x T 2 10.16 372.20 2.50 ' 1.30 5.41 39 .36 Error c 16 11.00 432.91 3.20 1.53 6.13 43.61 TABLE XI (Continued) 20:2 20:4 20:5 22:1 22:5 22:6 Pairs 2 0.48 0.18 270.26 16.07 4.61 150.27 Method 1 0.44 0.15 227.71 12 .67 3. 29 . 107.74 Error a 2 0.46 0.21 285.96 16.55 4. 69 148.06 Location 1 0.63 0.33 391.27 21.45 . 7.35 228.58 M x L 1 0 .45 0.16 235.33 13.46 3.44 111.47 Error b 4 0.46 0. 21 276.43 16.55 4.64 154.42 Time 2 0.46 0.27 383.62 18. 87 7.41 208.27 T x M 2 0.44 0.17 232.17 13.65 3. 64 112.25 T x L 2 0.45 0.23 328.47 17. 33 5.74 179.59 T x M x L 2 0.46 0.18 244.04 14.26 3.91 119.98 Error c 32 0..48 0.20 283.41 16.14 4.76 151.29 x A l l mean square values are multi p l i e d by 1.0 x.10" 7 Methods were tested by error a. Location of M x L were tested using error b. A l l other terms were tested by error c. * S i g n i f i c a n t at the 5% l e v e l (P < 0.05). ** S i g n i f i c a n t at the 1% l e v e l (P * 0.01). *** S i g n i f i c a n t at the 0.1% l e v e l (P £ 0.001). TABLE XII Method x loc a t i o n x time i n t e r a c t i o n means from the analyses of variance of the free f a t t y acids (expressed as ug per gram of neutral l i p i d ) of coho salmon. Free Length of ' Inside Muscle Outside Muscle Fatty Frozen Brine Plate Brine Plate Acid Storage Frozen Frozen Frozen Frozen 14 :0 10 weeks 466.7 1 649.2 238 .2 154.7 27 it • 540.7 598 .2 172 .0 174.9 78 n 26986.9 2509.5 522 .5 738.2 15 :0 10 weeks 41. 3 52.5 20.0 10 .7 27 tt 29.7 27.1 9.8 7.9 78 II 1082.2 115.4 27. 3 32.1 16 : 0 10 weeks 2590.4 2514.0 848.5 610.0 27 ti 2431.5 3180.3 803.1 787. 2 78 96914.8 12382 .6 2383.0 2683.0 16 :1 10 weeks 1126.3 1424.4 620.9 316.3 27 it 1661.2 2300.8 493.4 624.9 78 tt 53468.6 6404.1 1089.1 1654.0 16 :2 10 weeks 38.1 67.7 48.1 19.6 27 II 92.1 77.4 27.6 40.2 78 II 3522.6 233.4 69.0 163. 7 17 :0 10 weeks 94.7 169.9 72.1 38.7 27 tt 151. 3 196. 3 63.2 79.1 78 tt 10643.0 1066,7 231.9 299.8 18 :0 10 weeks 4 31.9 606.0 224 . 3 117.0 27 tt 385.6 462.6 130. 6 122.0 78 tt 22326.5 2985.8 496.8 1131.5 18 :1 10 weeks 3034.8 3605.5 1377.8 916.4 27 ti 3792 . 8 4866.1 1404.8 1281.8 78 tt 136144.9 16151.0 3160,7 4069.0 18 :2 10 weeks 187.5 262 .1 106.1 49,7 27 ti 4 31.7 430 . 2 121.1 174.7 78 II 11669.0 1803.7 314.7 403.7 18 : 3 10 weeks 150. 2 198.5 69. 0 40.5 27 ti 164.2 183.0 46.1 57.6 78 it 8052.3 980.2 164.3 271.4 TABLE XII (Continued) Free Length of Inside Muscle Outside Muscle Fatty Frozen Brxne Plate B r i n e Plate Acid Storage Frozen Frozen Frozen Frozen 18 :4 10 weeks 188. 0 304. 3 112.5 59.0 27 II 175.4 220.6 56.2 83.4 78 n 15982.1 14 6 5.4 283.7 393.2 20 :1 10 weeks 515. 2 807.2 274.7 170.8 27 it 605.6 859 .9 222 .9 246. 2 78 ii 42595.8 3593.4 597.0 958. 9 20 :2 10 weeks 7 8 . 1 . 43.9 9.7 19.9 27 II 246.7 287 .7 53.2 49.6 78 ti 4407.3 174.9 32.2 65.1 20 :4 10 weeks 119 .9 195.0 61.5 41.6 27 it 99 . 3 127.7 23.4 31.1 78 tt 3021.7 423.9 90.3 107.1 20 : 5 10 weeks 1822 .1 3061.3 997. 3 607.9 27 II 2726.6 2384.0 662 .2 772 .0 78 ii 110697.7 13572.0 . 2525.1 3596.3 22 :1 10 weeks 364.6 720. 0 198. 0 175.0 27 it 746.1 899.9 150.9 232 . 2 78 ti 25813.9 2448.7 331.1 617;9 22 :5 10 weeks 167.0 549 . 5 17-9 .4 117.4 27 it 517.3 .590.8 14 3.2 144.5 78 ti 145 7.3 2517.8 564.7 755.5 22 :6 • 10 weeks 1821.9 2 816.5 1025.5 502 .7 27 II 2526.3 2816 .5 530 .7 676.4 78 tt 80248.7 12753.5 1843.5 2784.8 Each value i s the average of 3 observations. TABLE XIII Analyses of v a r i a n c e o f t o t a l f r e e f a t t y a c i d s (expressed as ug f r e e f a t t y a c i d per gram of n e u t r a l l i p i d ) of P a c i f i c h a l i b u t , chinook salmon and coho salmon. P a c i f i c H a l i b u t -j Chinook Salmon Coho Salmon Source df M.S. df M.S. df M.S. P a i r s 2 49 7.51* 2 1.546** 2 1015.60 2 Method 1 781.96 1 1.102* 1 825.41 E r r o r a 2 54.37 2 0.041 2 1025.00 L o c a t i o n 1 24 54.80** 1 13.698** 1 1452.50 M x L 1 433.13 1 1.225 1 848.29 E r r o r b 4 83.88 4 0.257 4 1062.40 Time 4 217.85 4 2.6 0 3*** 2 1369.30 M x T 4 92.55 4 0. 234 2 851. 34 L x T 4 162.04 4 0.438 2 1191.40 M x L x T . 4 88.36 4 0.566 2 892.84 E r r o r c 32 128.75 32 0. 213 16 ; 1044.80 1 A l l mean . square values , are m u l t i p l i e d by 1. 0 x 10~ 6 Methods were t e s t e d by e r r o r a, L o c a t i o n and M x L were t e s t e d u s i n g e r r o r b. A l l o t h e r terms were t e s t e d u s i n g e r r o r c. *' S i g n i f i c a n t at the 5% l e v e l (P * 0.05) ** S i g n i f i c a n t a t the 1% l e v e l (P * 0.01) *** S i g n i f i c a n t at the 0.1% l e v e l (P * 0.001) TABLE X!V Method x loc a t i o n times time means from the analyses of variance of t o t a l free f a t t y acids (expressed as jig free f a t t y acid per gram of neutral l i p i d ) of P a c i f i c h a l i b u t , chinook salmon, and coho salmon-. Species Length of Frozen Storage Inside Muscle Outside Muscle Brine Frozen Plate Frozen Brine Frozen Plate Frozen P a c i f i c 14 weeks 5670.1 1934. 0 3705.8 498 . 9 Halibut 3.1 n 37222.4 7988 . 5 2967.4 1557.1 4b it 22538.6 20503. 3 4321.4 4507.1 62 tt 18090.5 5535. 3 3196.3 145 9.1 81 tt 266S1.0 11282. 7 5189.6 2125.5 Chinook 9 weeks 526.5 1238". 6 299 .2 412.5 Salmon 26 it 2016.4 1976. 8 415 .1 715.4 40 ti 2002.0 2628. 6 1060.3 1102.9 58 n 1699.7 1601. 5 10 81.8 927 .8 77 II 1483.5 3066. 5 1522.2 1146.8 Coho 10 weeks 1321.9 1804 . 7 648.4 396.8 Salmon 27 t! 1732.4 2050. 9 511.4 558 .6 78 tl 66814.9 815 8. 2 1472.7 2072.5 Each mean i s the average of 3 observations. TABLE X-V Correlation c o e f f i c i e n t s (r) f o r the correlations of pH, thaw d r i p , color (Hunter Rd, a, b, and a/b values) with each other f o r P a c i f i c h a l i b u t , Chinook salmon, and Coho salmon. PACIFIC HALIBUT Variable pH ,Thaw Drip pH Thaw Drip 1.000 -0.358* 1. 000 ' CHINOOK SALMON Variable pH Zh™ Rd Drip a b a/b pH 1.000 Thaw Drip 0.074 1.0 00 Rd -0.336 0.163 1.000 a -0.017 0.187 -0.232 1.000 b 0.290 0.021 0.417* 0.263 1.000 a/b -0.103 0.193 -0.398* 0.928**-0.112 1.000 COHO SALMON Variable pH Thaw Drip Rd a - - • b a/b pH . 1. 000 Thaw Drip -0.132 1. 000 Rd -0.033 0.124 1. 000 a -0.596** 0.041 0. 55 6* 1. 000 b 0.270 0. 237 0. 7 7 4** 0. 217 1 . 000 a/b -0.721** 0.201 -0. 062 0. 725*' *-0 .511 1. 000 Si g n i f i c a n t at the 5% l e v e l (P ^ 0.05). Si g n i f i c a n t at the 1% l e v e l (P ^ 0.01). TABLE XVI Correlation c o e f f i c i e n t s (r) f o r the c o r r e l a t i o n of pH, thaw dri p , c o l o r (Hunter Rd, a, b, and a/b values), TBA values, and free f a t t y acids with f l a v o r (number of correct i d e n t i f i c a t i o n s ) for P a c i f i c h a l i b u t , Chinook salmon, and Coho salmon. Variable P a c i f i c halibut Chinook salmon Coho salmon Variable P a c i f i c h a libut Chinook salmon Coho salmon pH -0.258 -0.102 -0.737 %C 22:1 -0.16 0 -0.338 0.223 Thaw Drip -0.198 0.116 . -0.136 %C 22:5 0, 012 0.113 0.075 Hunter Rd -0.375 0.455 %C 22:6 -0.077 0. 009 -0.240 Hunter a -0.042 0.653* FFA „ Hunter b -0.5 27* 0.059 C 14 F 0.016 0.046 0.038 Hunter a/b 0.024 0.147 C 15 F -0.051 -0.029 0. 039 TBA. -0.039 0.460** 0.152 C 16 F 0. 070 -0.059 0.031 FFA c 16 :1 F 0.028 -0.039 0.032 %C X 14 0.086 0.038 0.147 c 16 :2 F 0. 052 -0.127 0. 044 %C 15 0.114 -0.271 0.368 c 17 F 0 . 044 -0.024 0.032 %C 15 0.006 -0.099 0.261 c 18 F 0. 046 0.065 0. 031 %C 16 :1 0.086 -0.057 0 .176 c 18 :1 F 0.0 69 ' -0,119 0.038 %C 16 :2 0.109 0.058 -0.115 c 18 :2 F '0.030 -C. 09 3 0.020 %C 17 0. 215 0.142 -0. 040 r 18 : 3 F 0 .017 -0.069 0.028 %C 18 -0.130 0. 332 0. 216 c 18 :4 F 0. 261 0. 090 0. 041 %c 18:1 0.121 -0.045 0.515* c 20:1 F 0.017 0.023 0.044 %c 18:2 0.114 0.192 0. 308 c 20:2 F 0.0 24 -0 .102 0.053 %c 18 : 3 0 .076 0.110 0.117 c 20:4 F 0. 091 0.053 0.023 %c 18 :4 0.053 0.204 0.193 c 20:5 F 0. 05S -0.276 0.0 32 %c 20 :1 -0.315 -0 . 410* 0.141 c 22 :1 F 0. 247 -0.112 0.043 %c 20:2 0.064 0.15 6. 0. 060 c 22:5 F -0.0003 -0.0003 0.021 %c 20 :4 0. 096 -0.131 -0. 385 c 22: 5 F 0. 02 3 0.036 0.020 %c 20:5 -0.017 -0.256 -0.020 TF FA/GF 3 0.036 -0. 053 0. 031 1 Free f a t t y acids expressed as percent of t o t a l free f a t t y acids analyzed. 2 Free f a t t y acids expressed as jig free f a t t y acid per gram of neutral f a t . 3 Total free fatty acids expressed as ug t o t a l free f a t t y a c i d per gram of neutral f a t . * S i g n i f i c a n t at the 5% l e v e l . ** S i g n i f i c a n t at the 1% l e v e l . 

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