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UBC Theses and Dissertations

Physico-chemical changes occurring in fish flesh during freezing and thawing as measured dilatometrically Mahadevan, Vaidyanatha Iver 1948

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PHYSICO-CHEMICAL CHANGES OCCURRING IN FISH FLESH DURING FREEZING AND  THAWING AS MEASURED PILATOMETRICALLY by V. MAHADEVAN A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS IN THE DEPARTMENT OF ZOOLOGY THE UNIVERSITY OF BRITISH COLUMBIA APRIL, lolj-8 PHYSICO-CHEMICAL CHANGES •OCCURRING- IN  FISH FLESH DURING FREEZING AND THAWING AS MEASURED DILATOMETRICALLY  ABSTRACT The thesis deals with the use of a dilatometer in studying some of the physico-chemical phenomena occur-ring in fresh f i s h flesh when subjected to freezing at o o temperatures ranging from 0 C to -30 C. Two different kinds of f i s h flesh, marine lingcod (Ophlodon elongatus) and fresh water rainbow trout (Salmo galrdnerll) were used for comparison. True freezing point determinations of samples of fresh flesh cut from the above species of f i s h were made and found to be the same, viz, -1.5°C (29.1°F.). The percentage of water removed as ice at varying temperatures below the I n i t i a l freezing point were calculated by necessary adjustments of experimentally determined values. A permanent net decrease in volume accompanying freezing and thawing of the samples of flesh was observed and measured. This change in volume ls probably due to the denaturation of the proteins, and was found to be 0.075#. The coefficient of cubical expansion (oC ) of anhydrous f i s h muscle was measured for the f i r s t time and found to have the average value of 0.000118 over a temper-ature range from -30°C. to +20°C. CONTENTS INTRODUCTION . . 1 - 7 APPARATUS 8 - 9 FILLING THE APPARATUS 9 - 1 2 EXPERIMENTAL PART 13 - 1? EVALUATION OF RESULTS A* Ad Justments of Experimental Data ....... 1 8 - 2 5 B. Results from Adjusted Data 25 - 28 DISCUSSION 29 - 3^ SUMMARY 35 ACKNOWLEDGEMENT 36 - 37 LITERATURE CITED 38 THE PHYSICO-CHEMICAL CHANGES IN FISH MUSCLE DURING FREEZING AND THAWING AS MEASURED DILATOMETRICALLY INTRODUCTION I t Is a" common observation that fish after landing at the shore do not' keep fresh for long and begin to emit unpleasant odours due to many biochemical and biophysical changes setting in. During the past few years7 great strides have been made in order to arrest these changes, thus enabling the Industries successfully to distribute them to the' consumers without app'reciable loss in the nutritive value, taste and smell. Before a r t i f i c i a l freezing was used as a method of preserving fi s h , drying, salting and pickling and later canning were adopted; hut fish preserved by the above methods are greatly changed. They do not at a l l meet the demands for fresh f i s h . Freezing and re-frigeration are the only methods of preserving that keep f i s h in essentially i t s original condition over long periods. The problem of freezing is simply one of 2. detecting the effect of lowering temperature upon the rate at which these changes occur. Unlike the flesh of terrestial animals, fish flesh is much more tender, for the simple reason that i t lives in an entirely different environment (water) which supports i t by hydrostatic pressure. Hence fish flesh is much more prone to mechanical 'injury and bacterial invasion. It may be merely coincidental that the proteins of f i s h muscle much more sensitive to chemical and physical change, or i t may be in some obscure \fay related to the demands which environment makes upon i t . However, It ls definitely known that fish proteins undergo des-tructive alterations more quickly than do mammalian proteins such as those present in beef. Most people consider that f i s h is at i t s best from the point of view of flavour, consistency and pala-ta b i l i t y Immediately after i t is caught. Storage in cracked melting ice immediately after catching w i l l keep fis h in a marketable condition for some days. Cod and haddock carefully treated in this manner may be kept from i eleven to fourteen days, and halibut up to almost twenty days, depending upon the original state of infection, without significant change. But i t must be emphasized that once this i n i t i a l latent period is over, decom-position proceeds with rapidity and certainty as Beatty 3 . and Gibbons ( 1 9 3 5 ) have shown. This period of time is insufficient for any hut the most.proximate markets and freezing must be resorted to for the moi-e distant ones. The effects of freezing upon a tissue are due to two things, the removal of water as ice and the re-duction in temperature of the system. Both w i l l cause change in the rate of physical, chemical, and microbio-logical reactions taking place in the system, greatly inhibiting some and enhancing others. The degree of change vrt.ll depend upon the rate of freezing and the extent or degree of severity of freezing. A great deal of literature is available re-lative to the freezing of biologic tissue, but most of them have concerned themselves with the effect which the rate of freezing has upon visible structure. The "rapid freezing" technique has been evolved mainly out of these. The true implication of the phrase "rapid freezing" has not been correctly understood since the demarcation line between "slow" and rapid freezing had not been experi-mentally determined. Much of the credit for accumulating experi-mental evidence in this connection goes to Moran ( 1 9 3 2 ) , who by an ingenious method discovered that when the temperature of beef muscle was lowered from ^ 1 ° to 2-3°F ( 5 ° to - 5 ° 0 ) within an interval of from forty to f i f t y minutes, the rate of freezing was such that very l i t t l e , i f any, physical damage was done to the c e l l structure. He also showed the formation of large ice crystals with resultant damage to the tissue on sloxv freezing. He therefore designated the former rate as the c r i t i c a l rate of freezing. The success which attended the use of this method in the determination of the rate of freezing of beef led to numerous investigations' on fish flesh. Finn (1933) has shown that the maximum zone of crystal for-mation l i e s between 3l°F (-.5°C) and 23°F (-5°G), about 82/£ of water appearing as lce» Choosing this interval, Young (1935) calculated that the time for the c r i t i c a l rate of freezing for f i s h would be about thirty five minutes* Although rapid freezing has been evolved in order to preserve the original structure of the tissue., recent experimental work has indicated that the condit-ions of storage after rapid freezing are more important to the ultimate condition of the fish* Quite apart from this visible physical effect, the depth of freezing has been shown to be responsible for most of the changes in the frozen state. Proteins in their natural state are colloidal suspensions of albuminous material in aqueous salt sol-ution. The relationship \tfhich water bears to the system is very delicately balanced, and among other things, is dependent for i t s equilibrium upon certain definite concentrations of naturally occuring salts, acids and basic substances. When water is removed from the system as ice, salt and acid concentrations increase to a point where an irreversible disturbance in the equilibrium (called denaturation) takes place. When s t i l l further water is removed as ice by lowering the temperature, another zone of concentration is reached which does not have this effect, as shown by Finn (1932). In this zone the proteins may be stored for some months without apparent harm. It is necessary at this point to understand clearly what freezing and freezing points are. It is frequently but erroneously assumed that the f i s h flesh is "frozen" by the time they become^ rigid at a tempera-ture of about 25°F (-^°C). Finn (1933) demonstrated that 9% of the liquids of fish muscle juice could s t i l l be separated in the unfrozen.state at 1^°F (-10°C), but this method could not well he applied at much lower temperatures. Carter (19^) later demonstrated by a different method that about 11% of the liquids of halibut muscle juice remained unfrozen at -7°F (-21.5°C). This method, depending on the delicate measuring of the expansion i n volume caused by the f r e e z i n g of the l i q u i d , i s a p p l i c a b l e at s t i l l lower temperatures. C a r t e r and F r o s t (19^ 5) i n an a r t i c l e on "The Complete F r e e z i n g of F i s h F l e s h " summarize our present knowledge concerning the s u b j e c t , end proceed to a s c e r t a i n a t what temperature the f r e e z i n g of the f l e s h of f l o u n d e r i s a c t u a l l y complete. To f u r t h e r the knowledge of the physico-chemi-c a l e f f e c t s r e s u l t i n g from the f r e e z i n g of f i s h muscle, and to undertake a more c a r e f u l a n a l y s i s of the d i l a -tometric experimental procedure t h a t appears to be most s u i t a b l e f o r meaeuring these e f f e c t s to be examined, experiments I, II, III and IV below were conducted. As a p r e l i m i n a r y to these experiments, a some-what n o n - r e l a t e d experiment was undertaken to become f a m i l i a r w i t h the apparatus and method of i t s o p e r a t i o n . The r e s u l t s of t h i s experiment were d e s i r e d a l s o as pro-v i d i n g some da t a requested by the B r i t i s h Columbia f i s h i n g i n d u s t r y . I. The true f r e e z i n g - p o i n t range of n o n - o i l y marine and fresh-water f i s h f l e s h . II. The p r o p o r t i o n of aqueous l i q u i d i n such f l e s h as f r o z e n at d i f f e r e n t temperatures. III. The magniture of any n o n - r e v e r s i b l e , permanent change i n a p h y s i c o - c h e m i c a l c o n d i t i o n (e.g. 7 . volume) of the f l e s h a f t e r b e i n g f r o z e n and thawedo ' • I n c i d e n t a l to conducting the above e x p e r i -ments, one type of data not e x p l i c i t l y recorded In a v a i l a b l e l i t e r a t u r e had to be determined: IV. The c o e f f i c i e n t of cubic expansion of anhydrous f i s h muscle t i s s u e . 8. APPARATUS The apparatus used throughout t h i s i n v e s t i -g a t i o n was the same as, or s i m i l a r t o , that used by-C a r t e r and F r o s t (19^ 5) i n an e a r l i e r i n v e s t i g a t i o n of some phases of the same problem. The apparatus i s e s s e n t i a l l y a d i l a t o m e t e r , o p e r a t i n g on the same p r i n c i p l e as a thermometer, namely to measure changes i n volume of an enclosed substance ( f i s h f l e s h ) as the temperature of that substance a l t e r s . The change i n volume i s i n d i c a t e d by the change i n p o s i t i o n o f a column of mercury i n a c a p i l l a r y bore connected with the enclosed space i n which the substance i s c o n t a i n e d . The d i l a t o m e t e r was c o n s t r u c t e d of Pyrex g l a s s as shown d i a g r a m m a t i c a l l y i n f i g u r e I . A c y l i n d r i c a l b a r r e l A about 15 x 2 cm. d i v i d e d by a standard-taper ground-glass s l e e v e j o i n t J served to co n t a i n the sample of f i s h f l e s h . The bottom of A was c o n s t r i c t e d to be continuous w i t h a c a p i l l a r y stem K bent i n the shape shown to a f f o r d a h o r i z o n t a l p r o l o n g a t i o n of uniform bore a g a i n s t a m i l l i m e t e r s c a l e S. T h i s p r o -l o n g a t i o n was made h o r i z o n t a l I n s t e a d of v e r t i c a l to a v o i d undue mercurosta.tlc pressure b u i l d i n g up i n the b a r r e l A. During experiments, the b a r r e l A and the v e r t i -c a l p o r t i o n of the c a p i l l a r y tube K were immersed i n a Fig. 1. DILATOMETER (To face page 8.) cylindrical Pyrex glass jar (not shown in the diagram) containing about five l i t e r s of a 28$ calcium chloride brine. The level of immersion is shown by the line LL in the diagram. This volume of brine allowed satis-factory temperature control. Cooling of the brine bath was accomplished by lowering to i t s bottom a small wire containing lumps of "dry ice" whose generation of C0 2 gas also provided efficient s t i r r i n g of the brine. Other appurtenances to the apparatus are des-cribed in the following section. FILLING- THE APPARATUS To f i l l the dilatometer in readiness for an experiment, a known weight of clean mercury P was i n -troduced into the barrel A and through the stopcock F attached to the far end of the capillary K suction was applied u n t i l the mercury in the capillary reached a convenient reading near the left-hand end of the scale S. The stopcock F, lubricated with grease, was then closed. A sample of fresh fish flesh Q, x»/as cut with scissors or a knife from the fleshy body part of the fis h so that, its dimensions (e.g. l x l x 17 cm.) would allow insertion of the sample through J into the barrel A. The sample of flesh was carefully weighed (25 to ^0 10, gm.) and a corresponding symmetrical sample from the same f i s h was preserved f o r moisture d e t e r m i n a t i o n and p r e p a r a t i o n of anhydrous muscle t i s s u e (see l a t e r under phase IV of the experimental p a r t ) . The sample of f i s h f l e s h Q, was then Introduced i n t o the b a r r e l A by I n s e r t i n g i t through the opening J while p r o t e c t e d by a p i e c e of cellophane to prevent the ground-glass s u r f a c e of J from becoming contaminated by moist f l e s h samples. In some experiments, where as great a weight of f i s h as co u l d be accommodated i n A was d e s i r e d , a p o r t i o n of the weighed sample Q, was i n s e r t e d i n t o t h a t p a r t of A above the j o i n t J . Before c l o s i n g j o i n t J , a recorded volume of hexane (from petroleum, Ea,stman Kodak) s u f f i c i e n t to f i l l A from the s u r f a c e of the mercury to the bottom "of the j o i n t J xms i n t r o d u c e d d i r e c t l y from a b u r e t t e . By poking w i t h a wire around the s i d e s of the column of f i s h , any a d j e r i n g a i r bubbles were r e l e a s e d . The s p e c i a l l y - l u b r i c a t e d j o i n t J was then f i t t e d t o g e t h e r . The 50-ml. b u r e t t e B c o n t a i n i n g the hexane was now con-nected" by meajis of the s h o r t p i e c e of rubber t u b i n g R to the s h o r t g l a s s tube above the s p e c i a l l y - l u b r i c a t e d c l o s e d stopcock V. B e f o r e opening V, hexane was allowed to d i s p l a c e a l l a i r above V and the reading of the hexane on the b u r e t t e was recorded. By opening V and a p p l y i n g g e n t l e i n t e r m i t t e n t s u c t i o n to the top of the 11. "burette, hexane flowed down to f i l l the r e s t of the space In A* Any adhering a i r bubbles i n the upper p a r t of A were u s u a l l y e a s i l y removed by tapping, though sometimes strong s u c t i o n at the top of the b u r e t t e had to be a p p l i e d to expand the bubbles to cause them to r i s e . Any d e s i r e d f i n a l adjustment of the mercury column against the scale S was made a t t h i s stage by c a r e f u l l y opening stopcock F and l e t t i n g i n a l i t t l e a i r or a p p l y i n g a l i t t l e s u c t i o n , as r e q u i r e d , while the hexane i n A was s t i l l i n communi-c a t i o n w i t h the hexane i n the b u r e t t e . F i n a l l y the stop-cock V was t i g h t l y c l o s e d , the a d d i t i o n a l amount of hexane added to f i l l the upper p a r t of A was recorded from the b u r e t t e , and the bu r e t t e and rubber connection tube were removed. Stopcock F was now opened immediately and l e f t open to observe from any change i n the scale reading of the mercury whether the system was p r e s s u r e - t i g h t , also to permit volume changes i n the apparatus agai n s t atmos-ph e r i c pressure. Mercury was employed as a f i l l i n g l i q u i d f o r the c a p i l l a r y because i t does not wet the glass and would not evaporate through F; i t was not p r a c t i c a l , however, to use mercury to f i l l a l l the space i n A not occupied by the f l e s h sample Q, since Q, would be compressed i n t o the top p a r t of A, muscle j u i c e would be squeezed out, and remaining a i r bubbles could not be seen, Hexane was t h e r e f o r e chosen to f i l l the remaining space i n A s i n c e i t has a low f r e e z i n g p o i n t , i s non-misclble w i t h the aqueous l i q u i d s i n the f i s h f l e s h Q, and at the low tem-per a t u r e s used i n these experiments has a low s o l v e n t a c t i o n on any o i l i n the f l e s h sample Q,. Petroleum ether has a l e s s s o l v e n t a c t i o n hut i t s h i g h v o l a t i l i t y made q u a n t i t a t i v e f i l l i n g of A d i f f i c u l t . The use of hexane, however, i n t r o d u c e d d i f f i -c u l t y i n the choice of a s u i t a b l e l u b r i c a n t f o r J and V. Experience proved that g l y c e r o l had s u i t a b l e q u a l i t i e s . I t i s non-miscible with hexane, and when used s p a r i n g l y , allowed the ground-glass s u r f a c e s to be g e n t l y "wrung" toge t h e r whereas too greasy a l u b r i c a n t i n v i t e d the m e r c u r o s t a t i c pressure i n A to f o r c e J o i n t J a p a r t o r f o r c e out the p l u g of stopcock V. To avoid the b r i n e bath d i s s o l v i n g out the g l y c e r o l , the outer s u r f a c e s o f V and J were se a l e d w i t h a r i n g of " s i l i c o n e " water-re-p e l l i n g l u b r i c a n t or " p l a s t i c i n e " . EXPERIMENTAL PART 1. Determination Of The True Freezing Point Of Fresh Fish Muscle. (a) Llngcod (Ophlodon elongatus) muscle. The samples o f f l e s h used were cut from freshly-caught lingcod obtained d i r e c t l y from l o c a l f i s h handling plants. The columns of f l e s h were out from the fleshy "back well under the skin and were free from bone. Af t e r f i l l i n g the dilatometer as described, the b a r r e l was immersed i n the brine bath at 0°C. After allowing some f i f t e e n minutes f o r the apparatus and sample, to at t a i n bath temperature, the tempera.ture of the bath was then steadily and slowly lowered with dry ice at the rate of about 0.1 degree C. per minute. The slow and steady retreat of the mercury (contraction of the system) along the scale stopped almost immediately i f the temperature was held constant for a t r i a l i n t e r v a l of a minute, in d i c a t i n g that this rate of cooling was s u f f i c i e n t l y slow to allow substantially continuous equilibrium between the temperature of the contents of the b a r r e l and that of the bath as measured by a tested mercury, thermometer graduated i n tenths of a degree Centigrade. At some temperature a few degrees below -3°C ( v a r y i n g i n d i f f e r e n t experiments) , the r e t r e a t of the mercury halted.and a sudden advance (expansion due to formation of i c e i n the sample)' o c c u r r e d . I f the temper-ature was now h e l d constant, t h i s advance continued f o r many minutes due to i c e formation from the ( s l i g h t l y supercooled) aqueous l i q u i d s i n the f l e s h . F o r the purpose of t h i s experiment, however, once t h i s advance had commenced, i t was d e s i r e d to h a l t i t immediately by warming the bat h a few degrees u n t i l a r e t r e a t o f the mercury showed melting had commenced, but to a v o i d com-p l e t e m e l t i n g i n order t h a t some n u c l e i of i c e would remain as seeding c r y s t a l s to a v o i d subsequent super-c o o l i n g . Furthermore, i t was d e s i r e d to prevent as much as p o s s i b l e exposure of the f l e s h to s u b - f r e e z i n g temp-erat u r e In order to minimize any ch e m l c o - p h y s i c a l changes i n the f l e s h due to the b r i e f unavoidable f r e e z i n g r e -s u l t i n g from s u p e r c o o l i n g . While the r e t r e a t of the mercury i n d i c a t i n g thawing was s t i l l t a k i n g place,' the bath temperature was again lowered s l o w l y . T h i s l o w e r i n g e v e n t u a l l y overtook the thawing, and the temperature a t which r e -t r e a t of the mercury h a l t e d and ad.vanee again took p l a c e was taken as the true f r e e z i n g p o i n t of the f l e s h , s i n c e c r y s t a l s of i c e would s t i l l be prese n t to a v o i d super-c o o l i n g . 15 a (b) Rainbow Trout (Salmo galrdnerll) muscle. For the purpose of comparing the freezing point of the muscle of a freshwater f i s h with that of a marine f i s h , a sample of flesh from a freshly-caught rainbow, trout was used in an experiment duplicating (a) above. In experiments under both (a) and (b) above, the magnitude of the scale readings of the mercury as i t advanced or retreated had no significance since only the temperature in relation to point of inflection of the movement was desired. Hence no corrections for change in volume with temperature of the apparatus or indicating liquids were necessary as in subsequent experiments of a different nature, II, Freezing the Muscle Slowly In Different Stages to -30°C(-20OF) In doing this experiment, the bath was allowed to come to equilibrium at various temperatures below the freezing point of the flesh before reading the thermo-meter, and the dilatometer to aquire the bath temperature before reading the scale. The bath was continuously stirred. Both llngcod and rainbow trout flesh were used. The results obtained in these experiments are recorded in "Results from Adjusted Data", part II, 16. III. Permanent change In Volume of Fish Flesh Caused by Freezing and Thawing This experiment is essentially the continuation of experiment II. The apparatus containing the frozen fish flesh was brought to room temperature and allowed to remain so overnight to ensure complete thawing out of the frozen flesh. It was then immersed up to the line LL ( f i g . I) in the brine bath at exactly 0°C. un t i l equilib-rium was reached between the temperature of the bath and the contents of the apparatus. This was ascertained as usual by awaiting constancy of the reading (N2) of the mercury on the scale. This scale reading was subtracted from the already-observed reading (N-^ ) of the mercury at exactly 0°C. before the f i s h flesh had been frozen. The difference (N^-^) in scale readings corresponded to the permanent change in volume that occurred in the sample of f i s h flesh due to freezing and subsequent thawing. The results are shown in "Results from Adjusted Data", part I I I . IV. The Coefficient of Cubical Expansion of Anhydrous Fish Muscle The coefficient of cubical contraction of anhydrous f i s h muscle was determined using the same appar-atus and the same procedure. This experiment was made mainly with a view to providing data needed in making certain adjustments in other experimental data. These 17. data were obtained for the f i r s t time, since they were not found in any available literature. The following procedure proved quite satis-factory in obtaining the requisite quantity of anhydrous f i s h flesh. Ordinary methods of dehydrating, like keeping in hot oven or immersing in absolute alcohol were avoided since the former method denatures the flesh by cooking and the latter also denatures the protein and possibly also extracts some constituents* About 500 grams of fresh llngcod flesh was finely ground to a brei and then transferred to a 500-ml narrow neck, round bottomed flask f i t t e d with a one-hole rubber stopper through which passes a glass tube.' The flask was immersed in a water bath kept at 40°-50°C and evacuated by connecting to a w.aterpump. The flask was shaken by hand occassionally. After four hours a con-siderable amount of moisture was found to have been re-moved from the flesh. The contents of the flask were transferred to a mortar and ground well. The resulting sticky powder was spread on a petri dish and kept over night in a vacuum desiccator over P2°5« Next day the granular mass was ground and passed through a ^ O-mesh sieve and the resulting powder was kept for two days in a vacuum desiccator over ¥2^5' 18. EVALUATION OF RESULTS A* Adjustments of Experimental Data Before interpreting the results the following factors affecting the observed values were borne in mind and allowance was made for them. During the lowering of the temperature of the bath from 4°C down to the freezing point of the f i s h flesh the apparatus and a l l i t s contents are contracting except the muscle Juice, which commences to expand (since water expands on cooling below ^°C.) and continues to do so slightly through any supercooled conditions, u n t i l i t begins to freeze. At and below the freezing point to a certain definite temperature, the fish muscle Juice progressively freezes and hence expands greatly, but the rest of the material, v i z . (a) hexane, glass, the solids of the f i s h muscle and the ice already formed undergo contraction; (b) the as yet unfrozen supercooled liquid undergoes expansion; and (c) the mercury in the apparatus is another important factor to be taken into account. During an experiment, as the mercury moves to different readings along the scale which i s at room temperature, there w i l l be a varying amount of mercury subjected to the varying temperature of the bath. Hence the net change in volume of the system recorded on the scale (due to changes in Fl&» „2 i*!o faco page 19) 19. bath temperature) ls the net result of the expansion due to the freezing of the f i s h muscle Juice (and the super-cooling of unfrozen liquid), and the contraction due to the rest of the material. The position of mercury on the scale reaches a maximum at a particular, definite temperature (below 0°C) and as the temperature is lowered thereafter, contraction due is noted on the scale because the expansion/to the freezing of muscle Juice ls less than the contraction due to the rest of the material. 1. Adjustment of Readings for Change in Volume with  Temperature of Organic F i l l i n g Liquid. Fig. 2 gives the scale correction for volume change of ^0 ml. of hexane (from petroleum Eastman Kodak) as i t s temperature f a l l s below room temperature (20°C). This graph was constructed from data on the density of "hexane from petroleum", Hell (1932). To apply the cor-responding correction for any other volume V of hexane of o measured at room temperature 20 C as i t s temperature f e l l below this temperature, the result obtained from the expression V x Scale Correction, was added to the observed scale reading. 2. Adjustment of Readings For Change In Volume  with Temperature of Glass Apparatus One cc. of pyrex glass contracts 0.0000108 cc, for each degree of temperature drop. Therefore, 55 cc. 200 250 3 0 0 350 SCALE DIVISION READING NOMOGRAM FOR READING THE SCALE CORRECTION  DUE TO TEMPERATURE DIFFERENCES  OF 80 GRAMS OF MERCURY, (To face page 20) 2 0 . of volume enclosed by pyrex glass contracts 5*^ scale divisions when the temperature of the glass f a l l s from +20°C to -30°C. This correction is so slight that for inter-mediate temperatures the proper correction to be applied was readily estimated without a graph. 3. Adjustment of Readings for Change in Volume with  Temperature of Mercury Indicator Liquid A nomogram was constructed to apply the proper scale reading for change in volume of the mercury from one temperature to another. (Fig. 3) The following expression was derived for the above purpose using the recognized densities of mercury and certain ascertained characteristics of the apparatus. With the apparatus empty and with enough mercury in the capillary, the scale reading R at room temperature T was taken from the point L ( = average level to which the liquid in the cooling bath comes). Then the apparatus was t i l t e d gently so that this same volume of mercury lay, entirely in that portion of the capillary against the scale. The two scale readings RQ_ and R 2 opposite the ends of the mercury thread were then recorded, the mercury was emptied out, and weighed. Let length of mercury column within the capil-lary between ^ and R2 - (R 2 - Ri) scale readings at 21. temperature T°C. Weight of mercury = W grama (kept constant in several experiments) Temp, of mercury = T°C If density of mercury (from tables) at T°C = Dtp then volume of mercury = W Dip And value of one scale division in the bore of the capillary tube = W / (Rg-R-^ - w - Q DT D T (R 2 - Ri) C la constant as long as the same capillary tube is in use. Also; W - CR = E = volume of the mercury from D T point (L) to zero point on capillary scale. This value E always remains a constant during an experiment. Then, i f R^  i s the reading on the scale at any given temperature Tg at any stage of an experiment, the scale reading correction to be added is obtained from the expression: W - DmE - D TR 3 X D B - Dip D BD T where Dg s Density of mercury at the bath temperature Tg. This last expression was equated to successive integral values 1 to 8 scale reading corrections. For each of these corrections, a series of values of was 22* was found by substituting for Dg densities of mercury at a selection of temperatures from 4,20° to -38°C. Plotting these corrections for against Dg gave the family of lines shown in the nomogram (fig . 3 ) which was then of considerable value for ascertaining the proper scale read-ing correction for observed values of R^ and Tg. In some experiments, a different weight W of mercury was used in f i l l i n g the apparatus. In this case, a different nomogram had to be constructed, but the prin-ciple used in correcting observed scale readings was exactly the same as the above. i+. Adjustment of Readings for Change in Volume with  Temperature of Anhydrous Fish Muscle When 25.00 gm (= 19.5 cc.) of anhydrous lingcod muscle was used in the dilatometer to determine the co-efficient of expansion of the anhydrous flesh, i t was necessary for experimental reasons to use 30.26 cc, of hexane to f i l l the remaining space in the barrel. This relatively large volume of hexane in comparison with the volume of the anhydrous f i s h muscle caused inherent d i f -f i c u l t i e s in adjusting observed values of the volume of the system at different temperatures, owing to a slight uncertainty in the physical constants for the hexane. There were no uncertainties in the contraction constants for the glass and mercury. 23. After calculating the values for contraction of the hexane, and adding to these the contraction of the glass and mercury at the various temperatures from +20°C to -30°C, then using the totals as f i n a l adjustments for the observed values of the contraction of the system as a whole, a slightly irregular curve was obtained when plot-ting volume of the anhydrous muscle against temperature. The straight line best representing this curve was drawn, end from i t the appropriate values were read for evaluat-ing the expression for coefficient of expansion: V T = V t ( l + oC(T-t)) where = volume of anhydrous flesh at temp.T = +20°C = volume of anhydrous flesh at temp.t = -30°0 °C = coefficient of cubic expansion over the temperature range -30°C to +20° 19.50 = 19.39 f l + o C{20 - ( -30) } | whence oC z 0,000118 The graph prepared is not illustrated, since i t s nature and use were similar to that shown for the change in volume of hexane ( f i g , 2 ), Corrections to ob-served scale readings to compensate for changes In volume of the solid constituents of the flesh were read and com-puted directly from the graph. The corrections were quite small. 2k. 5. Adjustment of Readings for Change In Volume with  Temperature of Preformed Ice In Fish Muscle. Since any ice formed at a given temperature below the freezing point of the flesh would contract on further cooling, i t was necessary to know this contraction to be able to adjust the observed scale readings at the lower temperatures. But before this could be done, a f i r s t estimate or approximation of the amount of ice present at each temperature had to be secured. This was computed by f i r s t assuming that the known weight of water in the sample of flesh a l l turned to ice, and calculating the expansion that would have occurred. Then, from the adjus-ted observed expansion, the f i r s t approximation of the percentage of ice formed at each temperature was found. Finally, from data in the International C r i t i c a l Tables on the coefficient of cubic contraction of ice at different temperatures below 0°C, the contraction of the ice formed at one temperature as i t cooled to a lower temperature was read from a graph (not shown) and these values were used in obtaining final adjusted values, 6. Adjustment of Readings for Change in Volume with. Temperature of Supercooled Aqueous Liquid in  Fish Muscle. The procedure used here was similar to that described above for ice. Once the f i r s t approximation for 25. the amount of ice formed at each temperature waa obtained as mentioned, a f i r s t approximation for the amount of supercooled unfrozen liquid was known, since: % supercooled liquid = 100 - % water already frozen to ice. Then, from this f i r s t approximation of the amount of super-cooled liquid present, the amount of i t s expansion as i t became colder was obtainable. Unfortunately there appeared to be no data on the density or specific volume of water supercooled below - 1 3 ° C . in the literature, although water supercooled to - 7 2 ° C , is known. However, in a book hy Dorsey, an expression was found that warranted extrapol-ation to lower temperatures: e 3 * Vr c = ' u 7 _ _ _ _ _ — By allowing V^OQ s 1, a table was prepared showing specific volumes of 1 gram of supercooled water at temperatures below - 1 3 ° C . (e.g. specific volume of supercooled water at -30°C a 1,0171). From the table of specific volumes, the contraction of any f i r s t approximation to the weight of supercooled water as i t cooled s t i l l further could be found, and then these values were used in obtaining final adjusted values, B . Results From Adjusted Data, I (a) Freezing point of llngcod flesh. The I n i t i a l freezing point of different samples of llngcod flesh due to unavoidable supercooling varied 2 6 . from - 3 . 6°C ( 2 5°F) to -7.8°(18°F). ,But the true freezing point obtained aa described in the experimental part was the aame for a l l the aamples, namely -1 .5°C(29.1°F)• On thawing and re-cooling, the aupercooling was again observed to a varying degree. (b) Freezing point of Rainbow Trout flesh. The I n i t i a l supercooled freezing point of one sample of rainbow trout flesh was found to be -6.8°C, and the true freezing point -1 .5°C. II. Percentage of Water Frozen Out at Different  Temperatures. Tables 1 and 2 below show the f i n a l calculated values of the percentages of the total water present in f i s h flesh that is frozen out at different low tempera-tures. The i n i t i a l value for rainbow trout flesh ia at the true freezing point of -1.5°C, while the i n i t i a l value for lingcod flesh is given at the supercooled i n i t i a l freezing point. In each case, the i n i t i a l value is shown as zero to indicate the condition when freezing was Just on the point of beginning. A l l values have been subjected to a l l the six adjustments described^ 27 TABLE 1 Freeh Rainbow Trout Flesh Temp. °C. % Total Water Frozen Out TABLE 2 Freah Lingcod Flesh Temp. °C. % Total Water Frozen Out - 1 . 5 0 - 7 . 6 0 2 0 . 2 - 7 . 8 8 6 » > - 5 . 0 7 9 . 1 - 9 . 0 88 .0 - 6 . 0 82 .9 - 1 2 i 0 9 1 . 7 - 7 . 0 8 5 . 7 - l l f . O 9 2 ^ 8 - 8 . 0 8 6 . 9 - 1 6 . 0 9 ^ . 0 - 1 0 . 0 9 0 . 2 - 2 0 . 0 9 6 . 0 - 1 3 . 0 9 2 . 8 - 2 5 . 0 9 7 . 9 - 1 5 . 0 9 ^ . 8 - 3 0 . 0 9 9 . 7 - 1 9 . 0 9 6 . 8 - 3 1 . 6 9 9 . 7 - 2 3 . 0 9 9 . 3 - 2 5 . 0 9 9 . 5 - 2 6 . 0 1 0 0 . 5 -28.0 1 0 0 . 1 - 2 9 . ^ 1 0 0 . 7 -III. Permanent Change In Volume of Flah Flesh Caused by Freezing and Thawing. The number of scale divisions corresponding to the permanent change in volume was 5 . 5 . The weight of 28. fleah taken (llngcod) waa ^0.98 gms. Since the specific gravity of f i s h flesh ls very nearly 1, the volume of the flesh was taken to be tyO cc. The volume occupied by 1 scale division of mercury was known to be 0.005^6 ccs. Hence the volume occupied by 5*5 scale divisions of mercury s 5-5 x 0.005^6 ccs. = 0.03005 ccs. This change in volume is due to *K) cc. of f i s h flesh alone since the apparatus and the rest of the material Inside It have so far not been shown to exhibit such phenomena. No adjustment of observed values were necessary since a l l readings were at the same temperature. Hence the permanent change in volume that occurs In f i s h flesh during freezing and thawing = 0.03005 x 100 = 0.075^ IV. Coefficient of Expansion of Anhydrous Fish Muscle. This was one of the adjustments necessary in the determination of the percentages of water frozen out at different temperatures, as previously explained. The manner in which the coefficient was calculated has been described already in part k under "Adjustment of Experi-mental Data". Therefore, only the f i n a l result is repeated here: <£- = 0.000118 over the temperature range +20°C to -30°C 2 9 . DISCUSSION So far the methods used for freezing point determinations of fi s h muscle have yielded insufficiently accurate results. Whole fi s h were kept in temperature-controlled chambers. The f a l l in the temperature of fish muscle due to progressive cooling was measured by inserting thermo-electric couples or similar temperature-measuring devices inside the f i s h muscle. The freezing point was taken as that temperature at which the reading of the Instrument was constant for a considerable period due to giving up of the latent heat of freezing. The drawbacks of the above method were several, one in particular being the lack of a definite level plateau in the curve showing the relation between time and temp-erature. The method described in this thesis involves the use of the dllatometer by means of which the sudden expan-sion due to freezing can be easily observed, principally due to the fact that water expands some ^% of i t s volume on freezing. This is the f i r s t time that this apparatus has been used with a l l possible refinements to record the true freezing point of f i s h muscle and other data. The operation of the apparatus is simple and the results can be accurately reproduced. 30. Attempts were made to find out whether once frozen and thawed, examples of fish flesh exhibited any difference in the further magnitude of the freezing point range. It was found with certainty that the true freezing point can never be obtained in the i n i t i a l freezing without a seeding crystal of ice. In other words, there was a state of supercooling of the flesh before i t actually began to freeze. Experimentally, this was overcome as described by f i r s t supercooling to the point of Incipient freezing, then taking advantage of the ice crystals formed, to determine the true freezing point. Another important result obtained revealed that the true freezing point of^marine and fresh water f i s h flesh used in these experiments was substantially the same, though they showed varying degrees of supercooling. A non-oily type of fish like llngcod was chosen for the experiments described since presence of a high per-centage of o i l in the flesh would Interfere with the expansion due to water alone, since o i l has not the same coefficient of expansion as water, and the coefficient of expansion of o i l ls seriously disturbed by separation of solid fats (stearine) on cooling. Although Reay (1933) and Finn (1933) have ex-amined the effect of various degrees of cooling upon the flesh of f i s h and found that the degree of freezing and the temperature of storage have a pronounced effect on the 31. texture of the proteins in the muscle, l i t t l e quantitative data on the magnitude of observable physical effects accompanying the denaturatlon of the proteins of the fish flesh is available. In my investigation i t is found that there is a definite permanent decrease in volume after freezing and thawing, Since the quantity of f i s h used in this investi-gation had to be limited to small quantities (about kO gms.), and the duration of storage after freezing was also small, the magnitude of the change in volume was consequently small, nevertheless quite perceptible. It i s hoped to devise further experimental methods whereby larger quantities of f i s h flesh can be frozen and kept for a longer length of time, and the magni-tude of the change in volume determined under more varied conditions. In the experiments recorded herein dealing with the percentages of water frozen out at different tempera-tures below the freezing point, previously determined data such as those by Carter and Frost were confirmed, but the present values have been calculated from the observed data using refinements not taken into account by them in their preliminary work. However, some of the refinements are known to be s t i l l inadequate. The relatively large colume of hexane needed to f i l l space around the flesh sample, and the high 32. coefficient of contraction of hexane, both tended to intro-duce slight discrepancies that are rapidly magnified by unavoidable d i f f i c u l t i e s in manipulation. These discrepan-cies were kept to a minimum, but showed up particularly when determining the coefficient of contraction of the anhydrous flesh. The irregularities mentioned as having occurred in the curve plotted to show the volume variation with temperature were such as to indicate the adjustments of the observed readings for contractions of the hexane were inexkct. Discordant data in the literature for the coef-ficient of contraction of ice, and lack of definite values for the coefficient of expansion or relative volumes of water supercooled below -13°C, a l l contributed to the un-certainties surrounding the adjustments for these effects below -13°C. Fortunately, the percentage of supercooled water present was least at the low temperatures where the uncertainty regarding the correction was greatest. The f i r s t approximation method used for the percentages of ice formed and supercooled water remaining was essential; hut i t was considered unwarrantable to perform a second approxi-mation to these values before computing the f i n a l values given in tables 1 and 2. The values exceeding 100$ in table 1 are of course Impossible theoretically; but the slight excesses over 100$ are considered inconsequential in 33. view of the numerous adjustments that had to be made to the original observed readings. These slight excesses in no way detract from the value of the results, and were not present in the results in table Z. The chief interest in tables 1 and 2 l i e s in the percentages of water frozen out at around -20°C (-^°F) because modern recommendations for commercial storage and shipment of frozen fish point to a temperature of 6°F as being the highest temperature compatible with maintenance of prime quality of the frozen product. Tables 1 and 2 show that above this temperature there is more than 5$ of unfro-zen aqueous li q u i d present in the unfrozen product. It i s probably this liquid phase that allows such deleterious effects as rancidity, denaturation, and the l i k e , to progress more rapidly above 0°F than below 0°F, Finally, i t should be confessed that i t is realized there may be other slight uncertainties in the accuracy of the results in tables 1 and 2, This investi-gation could not be extended to explore f u l l y the probabil-ity that what has been called "ice'1 at low temperatures is not really pure water ice; li k e l y i t is a complex mixture of various eutectlcs of salts occurring in the flesh tissue Juices. Also, what has been called "supercooled water" is probably a dilute solution of salts. These would not have the same physical properties as ice and water, but a com-plete investigation of the properties would be very complex, and beyond the scope of this work. 35. SUMMARY 1* The true freezing points of llngcod (Ophlodon elongatus) and Rainbow Trout (Salmo galrdenerll) flesh were determined and found to be ~1.5°C (29.1°F). 2. The percentages of water frozen out from the flesh at temperatures below the freezing point were found to Increase rapidly for the f i r s t few degrees below the freezing point, then more slowly, reaching 9 5 $ at about -18.8°C (0°F), then approaching 100$ asymptoti-cally at s t i l l lower temperatures down to - 3 0°C. 3 . The permanent decrease in volume of f i s h flesh at 0°C. after freezing and thawing was observed to be of the magnitude of 0 . 0 7 5 $ based on the original volume of flesh used. This change in volume is most probably due to denaturatlon of the proteins of fish flesh. The Coefficient of Cubical Contraction of anhydrous fish muscle has been determined for the f i r s t time and found to be 0.000118 over the range *20°C. to -30°C. 3 6 . ACKNOWLEDGEMENT I hereby wish to express my deep gratitude to the chairman and members of the selection Board, Education Department, Government of India for the award of one of the Foreign Scholarships to pursue higher studies in fisheries technology with special reference to f i s h o i l s and processing.. My thanks are due to the University of British Columbia, Vancouver, B.C., for enabling me to register myself for the M.A. degree and the Pacific Fisheries Experimental Station, of the Fisheries Research Board of Canada, for laboratory f a c i l i t i e s afforded in conducting this investigation. I wish to express my sincere thanks to Dr. N.M. Carter, Director, Pacific Fisheries Experimental Station for his s i x g g esting the problem, and his kind help in details of the experimental work and his valuable advice and criticism during the course of my stay. The assistance of Mr. N.E. Cook, Chemist at the Fisheries Experimental Station, in modifying the apparatus used is gratefully acknowledged. My thanks are also due to Dr. W.A. Clemens, Head of the Department of Zoology, and Dr. W.S. Hoar, Professor of Zoology and Fisheries, University of Br i t i s h 37. Columbia, for their keen Interest in the above investigation and supervision of my academic work. Acknowledgment is made to Bri t i s h Columbia Provincial Game Department for kindly providing us with a sample of freshly caught Rainbow Trout. 38. LITERATURE CITED Beatty, S.A. and Gibbons, N.E. (1935), Atlantic Prog. Rep.. B i o l . Bd. Canada, No.15, 1935. Carter, N.M. (19^4). Unpublished. Carter, N.M. and Frost, P.J. (19^5), Prog. Rep. Pacific Coast Stations, Fisheries Res. Bd., Canada, No.62, 21, 19^5. Dorsey, M.E. (19^)» "Properties of Ordinary Water Sub-stance", Rheinhold Publishing Co. 19 -^0. Finn, D.B. (1933). Cont. Can. Biol. N.S. 8, No.25, 1933. Finn, D.B. (1932). Proc. Roy. Soc, B .3, 356, 1932. He'll, L.M. (1932). Phys. Rev. 39, 670, 1932. Moran, T. (1932).. J.S.C.I., 51, 16, 1932. Reay, G.A. (1933). J.S.C.I., _52, 265, 1933. Young, 0,C. (1935).. Pacific Prog. Rep. Biol. Bd. Canada, No.26, 1935. 


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