The Nutritive Value of Marine Products: XVI. The Biological Value of Fish Flesh Proteins

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  • The Nutritive Value of Marine ProductsXVI. The Biological Value of Fish Flesh Proteins

    Bv J. M. R. BBvBnncB

    Pacific Fisheries Experimental Stat'ion

    (Received. for publication June 20, 19/16)

    ABSTRACTThe biological values of the crude flesh proteins of lingcod, halibut, lemon sole, and white

    spring salmon have been determined by growth and .nitrogen retention methods using youngWistar rats as experimental animals. Additional valups obtained by the. growth method arereported for herring and red snapper. For comparative purposes, values were also secured oncrudebeeff leshproteins,eggalbumin,andcasein. Thef iguresobtainedfor thef leshproteinsfromthe four types of fish were of the samc order of magnitude, and'they were higher than thoseprocured for the other protein foods tested. An examin,atiori of the gains in weight was carriedout by the co-variance method in which the variance of the actual gains, freed from the effectsof varying food consumption by regression, was subjected to analysis. This test indicated thatthe differences in the average weight gains between the rats fed fish flesh and those fed theother protein foods were highly significant. Data on the effect of sek on the determination ofbiological values are, subrnitted along with an explanation which helps to resolve conflictingreports on th is point .

    Recent work demonstrating the-deleterious.effects of protein deficiency onthe healing of wounds (Thompson, Ravdin and Rhoads 1938; Morris, Dubnikand Dunn 1945), resistance to infeotions (Cannon et al. 1943, 1944), and preven-tion of chloroform liver injury (Nliller, Ross and Whipple 1940) has arousedincreased interest in the dietary significance of protqins. Several reviews pub-lished in the last year or two cleal adequately with these and other aspects ofprotein nutrition in health and disease (cf. Elman 1944; Stare, Hegsted andMcKibbin 1945; and Co Tui i946). These reports emphasize the desirabilityof having- data readily available on the nutritive value of the Tain protein foodstuffs. A relatively abundant literature exists regarding the proteins of beef,egg, milk, and cereals, but amongst the more important protein foods, least isgenerally known and published respecting the nutritive properties of fish fleshprotiens. Indeed in a number of reference books and reviews on protein nutri-tion no data whatsoever can be found on these nutrients. In order to supplythis information in greater degree the work here reported was conceived andcarried out.

    At the suggestion of Dr. N. M. Carter, Director of the Pacific FisheriesExperimental Station, four commercially important types of fish were chosen forthis studv: lingcod (Ophi,odonelongatus),halibut (Hippoglossus stenolepis),lemon

    J. FIsn. Rrs. Bo. CeN., 7 ( l) L947.Printed in Canada.

    35

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  • 36

    sole (Parophrys uetulus), and white spring salmon (Oncorhynchus tshawytscha)'Additional determinbtions were subsequently performed on herring (Clupea pal-

    lasi,i') and red snapper (Sebastodes ruberrimus). Other protein preparations in-cluded in the series for comparative purposes were those of beef flesh, egg albumin,and casein. The latter was a vitamin-free product, Labco brand, and the albu-min was a pan-dried product from the firm of Canada Egg Products. The beefflesh protein preparation was obtained by processing similar proportions of allthe wholesale cuts and was thus roughly representative of the whole steer: roundsteak 2.5 lb., sirloin 2.5 Lb., tenderloin 1.5 lb., stewing meat (f lank and shoulder)6 lb., prime rib roast 2.5lb. (1 lb. equals 0.a5 kg.)

    EXPERIMENTAL

    The fish were prepared in the form of fillets or steaks and cooked one hourat 99 to 100'C. in a steam autoclave. The cooked product was then pressed,minceci, and partially dehydrated in a warm air tunnel at 60'C. The cookingliquors were saved, separated from accompanying oil or fat, and evaporated to athick syrup in vacuo on a water bath at 40 to 50'C. The concentrate wasthen re-incorporated into the partially dehydrated minced flesh and the wholere-dried at 50oC. to a moisture content of.2 to 4 per cent. The preparations werethen ground into a fine ?neal. The beef flesh was treated in the same mannerexcept that as much as possible of the fat and discrete connective tissue werediscarded before cooking and any observed after cooking was also discarded'This was done since one of 'the intended features of the investigation was to testonly that part of the protein foods which is usually consumed by man. It wasassumed that the gross connective tissue did not come under that category. Thebooking liquors were retained in the protein preparation because such a procedure

    simulates best household practice.In the five feeding tests described below, fresh protein preparations were

    made for each trial, with the exception of the halibut of trial 5, and the lingcod,lem_on sole, and white spring salmon preparations listed in table lV. As a neces-sary preliminary task, moisture, nitrogen, and fat determinations were carriedout on each protein product. The diets (table I) were then made up on the basisof these figures to contain the s4me amount of crude protein (Nx6.25) and to beof isocaloric value. Nitrogen determinations were carried out on certain of thediets and amounts agreeing closely with the expected values were obtained.The rats were of the Wistar strain and in every case they were divided amdngstthe different groups according to weight, sex, and litter. They were horjsed in aroom at a controlled temperature of 20 to 22"C. in individual cages having ]-inchmesh screen floors.

    The ideal diet utilized to determine the biological value of proteins should beadequate for maximal growth except with respect to protein. Perhaps the mostdifficult problem confronting anyone attempting to determine the biologicalvalue of proteins is the provision of B-vitamins without the introduction ofprotein material other than that being tested. This obstacle has diminished tosome extent in recent years, owing to t\e preparation of certain of the B-vitamins

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    in pure form. Evidence is herewith presented to shorv that under the conditionsset forth in this paper, protein is just as efficiently utilized when the followingfive crystalline vitamins are supplied: thiamine, riboflavin, pyridoxine, calcittmpantothenate, and nicotinamide, as when yeast and liver concentrates, aresupplied. as sources of the B-vitamins. In order to demonstrate the adequacyof the basal .diet, control diets were fed containing adequate amounts of proteinfor .maximal growth. In three of the feeding trials herein reported, the testprotein was fed at a level of 8/6., and at 10 and 12/6in the remaining t5ials. Thelevel of 8To was chosen so as to allow a moderate degree of growth which wasdefinitely below the maximum. These conditions have been found to be rvellsuited to the type of work under investigation (Osborne, Mendel .and Ferry1919). Alevel of I2/s test protein was utilized in the fourth feeding trial todetermine whether or not the differences in biological values' observed at alevel of 8/6 protein were maintained.

    Table I shows the composition of the basal (A) and control diets uti l ized inthe feeding experiments.

    Tesr.o r. Percentage composition of diets

    Con'rponents G

    Test proteinCasein (a)Beef drippingCorn oil (b)Sucrose '

    Salts (c)AgarCod liver oil (d)Vitamin mixture (e)YeastYeast concentrate (f)Liver concentrate (g)Choline chloride

    6352I1

    202020

    0.25o.250.20

    20

    62.9tr

    2

    11

    8

    101062.3

    o

    21I

    0.25o.250.20

    t2

    101058.352

    It

    2020

    51o

    21I

    1

    0.1

    20101050.352I

    1

    20101048,.8

    2I1

    45D

    2I15

    1.00.1

    0.25 |o.2510.201

    110.2

    (a) Labco brand, vitamin-free. (b) Mazola. (c) (Beveridge and Lucas 1945). (d) Mead's,1800 I. U. of vitamin A per g. and i75 LU. of vitamin D per g. (e) The vitamin mixtureis made up'of powdered sugar and crystalline vitamins (obtained from Merck) so that 10 g,food supplied 257 thiamine hydrochloride, 407 riboflavin, 207 pyridoxine hydrochloride,100'y calcium pantothenate, 100"y nicotinamide. (f) Harris Laboratories, Tuckahoe, NervYork. (g) Eli Li[y and Co., Indianapolis, U.S.A. *Cholinc chloride included in vitaminmixture so that 10 g. food supplied 10 mg. choline chloride.

    FeBnrNc Tnret. 1

    The fact that biological values derived from iats with widely different foodintakes possess only doubtful comparative significance is one which is well knorvn(Osborne, Mendel and Ferry 1919). An attempt was therefore made to perform

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  • 38

    the experiment on a paired feeding basis. Since some preliminary ',i','ork had

    indicated that the lemon sole and lingcod diets were perhaps slightly less palatable

    than the other rations,.the animals were paired-fed on their mates eating the

    lingcod preparation

    Taer,p II. Biological values of proteins of fish and beef flesh and of egg albumin' Average

    ini t ia l wt . of rats:67.6 g. Range: 4O-97 g, . . Days on test d iets;28. No. of rats

    per group: 10. Basal'diet: A. The rats were paired-fed on the corresponding animals

    eating the lingcod diet which was given ad libitum.

    Source ofdietary protein

    LingcodHalibutLemon soleSalmonBeefEgg albuminControl diet E

    Daily foodintake

    (9. ,

    9.69.09.09.19.06 , 1

    9.0

    Average gain' in wt.

    (g ' )

    56.350.350.152.O46.645.676.6

    Gain in wt. perg. protein

    eaten, ( g . )

    2.622.472.502.562.312.34

    In the results (table II), an examination of the food intake reveals that the

    attempted paired feeding technique was not entirely successful. This was due

    to the fact that the palatabilities of the diets were essentially the same. The rats

    on the diet that was fed ad libitum, ate from 5 to LO/s more food than did those

    on the other rations and for that reason greater gains were made and higher

    biological values observed in this group (cf. Osborne et al. 1919). The gains and

    biological values induced by the three other fish flesh diets were alrnost identical

    and greater than those brought about by the beef flesh and egg albumin diets.

    It may be noted that the rats on control diet E, identical to the test diets except

    that it contained an adequate amount of protein for growth in the form of 2O/s

    vitamin-free casein, made an average gain of 76.6 grams. This indicated that

    the chief l imiting factor in the test diet was protein.

    FepnrNc'I 'nrer- 2The results of the first feeding trial indicated the difficult nature of the task

    of carrying out successfully the paired feeding technique on animals fed the diets

    under discussion. Further, it had become obvious that although results such as

    had been obtained were of some comparative value when arrived at consistently

    in lepeated experiments; nevertheless,'the large variations obseived in the valueswithin groups made it extremely difficult to demonstrate any clear cut superiorityof one protein food over another. The principal factor contributing to the large

    variation in biological values was found to be the wide range in initial weight.Accordingly rats weighing 50+2.5 grams were used in the subsequent feeding

    trials. In view of the similarity in palatability of the different diets, it was decided

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    to let the animals eat ad l ibitum except when any rat started to consume markedlymore than the others. In such cases the amount of food offered was lowered.Only one change, a trivial one to facilitate the preparation of the diets, was madein the 6asal diet;choline chloride was incorporated in the diet along with theother dry ingredients instead of being included in the vitamin mixture. Since'Mitchell and Carman (1926) have shown that the protein content of new tissueis not always the same on different diets, the nitrogen retention method ofMcCollum and Shukers (cf. McCollum and Simmonds 1929) was utilized. Acomparison was thus made possible between the two methods of determiningbiological values and in addition information was gained regarding the effects ofthe different diets on the composition of new tissue. Individual nitrogen deter-minations were performed on a control group of ten rats at the start and oneach test rat at the end of the experiment. The intestines were stripped of theircontents and the carcasses hydrolyzed with 8 to 10 N H2SO4 for 10 to 12 hours.Tl-re resulting hydfolysate was made up to volume and aliquots were thenpipetted for macro-kjeldahl determinations. A statistical analysis of the percent-age nitrogen compositions of the different groups revealed that there were nosignificant differences. Z value for p :0.05 was 0.8639, found: 0.3724. Theaverage values agreed within a narrow range (2.72 to 2.83/s). It thereforeappeared that under the conditions herein described, gain in body weightafforded a true indication of protein anabolism. Significant in this respect isthe close parallelism of the values obtained by the nitrogen retention andgrowth methods (table III).

    TesLa ll l . Biological values of f ish and beef f lesh proteins. Init ial wt. of rats:50 +2.5 g.' Days on test diets: 28. Number of rats per group: 10. Basal diet: B (same.as

    basal diet used in feeding trial no. I except that choline chloride was not included. in the vitamin mixture but added along with the other dry ingredients).

    Source ofdietaryprotein

    .LiirgcodHalibutLemon soleSalmonBeef

    Daily foodintake

    (g . )

    Av. gain

    in wt,(s.)

    Gains corr.for foodintake

    (g . )

    Gain in wt.per g'

    proteineaten

    /6 Nitrogenretained of

    nitrogen

    ingested.

    8.99 .19 .28.98.9

    61.864.463.062.556.3

    63.063.661.463.156.9

    3.113.153.043,122.80

    56.9D l . l

    55.455.050.6

    A statistical analysis of the gains was carried out by the covariance methodin which the variance of the actual gains, freed from the effects of varying foodconsumption by regression, was subjected to analysis. This procedure wascarried out in preference to an analysis of the gains in weight per gram of proteineaten, because, as Crampton (f93a) has pointed out, a statistical analysis of thelatter ratios includes "in addition to the experimental error, a systematic errorwhich may be of sufficient magnitude to seriously distort the result, and vit iate

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    the estimate of experimental error and hence any test of significance." The

    systematic error is of course owing to the fact that no allowance is made in the

    calculation of the biological values for the amount of food required for mainte...