CO~l lERCIAL FISHERIES REVIEW Vol. 20, No. 8

AMINO ACID COMPOSITION OF THE PROTEIN AND INORGANIC CONSTITUENTS OF THE ASH OF

POLLOCK FISH SCALES By Donald G. Snyder*

ABSTRACT

THE AMINO ACID COMPOSITION OF THE PROTEIN OF pOLLOCK FISH SCALES WAS DE­TERMINED BY PAPER PARTIT ION CHROMATOGRAPHY AND MICROBIOLOGICAL ANALYSIS. THE DATA I DICATE THAT THE PROTEIN OF pOLLOCK FISH SCALES IS PROBABLY A SCLEROPRO­TEIN OF THE COLLAGEN TY PE CONTAINING HIGH LEVELS OF GLYCINE AND AMOUNTS OF ARGI INE AND SERINE ABOUT EQUAL TO THAT FOUND IN THE PROTEIN OF WHOLE EGG. THE SCALE PROTEIN CONT AINS FAIR AMOUNTS OF ALANINE, PROLINE, AND HYDROXYPROLINE, A <D EXTREMELY SMALL AMOUNTS OF CYST (NE AND TRYPTOPHAN. ALSO, THE SCALE PROTEIN CONTAINS SLIGHTLY LESS ASPARTIC ACID, GLUTAMIC ACID, AND THREONINE, AND MUCH SMALLER AMOUNTS OF HISTIDINE! ISOLEUCINE). LEUCINE, LYSINE, METHIONINE, PHENYL­ALANINE, TYROSINE, AND VALIN ~ THAN DOES WHOLE-EGG PROTEIN, THE INORGANIC CON­STITUENTS OF THE ASH OF pOLLOCK SCALES WERE DETERMINED SEMIQUANTITATIVELY·BY SPECTROGRAPHIC ANALYSIS, THE DATA INDICATE THAT NO UNUSUAL KINDS OR QUANTITIES OF I ORGANIC CONSTITUENTS THAT MIGHT CAUSE BIOLOGICAL INJURY WHEN FED TO ANIMALS ARE PRESENT IN THE ASH OF THE SCALES, AN INTERPRETATION OF THESE FINDINGS IS MADE IN RELATION TO CONCLUSIONS OBTAINED FROM A PREVIOUSLY REr,oRTED RAT-FEEDIN~ STUDY ON THE COMPARATIVE NUTRITIVE VALUE, DIGESTIBILITY, AND BIOLOGICAL VALUE OF pOLLOCK-FISH-SCALE AND CASEIN-LACTALBUMIN PROTEINS.

INTRODUCTION

During the past few years, the fishing industry has had the increasingly oiffi­cult problem of annually disposing of thousands of tons of fish scales because fillet

d fish now have replaced unprocessed fresh fish in sales volume. Harbors that clos to plants normally provide an inexpensive area of scale disposal, but unless tidal flows are strong, pollution m y result.

Chemical engineers and pharmac utical houses have been

ontact d to obtain suggestions for utilIzing th scales, but thi,s

pproa h has been unsuccessful. ttempts to use the scales as

f rtlhzer on local farms also h not m t with success,

RESEARCH AND

2 August 1958 COMMERCIAL FISHERIES REVIEW 5

Recently, however, as part of a gener al study to d et e rmine whether scale s may have value as a supplemental source of prote in in the d iet s of animals, Snyder and Nilson (1957) conducted rat-feeding tests to compare the nutritive value for growth, the biological value for maintenance, and the d ige s tibility of pollock-fish scale and casein-lactalbumin proteins. The data from the s e studie s indicate that pollock-fish scale protein is digested about 80 percent, but i s u tili zed about 30 percent less ef­ficiently than is a protein supplement consisting of thr ee parts casein and one part lactalbumin (CL), The data also indicate that fish scal e s a s the only source of pro­tein in a diet containing 9 percent protein are inc ap able of supporting growth of young rats but can be utilized as a limited sourc e of prote in when supplemented with CL protein. Increased utilization of pollock-fish s c ale protein, in combination with stepwise higher levels of CL protein, indi cate th at n o toxic substances per se for growing rats are present in the scale s. - -

The nutritional inadequacy of pollock -fi sh scal e protein alone and the increased utilization of that protein in c ombination with stepwise high er levels of CL protein were interpreted as like ly due to a d efi cien cy and/ or imbalance of specific nitrogen nutrients in pollock-fish scales. This interpretati on was supported also by informa­tion obtained in the literature c onc e rning deficiency states caused by feeding similar types of waste protein that serve a protective function in the animal organism, such as wool (Routh 1942), hog hoofs (Wagner and Elvehjem 1942, 1943), and chickenfeath­ers (Wilder et a l 1955). Chemic al analy s es of similar proteins that have a support­ing function in the animal organism, such a s collagen (Bowes et al 1955) and elastin (Graham et al 1949) have indicated deficiencies of some "essential" amino acids.

Information on the amino acid-composition of pollock-fish scale protein was considered nece ssary, the r efore, to evaluate properly the conclusions suggested from the result s of th e rat - feeding study. A knowledge of the inorganic constituents present in the a sh al s o w as deemed desirable, since the scales contain nearly 40-percent ash, pre sumably as various apatites (Corti and Keller 1952), which might contain unusual inorganic constituents or great excesses of other constituents that could affe ct nutritive qu ality.

MATERIAL AND ANALYSIS

The fish s c ale s received at this laboratory for these studies were furnished by the staff of the Fishery Technological Laboratory, U. S. Bureau of Commercial Fish­eries, East Bo s ton, Mass . P o llock (Pollachius virens) were scaled by hand. The scales were washe d thoroughly with water, drained, and spre::td in pans to dry in an oven at 1000 C. They then were sh ipped to this laboratory, where they were ground, as finally as pos s ible, in a Hoba rt coffee grinder.

No great v ari ation was found in the moisture, protein (N x 6.25), fat, and ash of representative sampl es of scales from the various lots. The mean and ran.ges of mOisture content for 4 l ots of scales were 4.4 and 1.0-6.9 percent, respectively; the protein content fo r 11 lots was 59,5 and 56.6-62.5 percent; the fat content for 4 lots was 0.004 and 0.0-1.0 percent; and the ash content for 5 lots was 38.9 and 36.1-43.1 percent. The method s of analyses of the Association of Official Agricultural Chem­~ (1955) were u sed.

A re~resentative sample of ground scales was chosen ,randomly from one of the lots r ecelVed a t thi s L aboratory for use in the present stuay. .The means of three analyses each of this sampl e of scales on a moisture-free baSiS were 60.1 percent for prote in and 39.4 per cen t for ash.

. No Significant diff e ren ce was found between the nutritive value of the scale pro­tem from the lot of s cales from wh ich this sample was obtained and that of the va­n ous othe r lots of s c ales It can be concluded therefore, that the sample from thlS lot f . '. 11 dif a scales was representative of the scales of pollock III general. The sma -

6 MMI',I{ IAJ r·JSIIJ~IUI'. HI. II :.

ferenc s in moistur " pr ot tn, fat, nd' h lit! nts fouO( scal s probably ( an b<: attrlbu l d to till: llutntwrl'l s tu which the seal s w r" 01lL: t('d ( lshih I" 1 ( Ij 1).

Semiquantit tlV

same on

were missing or \ r

MICROBIOLOGI AL AL ' IS: Altho tained from chromatographlc an lys r precise quantitative data obtam fr m mlcr blOlogl 1 ply the tnformahon n d d on how 0 onduc furth l' f the nature of posslbl ammo aCld 1mbal­ances in the protem from fl h cal s. These data also would aId tn dl t rmmmg the nature and amount of amino aCid sup­plementation ne dect to enhanc th nu­tritive value of the pollock-fish scal protemandtoreduceimbalan s. sam-ple of the ground pollock fish scal s was sent to the \\ isC'onsin Alumni 1 s arch Foundation, Madison, Wis., to be assay d for 18 ammo acids by a microblOlogical method. The method of analysis used was that of Henderson and Snell (1948). A single media was employed that contain­ed al.: the nutrients required for growth by the various lactic aCid -producing test organisms used to conduct the assay.

The microorganisms employed were Lac~obac,illus ar,abinosus 17-5 for glu­tamiC aCid, leUCine, phenylalanine, valine, and tryptophan; Streptococcus faecalis R for arginine, hi stidine, methionine, and threonine, Latobacillus citrovorum for alanine; Lactobacillus casei for serine

anIn rgmm partlc aCld

Cystin Glutamlc a ld Glycin Hlshdm 1501 ucm L "ucin Lysin 1\Iethionin . Ph nylalanin Prohn erine ...

Threonine Tryptophan Tyrosme .. Valin

01 20, 0 8

37. 1. 6 2.5 3.5 4.4 1.8 2.7 3.5 9.9 4.1 0.5 2.3 3.4

rt U 10 of ft h from

-i~O 5. ; 2.3

12.6 3.; 2.i 7 7 9.2 7.5 4.0 6.3

21 7~5 5.0 1.7 4.5 7.8

and Leuconostic mesenteroldes P-60 for aspartic acid, lYSine, proline, isoleucine glycine, cystine, and tyrosine. '

l!A PPROXI MATELY E UAl TO PERCENT Of PROTEI~ . ~NO ANALYSES FOUND .

August 1958 COMMERCIAL FISHERIES REVIEW 7

The results of the microbiological assay, as grams of amino acid per 16 grams of nitrogen, and the amino acid composition of whole-egg protein (Hawk et al 1954) are included in table 1. Whole-egg protein is considered by many to be an ideal pro­tein and thus should be an excellent protein for comparison and reference.

The amino acid analyses show that the pollock-fish scale protein is rich in glycine and contains about the same amount of arginine and serine as does whole­egg protein. Pollock-fish scale protein contains slightly less aspartic acid, glutam­ic acid, and threonine, and mu ch smaller amounts of isoleucine, lysine, methionine, phenylalanine, tyrosine, and valine. Extremely small amounts of cystine and try­ptophan are present.

SPECTROGRAPHIC ANALYSIS: The inorganic constituents of the two samples of ashed scales from the same lots were determined semiquantitatively by spectro­graphic analysis in a Bausch and Lomb quartz spectograph with direct arc excitation. The data were kindly furnished by Maurice J. Peterson of the U. S. Bureau of Mines, College Park, Md. The first sample of scales was dry-ashed at 5500 q~, and the second sample was wet-ashed with sulfuric and nitric acid at about 200 C.

Each of these samples of ashed scales was mixed with graphite powder of high purity and the mixtures were placed in a graphite electrode. The mixture in the graphite electrode then was arced against a graphite counter-electrode, using direct­current arc excitation, with the current being approximately 12 amperes. The com­pleted vaporization of the sample required about 2.5 minutes. While the arcing was taking place, the spectrograph recorded on photographic plates the spectrum of the elements present in the sample.

Two diffelent types of Eastman plates were used to record the spectrum from 2400 to 8600 Angstrom units: A spectrum analysis number 2 plate was used for the ultra-violet andoblue region, and an I-L plate was used for the remainder of the blue region to 8600 Angstrom units.

These photographic records, or spectrograms, were compared visually with spectrograms of varing concentration standards of mineral elements, the wave-

- lengths of which were recorded under identical conditions on separate plates. The semiquantitative results of the spectrographic analyses are presented in table 2.

Table 2 - The Percentage Range 01" InorganIc Constltuents .t-'resent in the Ash of Pollock Fish Scales

Ca 1-' .Mg Mn J:t'e Al -C-u Ni Na Ba Sr Li K Si Dryashed >10 >10 0.3- .01- .03- .01- :005- -!! .03- .001- .3- .005- .01- .Ul-

3.0 .10 .30 .1 .05 - .30 .01 3.0 .05 .10 .10 twet ashed >10 >10 .3- .01- .03- .02- .005- .005- .03- .001- .3 - .005- .1- .05 -

3.0 .10 .30 .2 .05 .05 .30 .01 3.0 .05 1.0 .50 lINol OEl Eel ED.

anganese, copper, barium, and lithium were present in amounts of les~ than. 0 . .1 p ercent by weight in both samples of ashed scales. Nickel was present m a. sImilar sunount in wet-ashed scales but was absent in the dry-ashed scales. Alummum,

_};lotassiuffi, and silicon wer': present in amounts of less than 0.1 percent in the dry­~ IBshed,. but were present in slightly greater percentages in the wet-ashed sc~les. ~~tronhum and magnesium were present in amounts of less than 3.0 percent m both : samples . Calcium and phosphorus were present in amounts of more than 10 percent u:.-n both samples.

CONCLUSIONS

. Disposal problems associated with an accumulation of waste fish scales at fil­~Hhng plants have stimulated an investigation of the value of scales as a supplemen­. ;; al SOurce of protein in the diets of farm animals.

8 COMMERCIAL FISHERIES REVIEW Vol. 20, '0 . 8

As an initial study in this investigation, Snyder and Nilso.n (19.57) conducted rat­feeding tests to compare the nutritive value fO.r growth, the b.lOloglcal value for maintenance, and the digestibility of pollock-fIsh scale prote~n and a protein supple­ment consisting of three parts c ase in and one part lactalbumm (CL). They found the mean apparent digestibility of pollock-fish scale protein t.o be ne~rly 80 percent when fed to male and female r a t s at a 9.00-percent level m the dIet. Pollock-fish scale protem was found to be completely digested, as indicated by true digestibili ' values, when only enough was fed t o equal metabolic nitrogen. The initial amino ac'd analyses by paper-partition chromatography reported herein apparently supplies a reasonable explanation for this rathe r unexpectedly high level of digestibility of sc 1 protem. The presence in extremely small quantities of tryptophan and cystine and n large quantities of glycine and the p res en c e of fair amounts of hydroxyproline sug­

gest that pollock-fish scale protein i s not a keratin that would be digested with dif­ficulty, but probably is a scleroprotein of the collagen type that would be digested eastly.

This classification of fish-scale prote in is not necessarily original, ' since Blo('" et al m 1949, from chemical analyses alone , r eported that herring scales contained a collagen-like protein. The classification of s c ale protein as keratin has persisted, how ver, probably owing to anatomical and p r esumed embryological consideration . Also, the studie s of Block and his co-worker s may not have received sufficient pub­lictty, or perhaps, supplied enough evid en ce to warrant classification of fish-sca le prot ins as collagens. The results of t h e amino a cid analysis presented herein and the dlgestibility studies presented earlier indicate rather strongly, however, that fish -scale protein is most likely a sclerop rot ein of the c ollagen type and not a k ra ­tm. These conclusions are based on interpretations of results from pollock scali 5

only, but probably apply to scales from all species of fish.

The quantitative amino acid content found probably also explains why, in the rat­f ding study reported earlier, pollock - fish scal e protein alone was not adequate for growth and why the utilization of that protein was increased when combined with skp-

Ise higher levels of CL protein. The microbio logical as well as the paper chroma­tographiC analyses show that the pollock-fish sc ale p r o te in is deficient in all th 0 -

11 d essential amino acids, except arginine, a nd in some of the nonessential am no a lds wh n compared to whole-egg protein . These defi ciencies in essential amino

1 also would explain the decreased assimilation by rats of pollock-fish scale pro-n as ompared to CL protein.

Th fact that scale protem contains nearly 40 - per c ent glyc ine suggests a po -gl m tmbalance or toxicity when the scales ar e fed at high levels. Thl , m may caus the manifestatlOns of other nutritional inadequacies of pollock-fl'h prot n.

r sul+s of the spectrographic analyses suggest that no unusual inorgam n s, or great excesses, are present in pollo ck fish s c ale s that would cau

al mJ ry wh n fed to animals. This conclusion i s also in a ccord with th n ba d on r sults from the rat-feeding study; that is, that scale s contam

bstan s ~ se for growing rats,

August 1958 COMMERCIAL FISHERIES REVIE W

LITERATURE CITED

ANONYMOUS 1955. OFFICIAL METHODS OF ANA LYSIS. ASSOC • .Qf OFF.

~. CHEM., WASHINGTON , D. C .

WITH VARIOUS MICOORGANISMS. ,L • .!!.!.Q.!,. CHEM., VOL . 172, PP. 15-29.

9

BLOCK 1'1 CHARD J.; DURRUM, EMMErT L.; AN D ZWE I G, GUNTER i 955. A MANUAL OF PAPER CHROMAT OGRAPHY AND PAPER

NICHOLSL JOHN TREAOWELL

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POSITION OF THE PROTEIN OF HUMA N TEETH AN D FISH SCALES. :!.. DENTAL RES. , YOL. 2 8 , PP . 518-524.

BOWES JOANE H.i ELLIOTT, R . G.; AND MOSS , J.A. i 955 . THE COMPOSITION OF COL LAGEN AND AC I D- SO LUBLE

C~~~A~~~_~~O~OVINE SKIN. BI OCHEM . ~" VOL . 61,

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OF FISH SCALE HYDROLYS AT ES AND HYDROLYSATES OF SEVERAL OTHER FISH ORGANS . SCHWE I Z Z . HYDROL. VOL. 14, PP. 3 3 6-337. --- - --- ,

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GRA HAM, CLAIRE E . ; WAITKOFF , HE LEN K.; AND H I ER , STANLEY W. 1949 . THE AMINO ACI D CONT ENT OF SOME SC LERO PROTEINS .

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IlL SC I ENeE., VOL. 34, PP. HI- :24.

NORTH ATLANTIC SEA SCALLOP FISHERY

Most of th e scallop-fishing vessels out of New Bedford, Mass., use two dr edges, each 11 feet wide. They are towed together, one from each forward gallow s fr ame , and broughtup alternately to be dumped on deck. The dredges are very sturdy so that they will stand up under the rough use that they g et. A complete II-foot dredge with 3-inch rings weighs about 1,400 pounds wh en empty. Some of the smaller boats wlth less powe r use 8- or 10-foot dredges and l3-foot models have been tried on the larger boats but th e I I - foot dredge is now fairly standard in the New ~edford fle et . The r e is really no standard design for a dredge; each fl sh­lng captainhas his own ideas on what makes for efficient gear and modi­fies and alters the basic design to suit himself.

-- Fishery Leaflet 442, Sea Scallop Boats ~nd Q~ar, -- - August 1957 .