oil cake meal for preparation of protein hydrolysate

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BIOTECHNOLOGY AND BIOENGINEERING VOL. VII, PAGES 285-293 (1966) Oil Cake Meal for Preparation of Protein Hydrolysate C. R. KRISHKA MURTI, Central Drug Research Institute, Lucknow, India Summary With a view to preparing substitutes for liver or other animal protein hydrolg- sates used in the treatment of protein malnutrition, a process has been developed based on sesame and mustard cakes as the starting materials. The laboratory process has been successfully scaled up to pilot plant production to compute tentative costing schedule. Essentially the process consists in defatting the cake, isolating the protein by peptization and isoelectric precipitation, enzymatically digesting the protein isolate by papain, and concentrating the digest tinder re- duced pressure. The final product is a light brown fluffy powder, rich in almost all the essential amino acids. Growth experiments with rats have indicated that the product is comparable to commercial casein although supplementation with lysine could further enhance its biological value. INTRODUCTION Efforts to find a lasting solution to the protein malnutrition problem have been rightly assigned top priority in the national development plans of underdeveloped countries like India. The provision of good quality proteins in adequate amounts in the dietary of the people, particularly those subsisting on predominantly vegetarian diets, has thus become of vital concern to food technologists in India. In spite of a rich cattle wealth and miles of coastal waters forming a potential reservoir of an infinite variety of fish and fish products, proteins of animal origin are in short supply in India. Increased output of animal proteins, including milk proteins, is linked to develop- ments in agriculture, livestock breeding, fisheries, and indirectly to large-scale industrialization. The implenientation of the Third Five Year Plan is expected to increase the output of food by 1&15% but it is doubtful whether our productive capmity of essential foods 285

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Page 1: Oil cake meal for preparation of protein hydrolysate

BIOTECHNOLOGY AND BIOENGINEERING VOL. VII, PAGES 285-293 (1966)

Oil Cake Meal for Preparation of Protein Hydrolysate

C. R. KRISHKA MURTI, Central Drug Research Institute, Lucknow, India

Summary With a view to preparing substitutes for liver or other animal protein hydrolg-

sates used in the treatment of protein malnutrition, a process has been developed based on sesame and mustard cakes as the starting materials. The laboratory process has been successfully scaled up to pilot plant production to compute tentative costing schedule. Essentially the process consists in defatting the cake, isolating the protein by peptization and isoelectric precipitation, enzymatically digesting the protein isolate by papain, and concentrating the digest tinder re- duced pressure. The final product is a light brown fluffy powder, rich in almost all the essential amino acids. Growth experiments with rats have indicated that the product is comparable to commercial casein although supplementation with lysine could further enhance its biological value.

INTRODUCTION

Efforts to find a lasting solution to the protein malnutrition problem have been rightly assigned top priority in the national development plans of underdeveloped countries like India. The provision of good quality proteins in adequate amounts in the dietary of the people, particularly those subsisting on predominantly vegetarian diets, has thus become of vital concern to food technologists in India. In spite of a rich cattle wealth and miles of coastal waters forming a potential reservoir of an infinite variety of fish and fish products, proteins of animal origin are in short supply in India. Increased output of animal proteins, including milk proteins, is linked to develop- ments in agriculture, livestock breeding, fisheries, and indirectly to large-scale industrialization. The implenientation of the Third Five Year Plan is expected to increase the output of food by 1&15% but it is doubtful whether our productive capmity of essential foods

285

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286 C. R. KRISHNA MURTI

would be sufficient to meet even the basic niinimum demands of our growing population. It thus becomes imperative for India to press into service all the resources of her plant kingdom for obtaining the bulk of her protein requirements for some years to come.

High protein content, ready availability, and a gradually dwindling export market are the main factors that determined our recourse to oil seed cakes as the starting material for preparation of protein rich foods for human consumption. Laboratory scale studies were made first by Giri' to devise processing methods for preparation of a protein digest from several oil cakes. The problem of processing oil cakes on a large scale has been tackled by Subrahmanyan and colleagues a t b l y s ~ r e ~ ~ ~ with a view to preparing protein isolates for incorporation in the Indian Multipurpose food.4

In order to provide substitutes for liver and meat hydrolysates extensively used in the pharniaceutical industry as vehicles for liquid vitamin preparations and in the treatment of peptic ulcers and other disorders which require administration of a concentrated form of protein or protein digests, a simple method for processing sesame and niustard cakes has been developed in this 1aboratory.j This process has now been successfully scaled up to pilot plant production with the active cooperation of the Central Food Technological Research Institute, Mysore, India.6 Nutritive value of this product has been assessed by rat growth and amino acid analysis which indicate that on supplementation with lysine, the hydrolysate can support adequately the growth of young rats at rates comparable to those of coniniercial casein. A brief account of this work is presented in this communication.

METHODS

Materials

Sesame and mustard cakes were purchased from local markets and dispatched by rail to Mysore for large scale processing. In the laboratory process, the cake flakes were reduced to 100 mesh size and extracted with petrol ether (5MO"C.) prior to peptisation. In the large scale processing, protein isolation was attempted both before and after defatting with commercial solvents. Sun-dried papaya latex in the form of granules was obtained from N/s Tiuniaon Nursery, Kanigarh, Naini Tal, India. Vacuuni dried papaya latex in the form

BIOTECKSOLOGT A h D I3IOE&-GIhEERI&-G, VOL. VII, ISSUE 2

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PROTEIN HYDROLYSATE FROR4 OIL CAKE MEAL 287

of a light yellow powder was kindly provided by Dr. G. S. Siddappa, Central Food Technological Research Institute, Mysore. Hexane, caustic alkali, hydrochloric acid, and other chemicals used were of commercial grade.

Analytical Methods

Nitrogen estimation was carried out by Kjeldahl digestion followed Fat and ash contents were estimated Enzymatic hydrolysis was followed

by distillation of the ammonia. by standard A.O.A.C. methods. by formal titration of the amino nitrogen released.

Amino Acid Analysis

Quantitative two-dimensional paper chromatography was carried out on buffered Whatman No. 1 sheets employing butano1:acetic acid:water (4: 1: 1) and pheno1:water as the two developing solvent phase^.^ Microbiological assay of aniino acids was by the methods described by Barton-Wright.'O Methionine, histidine, and arginine contents were estimated colorinietrically,11-12 and lysine manometri- cally employing acetone dry cells of Hafnia alvei (Syn. Bacterium cadavaris NCTC 6578) as the source of lysine decarb~xylase.'~

RESULTS

The processing of sesame and mustard cakes for the preparation of the hydrolysate consists essentially of defatting the cake, isolating the protein by peptization in alkaline pH, followed by isoelectric precipitation at pH 4-5, enzymatic hydrolysis of the isolated protein, concentration of the digest under reduced pressure and finally dehy- dration of the concentrate under vacuum. The final product is obtained as a fluffy brown powder or granules, highly hygroscopic, and with a characteristic peptone odor. The preparation as such is bitter to the taste, but however, can be masked by suitable blending with sweetening agents. The Kitrogen content of the final prepara- tion was 13-14%, of which 9-10yo was in the form of aniino nitrogen. Ash content varied from 1@12% and phosphorus 1.5-2.~57~. The contents of individual amino acids were as follows: arginine, 3-470 (colorinietrically) ; histidine, 2 . 5 3 % (colorinietrically) ; niethioni~ie, 1.8-2.2% (colorinietrically); tryptophane, o.?-o.9y0 (inicrobiologi- cally) ; leucine and isoleucine, 6.2-6.8% (chromatographically) ;

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288 C. R. KRISHNA MURTI

valine, 3.E1-3.67~ (microbiologically) ; cystine, 1.5-1.9% (micro- biologically) ; phenylalanine, 4.5-5Y0 (chromatographically) ; and lysine 2.545% (bacterial decarboxylase).

Processing of Oil Cakes on a Pilot Plant Scale

Exploratory runs t,o investigate the feasibility of the method were made with 10-50 kg. batches of cake and the dat,a is summarized in Tables I and 11. The process consisted of the following operations in sequence:

1. Size Reduction. This was carried out. in an Ajax comminuting mill so as to pass a 50-100 Inesh screen.

TABLE I Analysis of Cake Powder and Itesidue

~~ ~

Cake powder Residue”

Nitrogen, Fat, Nitrogen, Fat, % /C % % u-

Mustard 5.5 11.2 2.8 6.8 Sesame 6.9 6.7 1.8 4 . 2

Residue left after peptization and centrifugation.

TABLE I1 Yield of Protein from Cake

Dry weight Input of of residue, cake, kg. kg. Protein, kg. Recovery, %,

Mustard 50 24 6 12 Sesame 50 23 17 34

2. Isolation of Pyotein. Peptization of the cake powder was carried out by dispersing it in 50 gal. stainless steel vessels (A. P. V. Engineering Company, Calcutta) fitted with a Newman power driven stirrer. Cake to water ratio was kept a t 1:lO and pH of slurry raised to 9-9.5 from the original 5-55 by graded addition of 40% sodium hydroxide. The slurry was stirred for 1 hr. at 1800 r.p.ni. and centrifuged in a Basket Centrifuge (American Alachine Tool Company, Hyde Park, Boston, U.S.A.) run at 2500 r.p.m. The

BIOTECHNOLOGY AND BIOENGINEERING, VOL. VII, ISSUE 2

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PROTEIN HYDROLYSATE FROM OIL CAKE MEAL 289

overflow was collected in stainless steel tanks, and a residue consisting inainly of starch and fiber was discarded.

The clear peptized liquor was stirred with commercial hydrochloric acid to shift the pH back to 4.54.6 and the precipitated proteins allowed to settle and freed from adhering salts by centrifugation in the Basket centrifuge.

The wet protein from the Basket centri- fuge was dispersed in water in a steam jacketted kettle, the pH of suspension adjusted to 5.5-5.6 and temperature raised to 55OC. An aqueous suspension of papain mixed with 0.01% sodium hydrosul- fite or versene was added, and digestion allowed to proceed for 5 hr. at 55°C.

5. Concentration of Digest. The digest was freed from unhy- drolyzed protein by spinning in the Basket centrifuge and the clear digest concentrated in a Forced Circulation Evaporator (constructed by the Engineering Division, Central Food Technological Research Institute, Mysore) from 5" to 65-70' Brix.

The syrup was dehydrated in a vacuum shelf drier or in a Stoke's Freeze Drier.

3. Precipitation of Protein.

4. Enzymatic Digestion.

6. Dehydration of the Concentrate.

Batch Processing of Sesame Cake

Certain practical problems were encountered in processing mustard cake, particiilarly in the isoelectric precipitation of the protein and its separation from the whey. Further studies were, therefore, con- fined to sesame cake in batches of 40 kg., and a total of 450 kg. was processed to work out provisional costing data. Since preliminary experiments had indicated that solvent extraction of cake prior to peptization resulted in the recovery of a final product with a lighter shade and a more acceptable odor, sesame cake was defatted with

TABLE I11 Solvent Extraction of Sesame Cake with Hexane

Solvent Weight of Batch Sesame cake Hexme Yield of recovered, defatted

110. taken, kg. added, kg. oil, kg. kg. meal, kg.

1 241 499 23 450 1% 2 121 36'2 12 360 102 3 121 43 1 12 346 104

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290 C. R. KRISHNA MURTI

commercial hexane by two stage extraction in a vertical extractor followed by drying in a rotary extractor. The relevant processing data is summarized in Tables 111-VI. About 3o-35yO of the nitrogen

TABLE IV Protein Tsolation from Defatted Sesame Meal

Weight Weight of proteiii Weight. of residiie of meal

Batch taken, Wet wt., Dry wt., Wet wt., Dry wt., no. kg . kR . kR. kg. kp .

~

1 2 3 4 5 6 7 8 9

10

10 40 40 40 40 40 40 40 40 40

7.7 33.0 31.5 32.7 34.0 29.5 29.6 29.3 32.0 28.8

1.9 8.3 7.2 8.2 8.9 8 .9 8.5 8.8 9.6 7.2

27.4 112.0 112.3 113.0 112.0 112.0 101 .o 98.0

102.0 103.0

7.9 30.2 29.4 30.0 29.8 24.6 26.5 29.4 26.5 30.9

TABLE 'I: Nitrogen Recovered during Peptization of Sesame Cakes

Batch no.

Cake taken,

kg. Nitrogen input, g.

Nitrogen recovered as protein isolate,b g.

10 40 40 40 40 40

800 3200 3200 3200 3200 3200

250 (31) 1120 (35) 990 (30)

1070 (33) 1110 (35) 1160 (30)

Nitrogen lost in residue,b g.

440 (55) 1700 (53) I510 (49) 1670 (52) 1510 (50) 3250 (40)

a Nitrogen content of defatted cake was 8.0%. b Figures in parentheses represent recovery of nitrogen.

of the defatted cake could be accounted for in the protein isolates, of which about 75y0 was recovered in the final hydrolysate. The cost of production of the dehydrated granular form of the hydrolysate was tentatively computed as Rs. 10.4/kg.

BIOTECHNOLOGY AND BIOENGINEERING, VOL. VII, ISSUE 2

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PROTEIN HYDROLYSATE FROM OIL CAKE MEAL 291

TABLE VI Papain Hydrolysis of Sesame Protein Isolate

Dry weight

Weight of Weight of Weight of of

no. for hydrolysis, kg. protein,a kg. digest,b kg. kg. Batch protein taken unhydrolysed concentrate of hydrolysate,

~~~~ ~ ~ ~~

1 7.5 1.2 6.0 (70) 4 2 2 7.7 1.3 6.2 (70) 4.3 3 7 . 1 0.8 6.8 (64) 4.3 4 7.2 0.4 7 S(58) 4 .5 5 8.5 1.1 8 2(61) 5.0 6 9.3 1.3 6.3 (66) 4.2

The unhydrolyzed protein represenh the sediment left by centrifugation of the papain digests of protein isolate.

b Figures in parentheses indicate % solids measured by refractometer.

TABLE VII Biological Evaluation of Protein Hydrolysate by Rat Growth Method

Initial Final Protein weight, weight, intake,

Source of protein g- g. g. PER"

Casein M (5) F (5)

Protein M (5) hydrolysateb F (5)

Sesame protein M (6)

Sesame protein M (6) hydrolysate

hydrolysate Plus 2% lysine

7 weeks 39.3 130.0 64 1.4 37.3 114.0 54 1.2 39.0 142.0 64 1.6 36.6 106.0 58. 1.2

4 weeks 39.0 47.2 21.3 0.4

37.0 69.0 27.4 1.2

PER stands for Protein Efficiency Ratio or weight increase in g./g. of protein

b Protein hydrolysate used was made from a blend in equal proportions of Sesame protein

consumed.

mustard and sesame protein isolate and contained 6.2% lysine. hydrolysate used in the supplementation experiment contained 3.2% lysine.

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292 C. R. KRISHNA MURTI

Biological Value of the Hydrolysate

The Protein Efficiency Ratio of the hydrolysate was assayed by the rat growth method using a semisynthetic diet in which the nitrogen was supplied by either casein or the protein hydrolysate. Effect of supplementation of the hydrolysate with L-lysine hydrochloride at 2% level on the protein efficiency ratio was studied in parallel experiments. The results are summarized in Table VII. The data of evaluation for nitrogen utilization and retention by the balance sheet method in adult rats is summarized in Table VIII.

TABLE VIII Biological Evaluation of Protein Hydrolysate

by Nitrogen Balance Sheet Methods

Source of Nitrogen Digestibility Biological Nitrogen protein utilization, coefficient, value, retention, in diet /o 7% % % 0-

Casein 91.0 (0.43) 95.0 (0 5) 61.5 (3 .0) 45.7 (2.6) Protein

hydrolysate 92.0 (0.45) 96.0 (0.4) 69.9 (2.9) 55.1 (2.9)

* Figures in parentheses indicaQe standard errors of mean.

DISCUSSION

The main drawbacks to vegetable materials as sources of proteins for human consumption are their low biological value and the in- herent processing difficulties involved in separating the protein from the associated crude fiber. While the biological value of the vegetable proteins can be significantly raised by adequate supplementation with amino acids, the probleiii of preparing protein isolates from materials like the oil seed cakes requires further investigations in order to obtain maximum recovery of the protein. In the present study only 3540% of the nitrogen present in the defatted cake could be recovered in the form of the isolate; improved methods of peptization and separation could presumably raise the recovery of nit,rogen.

The amino acid deficiency of the mustard and sesame protein appears to be corrected by lysine alone to an appreciable extent. In feeding experiments supplementation with tryptophane did not result in any increase in protein efficiency ratio over the controls although the amino acid composition of the hydrolysates indicated that trypto-

BIOTECHNOLOGY AND BIOENGINEERING, VOL. VII , ISSUE 2

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PROTEIN HYDROLYSATE FROM OIL CAKE MEAL 293

phane may be a limiting amino acid. Since in the therapy of protein malnutrition in humans the emphasis is on a concentrated source of protein, the results of the present study reveal the usefulness of a method of processing as outlined above of oil cakes to obtain a pro- tein digest concentrate rich in almost all the essential amino acids and which can be blended with minerals and vitamins and amino acids.

References 1. Giri, K. V., J . Indian Med. Assoc., 17, 165 (1948). 2. Subrahmanyan, V., D. S. Bhatia, S. S. Kalbag, and N. Subramanian, J .

3. Anantharaman, K., N. Subramanian, D. S. Bhatia, and V. Subrahmanyan,

4. Subrahmanyan, V., G. Rama Rao, S. Kuppuswamy, M. Xarayana Rao,

5. Krishna Murti, C. R., Res. Ind. (New Delhi), 2, 285 (1957). 6. Krishna Murti, C. R., Res. Ind. (New Delhi), 6, 430 (1961). 7. Singh, C., and C. R. Krishna Murti, J . Sci. Ind. Res. (India), 19C, 87

8. Rastogi, IM. K., and C. R. Krishna Murti, Ann. Bwchem. Exp. Med.

9. Subramanian, N., and $1. V. L. Rao, J . Sci. Ind. Res. (India), 14B. 56

10. Barton-Wright, E. C., Mirrobwlogical Assay of Vitumins and Amino

11. Macarthy, T. E., and M. Sullivan, J . Bwl. Chem., 36,871 (1941). 12. Macpherson, H. T., Biochem. J., 36, 59 (1942). 13. Najjar, V. A., Methods in Enzymology, Val. I I , S. P. Colowick and N. 0.

Kaplan, eds., Academic Press, New York, 1955, p. 185.

Received December 23, 1964

Amer. Oil Chemists SOC., 36, 66 (1959).

Indian Oilseeds J., 3, 85 (1959).

and M. Swaminathan, Food Sea., 6, 76 (1957).

(1960).

(Cakutta), 22,51 (1962).

(1955).

Acids, Pitman, London, 1952.