Amino Acid Composition of the Protein from a Mushroom(Pleurotus sp.)

ZAKIA BANO, K. S. SRINIVASAN, AND H. C. SRIVASTAVA

Central Food Technological Research Institute, Mysore, India

Received for publication 29 November 1962

ABSTRACT

BANO, ZAKIA (Central Food Technological ResearchInstitute, Mysore, India), K. S. SRINIVASAN, AND H. C.SRIVASTAVA. Amino acid composition of the protein froma mushroom (Pleurotus sp). Appl. Microbiol. 11:184-187.1963.-Approximate analyses of mushroom protein(Pleurotus sp.) revealed that it contains 2.78/7o proteinand 0.14 % nonprotein nitrogen on a fresh-weight basis.A total of 17 amino acids, including all the essential aminoacids, were qualitatively identified. Quantitative estima-tion of essential amino acids showed that, except formethionine and phenylalanine, all are in fairly highconcentration. From these studies, it was concluded thatthe supplementation of this mushroom with cereal dietwould help to overcome lysine deficiency.

Edible mushrooms have been recognized from timeimmemorial. During World War II, yeasts were usedextensively as food and fodder supplement in Germanyand Jamaica (Prescott and Dunn, 1949). Torula speciesfrom waste by-products have been used for human con-sumption (Reiser, 1954; Prescott and Dunn, 1949; Wileyet al., 1950; Wiley, Holderby, and Fries, 1953). Attentionhas been paid to scientific cultivation of mushroomssince the 17th century, and it is reported that mushroomsare being used extensively in many countries for food andfodder (Botticher, Pannwitz, and Nier, 1941; Andersonand Fellers, 1942; Gilbert and Robinson, 1957; Giacomini,1957). Mushrooms have better flavor and taste, and thesame nutritive value as do Torula species (Block et al.,1953). They possess extensive enzyme complexes whicheniable them to flourish successfully on a wide variety ofiniexpensive substrates, such as lignin, cellulose, hemi-celluloses, pectin, and other industrial wastes which arenot suitable even for animal feed (Waksman, 1944;Reusser, 1956). Mushrooms represent one of the world'sgreatest untapped resources of nutritious and palatablefoods.

Studies on the nutritive value and composition of afew species of mushrooms (Bares, 1927; Gudlet, 1933;Kizel and Konovalov, 1937), and recent investigations byLintzel (1941), Fitzpatrick, Esselen, and Weir (1946),Esselen and Fellers (1946) have shown that in additionto the flavoring properties the proteins of some mush-rooms are equal to muscle protein in nutritive value. Other

recent investigations have shown the amino acid com-position of some important edible mushrooms (Seelkopfand Schuster, 1957; Reusser, Spencer, and Salans, 1958;Yokohata and Yamauchi, 1959; Touze, 1961; Orillo andCarnagal, 1961). According to Robinson and Davidson(1959), the efficiency of protein production, from a givenquantity of carbohydrates, in mushrooms and otherhigher fungi is about 65 % compared with about 20 % forpork, 15 % for milk, 5 % for poultry, and 4 % for beef.

Pleurotus (oyster mushroom) grows abundantly on deadand decaying wood in clusters, and is eaten by the majorityof people in India. This fungus is able to compete success-fully with other fungi on undecomposed paddy straw andcan be cultivated at a wide range of temperatures (21 to33 C, relative humidity 67 to 72 %; Bano and Srivastava,1962) unlike field mushrooms which need exact conditionsfor their cultivation. The ease with which this mushroomcan be cultivated economically on a large scale, within aperiod of 3 weeks, offers the possibility of its use in diete-tics. With this object in view, it was of interest to findout the amino acid composition of Pleurotus. This paperdeals with the qualitative and quantitative analysis ofamino acids of this mushroom.

MATERIALS AND METHODS

Cultivation of mushrooms. Pleurotus was grown on paddystraw beds prepared from paddy straw soaked in water for15 hr. The size of the paddy straw beds might vary, butthe best results were achieved in beds of 1 ft2 and 9 in.in thickness. The beds were kept on a raised platformunder shade. Spawns of Pleurotus species were preparedby inoculating sterilized paddy straw in a bottle; 4-month-old spawns were used for inoculating the beds.Cajanus cajans (red gram) powder (40 mesh) was thebest source of nutrient in the beds. The beds were wateredtwice a day, and the mushrooms appeared 20 days afterinoculation. The yield of mushrooms was about 150 to 200 gper bed (Bano and Srivastava, 1963). Fresh mushroomswere taken and dried in a desiccator (over P205) to constantweight. Samples for analysis were prepared as describedbelow.

Preparation of free amino acid fraction. Dry mushrooms(10 g) were ground for 10 min with 100 ml of 70 % (v/v)ethanol in water in a glass mortar. The suspension wascentrifuged, and the clear supernatant was collected in aconical flask. The solid residue was re-extracted three

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times. After centrifugation, all the clear supernatants werepooled and evaporated to dryness on a water bath. Theresidue was extracted with absolute alcohol to removesalts and other interfering material, was filtered, and thenwas evaporated to dryness. This residue was dissolved inS ml of 10 % (v/v) isopropanol in water and was used forthe analysis of free amino acids.

Hydrolysate. The residue after extraction by 70% (v/v)ethanol in water was dried to constant weight in a desic-cator, and a 1-g sample was hydrolyzed by autoclavingwith 25 ml of 6 N HCl at 15 lb of pressure for 8 hr. Thehydrolysate was filtered, and the filtrate was distilledunder vacuum to remove excess acid. The residue in theflask was treated with 10 % (v/v) isopropanol in water toa final volume of 5 ml. This solution was used for chromato-graphic analysis.The hydrolysate for the microbiological assay of amino

acids was prepared according to the method described byBarton-Wright (1952). Defatted material (1 g) was auto-claved for 8 hr with 2.5 N HCl; after addition of 2.5 Msodium acetate, the solution was adjusted to pH 4.5 andmade up to a known volume. For the assay, the pH of thesolution was adjusted to 6.8.For tryptophan estimation, the material was subjected

to alkaline hydrolysis according to the method of Bala-subramanyan et al. (1952).Paper chromatogr-aphy. The circular paper chromato-

graphic technique developed by Giri and Rao (1952) wasused for the identification of amino acids, using butanol-

acetic acid-water (40:10:50) as solvent. Whatman no. 1filter paper (31 cm diameter) was used. Standard aminoacid mixtures were also runi simultaneously for identifica-tion of amino acids. Chromatograms were developed with0.4% ninhydrin. Final confirmation of amino acids wasdone by means of specific tests.

Microbiological assay. Quantitative estimation ofessential amino acids was done by microbiological assay,according to the method of Barton-Wright (1952). Thefollowing organisms were used for the assay of differentamino acids: Lactobacillus arabinosis 17/5, for leucine,isoleucine, valine, and tryptophan; Leuconostoc mesenter-oides P. 60, for lysine, histidine, and phenylalanine;Lactobacillus fermenti 36, for methionine; and Streptococcusfaecalis R, for arginine and threonine.

Approximate analyses of mushrooms were done ac-cording to methods of the Association of Official Agri-cultural Chemists (1960).

RESULTS AND DISCUSSION

Approximate analyses of the mushroom (Table 1) showthat it contains 2.78 % protein on a fresh-weight basis anidabout 0.14 % in the form of nonprotein nitrogen. Thecircular paper chromatography of the free amino acid frac-tion shows the presence of 17 amino acids in addition toone peptide (Fig. 1). Methionine and tryptophan werepresent in traces in the free amino acid fraction. Qualita-tively, the amino acids of protein (Fig. 2) identified in the

FIG. 1. (left) Paper chromatogram showing the free amino acids. Symbols: L IL = leucine and isoleucine; Ph Al = phenylalanine;Al = alanine; Glu THR = glutamic acid and threonine; SE GLY ASP = serine, glycine, and aspartic acid; ARG = arginine; PEP =

peptide; Ly Hi = lysine and histidine; Cy = cystine; Ml, M2, and M3 refer to amino acid mixtures; UH = free amino acid fraction;H = hydrolyzed free amino acid fraction.

FIG. 2. (right) Paper chromatogram showing the amino acids in the mushroom protein hydrolysate. Symbols as in Fig. 1.

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BANO, SRINIVASAN, AND SRIVASTAVA

TABLE 1. Approximate composition of the mushroom (Pleurotus)

Constituent Per cent

Moisture ............................... 90.95Ash .................................... 0.974Protein ............................... 2.78Nonprotein nitrogen ........ ............ 0.14Fat (ether extract) ...................... 0.165Crude fiber ............................. 1.08

TABLE 2. Essential amino acid composition (g per 100 g of protein)of proteins of mushroom and egg

Amino acid Mushroom Egg Proportion*

g g

Arginine ............... 6.7 6.4 1Histidine .............. 2.1 2.1 2Lysine ................. 5.0 7.2 5Tryptophan ........... 0.9 1.5 1Phenylalanine ......... 2.0 6.3 3.5Methionine ............ 1.26 4.1 3Threonine ............. 4.2 4.9 2.5Leucine ............... 4.4 9.2 4Isoleucine ............. 5.8 8.0 2.5Valine ................. 4.65 7.3 3.5

* As proposed by Rose (1937).

acid hydrolysate were similar to the free amino acids inthe alcohol fraction with the exception of one peptide.Mushroom as compared with fruits and vegetables is a

better source of protein, containing lysine, arginine, histi-dine, and threonine in high concentrations. The essentialamino acid composition of protein (Table 2) shows thatmushroom is primarily deficient in phenylalanine andmethionine, when compared with egg protein (Block andMitchell, 1946) At the same time, when compared withthe proportions of essential amino acids required for satis-factory mammalian growth, as proposed by Rose (1937),using tryptophan level as unity, the amino acid pattern ofthe mushroom protein appears to be adequate in all otheramino acids, except phenylalanine and methionine. Supple-mentation of mushroom protein with phenylalanine andmethionine would be necessary, when used as a sole source

of protein in diet, to promote adequate growth. Thecomposition of protein of this mushroom is approximatelysimilar to that of Agaricus campestris (Esselen and Fellers,1946) except for the tryptophan content, which is higherin Pleurotus species.

Organoleptic tests of this mushroom showed it to haveacceptable flavor and biting properties. This mushroom isbeing utilized by people in different areas, and has beenfound to be nontoxic. Since mushrooms are considered as

delicacies, their supplementation with a cereal diet may

help to overcome lysine deficiency. Further work on thebiological value and protein efficiency ratio might throwmore light on the nutritive value of the protein.

ACKNOWLEDGMENT

The authors are grateful to V. Subrahmanyan and A.Sreenivasan for their keen interest and valuable guidanceduring the course of this investigation.

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