Production of Mushroom Mycelium as a Protein and Fat Source in Submerged Culture in Medium of Vinasse

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1 Production of Mushroom Mycelium as a Protein and Fat Source in Submerged Culture in Medium of Vinasse H. FALANGHE1 Chemistry Department, Instituto Zinotkcnico, Escola Superiot de A gric oltitua "Lu iz de Queiroz,"' University of Sao Paulo, Sao Pautlo. Brazil Received for publication June 11, 1962 ABSTRACT FALANGHE, H. (University of Sao Paulo, Sao Paulo, Brazil). Production of mushroom mycelium as a protein and fat source in submerged culture in medium of vinasse. Appl. Microbiol. 10: Of ten mushroom cultures investigated, only Agaricus campestris, Boletus indecisus, and Tricholoma nudum were capable of growing in submerged culture in medium of vinasse with added salts. Higher fermentative efficiencies were found under these conditions than in medium containing molasses or waste sulfite liquor. A. campestris showed a better capacity to produce protein but, since B. indecisus is capable of developing greater mycelium weight, its fermentative efficiencies are comparable. Both microorganisms could be grown in medium of vinasse with greatly varied amounts, producing higher mycelial weight in media with greater vinasse. The capacity of B. indecisus and A. campestris to utilize the noncarbohydrate fraction in total solids, instead of the total carbohydrates when they are in smaller amount, was observed in medium containing vinasse. B. indecisus and A. campestris were easily separated by filtration from the medium, although T. nudum was difficult to separate by this procedure. In experiments with A. campestris, the adaptative capacity of the organism to vinasse was demonstrated. Production of the mushroom Agaricus campestris in submerged culture was investigated for the first time in 1948 and showed possibility for application in the manufacture of several mushroom foods (Humfeld, 1948). The essential nutritional requirements of this microorganism were found to be comparatively simple (Humfeld and Sigihara, 1949) and they were later studied extensively (Humfeld and Sugihara, 1952). Interest in this subject led to a study of the possibility of growing different strains of A. campestris in several natural substrates (Humfeld, 1951), and a process was reported for the growth of this organism in citrus press water in Florida (Szuecs, 1950). Later, several mushrooms were tested for their ability to grow in submerged culture (Humfeld and Sugihara, 1954); and the developmental capacity, the protein content, and the fat content of the mycelium of ten mushrooms grown Present address: Northern Utilization Research and 1)evelop- ' ment Laboratory, U.S. Department of Agriculture, Peoria, Ill. in molasses and waste sulfite li(luor were compared (Reusser, Spencer, and Salans, 1958a) Mushroom mycelium was found to have approximately the same nutritive value as fodder yeast, when grown either in waste products of the citrus industry (Block et al., 1953) or in molasses and waste sulfite liquor (Reusser, Spencer, and Salans, 1958b). Eddy (1958) also concluded that higher fungi grown in submerged culture were capable of producing materials with good nutritive value, and Cirillo (1960) suggested mushroom mycelium, produced in waste sulfite liquor, as a protein supplement for human food or as an animal feed. The disposal of vinasse, the waste product from the distillation of fermented sugar cane juice in the "aguardente" (regional brandy) industries and of molasses in the alcohol industries, is an important industrial problem, previously studied by Almeida (1952), who suggested its use as efficient fertilizer for poor soils. The purpose of this work was to investigate the suitability of vinasse as a substrate for the production of mushrooms in submerged culture as a source of protein and fat. MIATERIALS AND 1\IETHODS This work was done with ten mushroom cultures: M1orchella hibryda Gray 149, Boletus indecisus Gray 168, and Xilaria polymorpha Gray 154, obtained from W. D. Gray, Department of Botany and Plant Pathology, Ohio State University; A. campestris (L) Fr. CBS, obtained from Centraalbureau voor Schimmelcultures, Baarn, Netherlands; A. camtpestris NRRL 2334, A. campestrlis NRRL 2335, A. campestris NRRL 2336, Tricholoma nudum NRRL 2371, Cantharellus cibarius NRRL 2370, and Collybia velutipes NRRL 2367, obtained from C. W. Hesseltine, Northern Utilization Research and Development Division, Peoria, Ill. These cultures were maintained on malt agar slants at 15 C in the dark. All the inocula were produced by the transference of mycelial bits from the agar slants to 250-ml Erlenmeyer flasks containing 40 ml of a 5 %( malt-extract broth. The inoculum was grown for 10 days resting at 22 C in the dark. After its development, the mycelium was washed with sterile distilled water and transferred to 250-ml glassstoppered Erlenmeyer flasks containing glass beads. Sterile distilled water (40 ml) was added asceptically to the flasks, and the mycelium was broken up by strong 572

2 196)(2] GROWTH OF MIUSHROONIS IN SUBAIERGED CULTURE 15)73 shaking (Reusser et al., 1958a). A 2-nil amount of the resultant suspension was inoculated into the medium. This procedure was used for all the experiments except one in which the inoculum was grown in medium of vinasse under agitation. In this case, 2 ml of mycelial suspension were inoculated into the fresh medium of vinasse. The media of vinasse, vinasse concentrate, and diluted vinasse each contained 4 g of ammonium sulfate, 0.1 g of monopotassium phosphate, 0.5 g of magnesium sulfate heptahydrate, g of ferrous sulfate heptahydrate, and g of zinc sulfate heptahydrate per liter of medium, except in the experiments in which the amounts of ammonium sulfate, monopotassium phosphate, and magnesium sulfate were varied. Media were sterilized in 500-ml Erlenmeyer flasks containing 200 ml of medium. After inoculation, the flasks were placed at 22 C in the dark on a rotary shaker developing 240 rev/min with an eccentricity of 1 in. The vinasse used in this work was obtained from the "aguardente" Pilot Plant of the Instituto Zimotecnico. Total carbohydrates in the medium were determined colorimetrically by the anthrone method (MIorris, 1948), and reducing sugars were colorimetrically determined with the alkaline copper reagent of Somogyi (1945) and the arsenomolybdate reagent of Nelson (1944). Total solids were determined by evaporating samples of media to dryness at 110 C. The mycelium was separated from the medium by filtration under vacuum through a fine screen and washed with distilled water. The dry weight of mycelium was obtained by drying the mycelium at 60 C under vacuum. Total nitrogen in the dried mycelium was determined by the micro-kjeldahl method, and the factor 6.25 was used to calculate the crude protein. Fats were estimated gravimetrically in the dried mycelium after 16 hr of continuous ether extraction. All data in this work are expressed as an average of the determinations made in three culture flasks. RESULTS ANI) DISCUSSION A. campestris was found to reach its maximal growth in a medium of vinasse after 288 hr, when the protein content of the mycelium was high and the fat content low (Table 1). The consumption of reducing sugars, total carbohydrates, and total solids increased up to 192 hr; from then on, only the consumption of dry matter occurred. B. indecisus and T. nudum (Table 2) showed a higher mycelium weight than A. campestris, but had a lower protein content. The fat content of the mycelium was comparable for the three microorganisms. The efficiency (g of protein per 100 g of reducing sugar) of B. indecisus was comparable with that of A. campestris, and the efficiency of T. nudunt was lower. The other seven microorganisms were incapable of growing in medium containing vinasse and in medium with vinasse diluted from 1 liter to 1.5 liter with distilled water. The adaptative capacity of A. cainpestris to the medium of vinasse is shown in Table 3; at 96 hr, the organism reached the same state of development that it was capable of reaching only after 192 hr when nonadapted (Table 1). The growth of A. campestris and B. indecisus in media of vinasse with varied composition is shown in Tables 4 and 5. The consumption in per cent of reducing sugars and total carbohydrates in the different media (Table 4) does not show the expected variation, although the consumption curve in percentage of total solids in medium with vinasse increased as the concentration of vinasse decreased. This observation indicates that, when the medium decreases in its total carbohydrates content, the organism will use more of the noncarbohydrates fraction of total solids. The higher production of mycelium per 100 g of total carbohydrates used, in the most concentrated medium, appears to show that carbohydrates are preferable for mycelial production. It is important to observe here, however, that, since the media with the greatest vinasse concentration were initially less favorable to A. campestris (as shown by the comparatively reduced growth of the microorganism in medium with vinasse concentrated from 1.8 to 1 liter), the growth of the microorganism in the more concentrated medium was delayed. Both organisms were unable to grow in medium with vinasse concentrated from 2 to 1 liter. The reason why the microorganism produces greater mycelial weight in a medium of vinasse less rich in total carbohydrates and reducing sugars (Tables 4 and 6) than in a medium with greater total carbohydrates and reducing TABLE 1. Growth of Agaricus campestris (L) Fr. CBS in medium containing vinasse* Incubation Dry wt of Dry wt of Crude protein Crude fat Reducing sugars Total carbohy- Dry matter time myceliumt mycelium (dry matter) (dry matter) used drates used used Sugar used Efficiency t Original sugar hr glliter % % % _ * Vinasse with 1.36%7c reducing sugars, 2.44t% total carbohydrates, 4.15%7 total solids including added salts; ph adjusted to 5.00 with 75% NaOH.

3 574 FALANGHE [VOL. 10 TABLE 2. Growth of Tricholoma nudum and Boletus indecisus in medium containing vinasse* Crude Crde oa Efficiencyt DytDry wt pro- fa R crb_ Dry Seis of my- ofrywttein ftducing cro-matcel- ofm-(dry (dry suasdhy- tr Oii humf celium mat-sugars drates esrd Sugar Orlg te)ter) used used sugar T. nudum NRRL B.indecisus Gray * Vinasse with 1.41% reducing sugars, 2.55% total carbohydrates, 4.23% total solids, including added salts; ph adjusted to 5.00 with 75% NaOH. Determinations were made at 288 hr. TABLE 3. Growth of Agaricus campestris (L) Fr. CBS in medium containing vinasse * Crude Redu Total Efficiencyt Incu- Dry wt D.ry wt protdn fat carbo- Dry bation of my- ofmypyrotein (dry ing hy- matter time celiumt celium (dry mat- sugars drates used matter) ter) used used Sugar Original used sugar hr * Vinasse with 1.37% reducing sugars, 2.55% total carbohydrates, 4.13% total solids, including added salts; ph adjusted to 5.00 with 75% NaOH. Inoculum grown for 12 days in a medium of vinasse on a rotary shaker. t Ratio of g of protein/100 g of reducind'g sugars. sugars (Tables 1 and 2), consuming the same percentage of total solids, is being studied in this laboratory. The higher efficiencies together with the greater mycelium weight per 100 g of total carbohydrates obtained in media with 1 liter of vinasse diluted to 1.2, 1.5, and 1.8 liters (Table 5), when compared with undiluted vinasse (medium 1 in Table 4), show that media with diluted vinasse are more favorable for rapid growth of the microorganism. Because of this faster growth in diluted media (Table 5), autolysis is also greater; and this may account for the decreasing mycelial weight per 100 g of total carbohydrates, the decreasing protein content, and the increasing percentages of total solids determined in the diluted media. The results in Table 6 show the effect of varying the amounts of ammonium sulfate, magnesium sulfate, and monopotassium phosphate in medium containing vinasse on the development of A. campestris. Very little or no improvement in the mycelial weight or in the protein content was found by varying the amount of monopotassium phosphate and magnesium sulfate. However, raising the ammonium sulfate from 2 to 4 g per liter of medium, led to an increase in the weight of the mycelium and a greater protein content. No improvement could be detected when the amount of ammonium sulfate was further raised from 4 to 6 g per liter of medium. The growth of an A. campestris (L) Fr. CBS mutant culture (Falanghe and Bobbio, 1962) in medium containing vinasse and in media with vinasse diluted with distilled water, under the usual conditions of agitation and temperature, gave lower mycelium weight than the standard culture. A blue pigmentation was produced by this organism under these conditions, and the pigment was identified as indigo as previously reported (Falanghe and Bobbio, 1962). TABLE 4. Growth of mushrooms in media with varying amounts of vinasse CCrude rdfa Reuig Total Dy Efficiencyl Me- Dry wt of Dry wt of protein Crude fat Redscing carbohy- matery Species dium* myceliumt mycelium (dry mattr) suseds drates ustedr ua Oiia ugaroiia mte)matter) used used ssd Agaricus campestris (L) Fr. CBS A. campestris (L) Fr. CBS A. campestris (L) Fr. CBS A. campestris (L) Fr. CBS Boletus indecisus Gray B. indecisus Gray B. indecisus Gray B. indecisus Gray * Medium 1.8, 1.5, and 1.2 denotes vinasse concentrated from 1.8, 1.5, and 1.2 liters to approximately 1 liter. In medium 1, the vinasse was not concentrated. Medium 1.8, 1.5, 1.2, and 1 contained, respectively, 1.11, 0.98, 0.89, and 0.68% reducing sugars; 2.13, 1.56, 1.44, and 1.42% total carbohydrates; 6.36, 5.52, 4.74, and 4.09% total solids. All media contained added salts. The ph was adjusted to 5.00 with 75% NaOH. Determinations were made at 288 hr. t Expressed as, g/100 g of total carbohydrates used. I Ratio of g of protein/100 g of reducing sugars.

4 1962] GROWTH OF MUSHROOMS IN SUBMERGED CULTURE 575 A. campestris, B. indecisus, and T. nudum are thus capable of growing in submerged culture in media containing vinasse. The ability to grow in media with varying concentrations of vinasse was shown by both A. campestris and B. indecisus, which produced greater mycelial weight at the highest concentration. An adaptative capacity to medium with vinasse was shown by A. campestris. The ability to utilize total solids in increasing percentage, when total carbohydrates in the medium are in decreasing amount, was observed. From the viewpoint of mycelium richness in protein, A. campestris was found to be the better organism; however, B. indecisus, although developing a mycelium with a lower percentage of protein, because of its greater capacity to produce mycelium was comparable to A. campestris in its efficiency. From the viewpoint of the separation of mycelium from the medium, A. campestris and B. indecisus could be easily separated by filtration, but T. nudum was not readily separable by this procedure. The mycelium of B. indecisus and A. campestris grew in a sphere-shaped form, and T. nudum developed in a form similar to a yeast. A. campestris, B. indecisus, and T. nudum are capable of higher fermentative efficiencies in a medium of vinasse than have been reported in a medium of molasses or waste sulfite liquor (Reusser et al., 1058a). ACKNOWLEDGMENTS The writer wishes to express his grateful acknowledgment for grants from the Rockefeller Foundation and Conselho Nacional de Pesquisas. The competent technical assistance of Thereza D. Nogueira is acknowledged with pleasure. LITERATURE CITED ALMEIDA, J. R problema da vinhaga em Sao Paulo. Univ. Sao Paulo, Bol. Inst. Zimotec., No. 2. BLOCK, S. S., T. W. STEARNS, R. L. STEPHENS, AND R. F. J. Mc- CANDLESS Mushroom mycelium, experiments with submerged culture. J. Agr. Food Chem. 1: CIRILLO, V. P Fermentation process for producing edible mushroom mycelium. U.S. Patent 2,928,210. EDDY, B. P Production of mushroom mycelium by submerged cultivation. J. Sci. Food Agr. 9: FALANGHE, H., AND P. A. BOBBIO Identification of indigo TABLE 5. Growth of mushrooms in media with varying diluted amounts of vinasse CCrude rdfa Reuig Total Efficiencyt Me- Dry wt of Dry wt of protein Reducing carbohy- Species dium* myceliumt mycelium used Sugargars Originalte matter) matter) used used used Susar Original Agaricus campestris (L) Fr. CBS A. campestris (L) Fr. CBS A. campestris (L) Fr. CBS A. campestris (L) Fr. CBS A. campestris (L) Fr. CBS Boletus indecisus Gray B. indecisus Gray B. indecisus Gray B. indecisus Gray B. indecisus Gray * Media 1.2, 1.5, 1.8, 2, and 3 denote vinasse diluted from 1 liter to 1.2, 1.5, 1.8, 2, and 3 liters with distilled water. Media 1.2, 1.5, 1.8, 2, and 3 contained, respectively, 0.55, 0.43, 0.35, 0.33, and 0.21% reducing sugars; 1.07, 0.87, 0.76, 0.67, and 0.45% total carbohydrates; 3.27, 2.58, 2.21, 1.92, and 1.36% total solids. All media contained added salts. The ph was adjusted to 5.00 with 75% NaOH. Determinations were made at 288 hr. TABLE 6. Growth of Agaricus campestris (L) Fr. CBS in medium of vinasse* with varied amounts of added salts Ammo- Dry wt of Crude protein Crude fat Mono- Dry wt of Crude protein Crude fat Magnesium Dry wt of Crude protein Crude fat sulfate mycelium (dry matter) (dry matter) potassium sulfate mycelium (dry matter) (dry matter) sulfate mycelium (dry matter) (dry matter) ~~~~~~~~~phosphate glliter glliter % % glliter glliter % S glliter glliter N S * Vinasse with 0.15% reducing sugars, 0.37% total carbohydrates, and 3.94% total solids. The ph was adjusted to 5.00 with 75% NaOH. Determinations were made at 288 hr.

5 576 FALANGHE [VOL. 10 produced in submerged culture of Agaricus campestris, mutant culture. Arch. Biochem. Biophys. 96: HUMFELD, H The production of mushroom mycelium (Agaricus campestris) in submerged culture. Science 107:373. HUMFELD, H., AND T. F. SUGIHARA Mushroom mycelium production by submerged propagation. Food Technol. 3: HUMFELD, H., AND T. F. SUGIHARA The nutrients requirements of Agaricus campestris grown in submerged culture. Mycologia 44: HUMFELD, H Production of mushroom mycelium. Yearbook of Agr., , p HUMFELD, H., AND T. F. SUGIHARA Submerged culture of the mycelium of various species of mushroom. Appl. Microbiol. 2: MORRIS, D. L Quantitative determination of carbohydrates with dry Wood's anthrone reagent. Science 107:254. NELSON, N A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153: REUSSER, F., J. F. T. SPENCER, AND H. R. SALANS. 1958a. Protein and fat content of some mushrooms grown in submerged culture. Appl. Microbiol. 6:1-4. REUSSER, F., J. F. T. SPENCER, AND H. R. SALANS. 1958b. Tricholoma nudum as a source of microbiological protein. Appl. Microbiol. 6:5-8. SOMOGYI, M A new reagent for the determination of sugars. J. Biol. Chem. 160: SZUECS, J Essence of mushroom and its preparation. U.S. Patent 2,505,811.