Adaptability to submerged culture and amtno acid contents of certain fleshy fungi common in Finland

Mar j a L i is a H a t t u l a and H . G. G y ll e n b e r g Department of Microbiology, University of Helsinki, Finland

The literature on the possibilities of sub­ must be considered when the suitability of merged culture of macrofungi is already fungi for submerged production is evaluated. voluminous. However, the interest has merely The present work concerns a collection of been restricted to the traditionally most va­ fungi common in Finland which have been lued fungi, particularly members of the gene­ investigated according to the criteria descri­ ra and M orchella (HuMFELD, 1948, bed above. 1952, HuMFELD & SuGIHARA, 1949, 1952, Su­ GIHARA & HuMFELD, 1954, SzuEcs 1956, LITCHFIELD, 1964, 1967 a, b, LITCHFIELD & al., MATERIAL 1963) . The information obtainable from stu­ dies on these organisms is not especially en­ The material consisted of 33 of fun­ couraging because the rate of biomass pro­ gi, 29 of which were obtained from the duction seems to be too low to compete fa­ Department of Silviculture, University of ,·ourably with other alternatives of single cell Helsinki, through the courtesy of Professor P. protein production (LITCHFIELD, 1967 a, b). Mikola and Mr. 0 . Laiho. Four species were On the other hand, the traditional use of fun­ isolated by the present authors by taking a gi as human food all over the world is a sterile piece of the fruiting body at the point significant reason for continued research on where the cap and the stem are joined and the submerged production o.f fungal mycel­ by transferring it on a slant of suitable agar. ium. If the fungal mycelium is comparable The species considered a!'e listed below fol­ with other single-cell protein preparations as lowing the nomenclature of M. MosER to its protein content and amino acid com­ (1967). (For the species indicated by an position, it seems to gain preference over asterisk fruiting bodies were collected for other alternatives due man's traditional know­ comparison whereas the species with the ledge and experience concerning the toxicity + label were isolated in pure culture by characteristics and other conditions influen­ the pl'esen t authors. ) cing the edibility of fungi. A particular aspect requires attention in this connection. Clavaria ligula The traditional «« used a.s food · cibarius * concerns the fruiting bodies whereas the sub­ Suillus grevillei ( elegans) merged cultivation produces preparations of S. luteus * mycelium. Therefore it seems that besides the S. bovinus * adaptability to submerged growth the simi­ S. variegatus * larity in composition of the mycelial pre­ edulis * + paration to the corresponding fruiting bodies L eccinum testaceo-scabrum * +

39 L . sp. («Boletus versipellis«) how rapidly the fungi used the sugar in the L. scabrum * medium. The first screening experiment to Paxillus incolutus * omit the most slowly growing species was P. atrotomentosus made by growing the mycelia in the three aurantiaca («Cantharellus following media: a. «) a ) 5 % malt extract, Tricholomopsis rutilans (Tricholoma r.) b) Modess.' medium (MoDEss 1941) , Tricholoma flavobrunneum c) Reusser's medium (REusSER & al., 1958) T. imbricatum the composition of which is as follows: Lyophyllum loricatum Armillariella mellea (Armillaria m.) * Glucose 40 g, Ammonium tartrate 8 g, KH2P04 Collybia dryophila * 0.5 g, MgSo ·7 H 0 0.,5 g, CaC1 50 mg, FeS04 ·7 C. butyracea 4 2 2 H 2 0 2 mg, MnS03 • H 20 2 mg, Pyridoxine 1 mg, A1icromphale perforans ( M arasmius p.) p-Aminobenzoic acid 1 mg, Thiamine 0.5 mg, Marasmius androsaceus Distilled water 1000 ml. The glucose was sterilized procera ( p.) separately and the vitamins were added in separate Agaricus arvensis * + solution which was sterilized by filtration. All three amianthinum media were bottled in 250 ml ·Erlenmeyer-flasks, * + 80. m1 in each, and they were sterilized at ll6°C capnoides for 20 minutes. mutabilis (Pholiota m.) The inoculation was made by transferring C ortinarius hemitric h us two agar slants of every species into 80 ml Lactarius rcpraesentaneus ·* 5 % malt extract. The flasks were incubated L. necator* 10 days at room temperature as stationary L. deliciosus aggr. * cultures. The grown mycelium was washed L. rufus* with sterile distilled water and was then broken by use of a sterile Ultra-Turrax­ The pure cultures were maintained on Modess' machine. The homogenized mycelium was agar the composition of which is the following suspended into 80 ml of sterile water and 10

(MonEss, 1941): KH2 P04 0.5 g, MgS04 ·7 H 2 0 ml of this suspension was used to inoculate

0,5 g, NH4 Cl 0,5 g, Malt extract 5 g, Glucose 5 g, each flask. Because of the different amount of , FeCl3 1 % sol. 0.5 ml, Agar. 15 g, Distilled water growth of the test organisms the suspensions 1000 ml. The medium was sterilized at 116 o C for employed as inocula were made visually as 20 minutes. similar in density as possible. Every organism The mycelia were inoculated on new agar slants and medium was tested in duplicate. The once a month and the tubes were kept four weeks flasks were incubated for two weeks in a at room temperature (22-24°C) after which they rotary shaker (200 rpm) at 28 °C. After this were transferred to refrigerator temperature. For period the growth was scored visually. Table sixteen of the test organisms (indicated by in the 1 shows the results of the experiment. above list) fruiting bodies were collected. These On the basis of the results of the experi­ were cleaned of dust and sand by scraping and no ment indicated in Table 1, the following water was used for cleaning. The fruiting bodies species were omitted from further study: were cut into small pieces and dried at 40°C until , P. atrotomentosus, Tricho­ they were easy to grind to fine powder. The powder loma flavobrunneum, Tricholomopsis ru­ was kept in a closed wessel in tfue refrigerator. tilans, Lactarius repraesentaneus, L. deli­ ciosus, Mar.asmius perforans, M . androsaceus, , and RESULTS Boletus «versipellis«. An experiment concerning the sugar con­ Adaptability to submerge d culture sumption ·was made with the remaining species in order to evaluate the utilization of The adaptability to submerged culture was carbon and the ylield of mycelium per quan­ investigated by some experiments where the tity of sugar used. Reusser's medium, which mycelia were grown on different media. The contains glucose, and Modess' medium, which primary criterion in these experiments was contains maltose in addition to glucose, were

40 Table I. Submerged growth of fungi in different Table 2. The efficiency of mycelium production, media. + + + growth excellent, + + good gram of mycelium/ I 00 grams of mgar. growth, + weak grofth, - no growth (the amount mycelium equal to the inocolum). Mushroom Reusser's Modess' medium medium Cantharellus cibarius 54.6 46.7 Suillus grevillei 42.7 42.2 "'8 ..;: 8 ~ ::l 1;l ::l Suillus luteus 12.9 24.3 Species V>·~ "8~ ;:l"d Suillus bovinus 53.3 22.6 ~8 ..::"' 8"' Suillus variegatus 42.4 16.9 Clavaria ligula ++ +++ +++ testaceo-scabrum 36.4 36.2 Cantharellus cibarius +++ +++ +++ Leccinum scabrum 1.5 3.7 Suillus grevillei ++ ++ ++ Hygrophoropsis aurantiaca 26.8 33.5 Suillus luteus ++ + ++ Tricholoma imbricatum 1.0 5.4 Suillus bovinus +++ ++ +++ Lyophyllum loricatum 48.7 36.7 Suillus variegatus + + + A rmillariella melle a 43.8 32.4 Boletus edulis + + Collybia dryophila 8.8 24.5 Leccinum testaceo-scabrumt- + + +++ +++ Collybia butyracea 1.3 8.2 «Boletus versipellis« ++ 12.1 Leccinum scabrum ++ ++ Agaricus arvensis 1.8 6.9 Paxillus involutus + + + Coprinus comatus 0.8 2.6 Paxillus atrotomentosus 3.9 4.8 Hygrophoropsis 6.0 28.0 aurantiaca + + + + Cortinarius hemitrichus 57.4 50.2 Tricholomopsis rutilans -+ ++ Lactarius necator 4.5 13.4 Tricholoma Lactarius rufus 0.7 2.5 flavobrunneum + Clavaria ligula 1.0 1.9 Tricholoma imbricatum ++ Lyophyllum loricatum + + + the suspension was inoculated into 80 ml of Armillariella mellea +++ +++ +++ the medium in a 250 ml Erlenmeyer flask. Collybia drJ•ophila + + + +++ The flasks were incubated in a rotary shaker Collybia butyracea + + ++ ++ (200 rpm) at 28°C. Incubation was con­ Micromphale perforans + + + tinued until a considerable amount of rapidly Marasmius androsaceus + + + growing mycelium had developed. During the M acrolepiota pro cera + + ++ ++ incubation the volume of the medium was Agaricus arvensis + + + controlled by adding distilled water to com­ Cystoderma amianthinum + + pensate for evaporation losses. The mycelium Coprinus comatus + + + was separated by Buchner-filtration, and was Hypholoma capnoides + + ++ + then dried to constant weight. Table 2 shows Kuehneromyces mutabilis + + + the efficiency of submerged growth expressed Cortinarius hemitrichus - ++ +++ as grams of mycelium per 100 grams reducing Lactariusrepraesentaneus - sugar. Lactarius necator ++ ++ ++ The results clearly indicated that several of Lactarius deliciosus the species included in this experiment grow aggr. too slowly to be useful for submerged my­ Lactarius rufus + ++ celium production. The growth rate seemed to depend on the medium as well as on the chosen for this experiment. Sugar deter­ species. However, both the media emploved minations were made (a) before inoculation are commonly used in the submerged culti­ and (b) at the time the incubation was in­ vation of fungi. In either or both of the terrupted according to the method of Ber­ media the following ten species yielded trand ( GR:oSSFELD, 1935). A two weeks old mycelium to an amount higher than 25 % stationary culture of mycelium was used as of the weight of the utilized sugar and might inoculum. The mycelium was broken by thus be worth further attention: C antharellus Ultra-Turrax into sterile water and 10 ml of cibarius, Hyprophoropsis aurantiaca, Suillus

41 bovinus, S. variegatus, S. grevillei, Leccinum stationary cultures of the corresponding fungi. testaceoscabrum, Armillarie!la mellea, Lyo­ The mycelia were washed with water, broken phyllum loricatum, Kuchneromyces mutabilis with Ultra-Turrax and suspended in sterile and Cortinarius hemitrichus. · water. These suspensions were used as inocula. The inocula were made visually as similar in density as possible. The growth was fol­ Amino acid content lowed by taking twice a week a sample for sugar and ammonium nitrogen determin­ For the production of mycelium for amino ations (GROSSFELD 1935, CoNWAY 1957). The acid analysis the fungi were grown in sub­ cultivation was considered finished when all merged culture in 2 liter fermentors which the sugar was consumed. The grown myc­ contained 1 liter medium. The temperature elium was separated by Buchner filtration was 28°G, agitation 280 rpm and the aeration and dried at room temperature. The dry rate was 1 1 air /min/1 medium. Although the mycelium was ground to fine powder and first experiments had shown that Reusser's the determinations of moisture and ash medium would not be optimal for all the test content (A.O.A.C. 13 004, 13 006, 1960) a~ organisms this medium was used in these ex­ well as of nitrogen content (WILSON & Wu­ periments because its composition is exactly soN, 1960) were made from the powder. Tl.e defined. The first cultivations were made total protein content was calculated by m1,l. with the species which had the greatest yield tiplication of the nitrogen value obtained by coefficients in the sugar consumpiton expe­ the micro-Kjeldahl method by 6.25. Two riments. In addition mycelia of those species species, Collybia dryophila and Paxillus invo­ for which fruiting bodies were available were lutus were cultivated in Erlenmeyer flasks in also produced. a rotary shaker because the culture in fermen­ The inocula were made from two weeks old tors did not succeed due to contamination.

Table 3. Growth of the mycelia in submerged culture and comparison of the mam constituents of the mycelium to the corresponding figures for the fruiting bodies.

Mycelium Fruiting bodies

'- '. .... 8 "' c: u c 0 ;:l d(d ~ 0 .9 '0 c·~ '"d"'""' .6-JQ.) S.g <; .... a ~-.; ~·5 ..c:'Otlso 0 ;:l '0 "@ "' ~~~ c: j;'_ ·c: c:-:.i ·u"' ~ -~~ ~ g ~b/J~ ~il 'E a ~ 'E a p. e&sabb eg bt) ..c:j;'_ ..c: "' "' 0"' 0 o .... 2 c c'ol e c CfJ ~ u u 0 bt)~.s ~ 8 (:1.'0 ~ '0 8 P.'O Cantharellus cibarius 17 23 .1 3.91 28.5 9.44 21.5 Suillus grevillei 16 20..3 6.35 36.4 Suillus luteus 20 62.0 4.25 23.8 5.85 16.4 Suillus bovinus 18 41.4 3.26 25.9 5.04 20.9 Suillus variegatus 35 15.9 3.67 30.3 5.36 9.8 Boletus edulis 50 57.9 4.98 37.6 7.66 33.8 Leccinum testaceo-scabrum 14 43.3 3.53 38.5 5.84 28.7 Leccinum scabrum 19 21.3 4.46 47.1 5.28 32.0 Paxillus involutus grown m shaker 3.50 30.4 10.96 29.7 Hygrophoropsis aurantiaca 18 38.4 2.09 23.2 Lyophyllum loricatum 7 32.3 4.01 38.3 A rmillariella melle a 17 42.1 8.97 18.9 8.66 21.0 Collybia dryophila grown in shaker 1.95 22 .1 8.81 30.4 Agaricus arvensis 53 20.8 3.97 42.4 11. 24 55.1 Coprinus comatus 52 13 .1 4.70 39.2 9.28 23.0 K uehneromyces mutabilis 21 22.3 7.08 49.7 Cortinarius hemitrichus 17 31.8 3.74 40.9 Lactarius necator 49 45.9 2.81 21.4 8.82 22 .3 Lactarius rufus 29 46.3 2.25 17.8 7.84 34.2

42 The growth of the mycelia is presented in Rotawapor evaporator at 40°C . Distilled water Table 3 where the main composition of the was added and evaporated until the pH of the mycelia is also compared with the corres­ solution was about 2. One drop of 30 % H 2 0 2 ponding data for the fruiting bodies. The and some ml:s of water were added to the dry results show that the growth rate of the fungi amino acids to oxidize cysteine to cystine and the was very low. Compared with microorganisms solution was allowed to stand for one hour at room grown in submerged culture even the shortest temperature. The amino acids were diluted with cultivation time with Lyophyllum loricatum, 12.5 % sucrose soiution, the pH was adjusted to i.e. seven days, must be considered far too 1.9- 2.1 and the final volume of the solution was long. The average growth time in the medium 25 ml, one ml of which was analyzed by Technicon used was 27 days. The average protein con­ Auto Analyzer. tent in the mycelia (32.2 %) cannot be There are notes in the literature on losses of regarded as high but it was higher than in amino acids during hydrolysis, but, on the other corresponding fruiting bodies (27 .1 %) . hand, according to a number of investigators valine The amino acid determinations were carried will not be liberated at the same time with other out in the following way: The dry powdered pre­ amino acids. To obtain reliable results the mush­ paration of either mycelium or fruiting bodies was room material was hydrolyzed for different periods heated at 103 °C to constant weight and 100· mg of time, 12, 20, 24 and 72 hours. After 12 hours of this material was weighed into 100 ml of 6-n hydrolysis only a -e-diaminopimelic acid was HCI. Nitrogen gas was bubbled through the so­ released quantitatively. There was no evident diffe­ lution before hydrolysis. The hydrolysis was made rence in the amino acid composition of the material under reflux at 180°C and nitrogen was conduc­ hydrolyzed for 20 and 24 hours, and the 20 hours ted into the vessel through the condenser. After hydrolysis was chosen as the zero time. No rise hydrolysis the solution was filtered to remove in the valine content occured after 72 hours humin and the hydrocloric acid was removed in hydrolysis even if a metal catalyst was used, but

Table 4. Amino acid composition of Leccinum scabrum, Cantharellus cibarius and Armillariella mellea and the average composition of the amino acids of the mycelium and the fruiting body prt> parations in- vestigated. Amino acids are given as grams per 16 gram protein nitrogen. Amino acid Leccinum scabrum Cantharellus A rmillarie lla Amino acids cibarius melle a average fr. body my c. fr. body my c. fr. body myc. fr. body my c.

Aspartic acid 6.88 6.89 7.04 8.32 10.09 6.52 7.30 7.19 Threonine 3.81 3.51 3.21 5.27 4.70 4.33 4.02 4.28 Serine 3.59 3.58 3.40 4.14 5.70 3.99 3.92 4.07 Glutamic a:cid 11.88 11.47 10.24 10.73 14.52 7.51 11.03 9.65 Proline 3.27 3.55 2.79 3.76 3.84 2.79 3.70 3.80 Glycine 5.55 3.94 3.24 4.33 3.96 2.96 4.27 3.85 Alanine 6.31 5.78 3.85 5.57 2.83 4.05 4.62 4.94 Valine 0.27 0.23 0.63 1.57 0.36 0.53 0.53 0.31 Cystine 4.39 4.93 3.56 5.04 5.17 3.98 4.41 4.82 Methionine 0.30 0.88 0.80 1.00 0.24 0.34 a-e-diaminopimelic acid 18 .83 9.76 15 .85 12.61 11 .86 10.47 14.70 13 .43 Isoleucine 2.51 7.36 5.42 5.47 4.65 5.74 3.32 5.08 L eucine 3.57 6.11 4.96 6.67 5.57 5.18 4.85 5.89 Tyrosine 1.36 2.85 1.95 2.79 2.76 2.19 2.51 2.73 P henylalaninc 2.87 3.46 2.67 3.02 1.76 2.75 2.60 3.10 y-aminobutyric asid 0.59 0.07 0.56 0.60 0.78 2.82 1.59 1.13

Ammonia (NH4 ) 2S04 11.12 10.36 13.54 8.13 10.02 18.86 12.63 10.62 Ornithine 0.93 0.09 3.93 0·.38 0.40 OAO 0.64 0.30 Lysine 3.68 4.31 3.55 5.34 4.44 4.43 3.88 4.80 Tryptophan 1.46 1.63 1.28 3.12 1.71 3.24 1.79 2.19 Histidine 2.58 2.61 2.26 2.62 1.91 2.23 2.49 2.54 Arginine 4.73 6.19 5.20 4.87 5.31 5.05 5.04 5.01

43 Table 5. The range of variation in the amounts of different mycelia and 14 preparations of amino acids in the protein of different fungi fruiting bodies, three species are chosen to compared to the FAO recommendations, ammo give a representative picture of the amino acids g/16 g protein nitrogen. acid composition. The fruiting bodies of these species are commonly used as food and the Amino acid Fruiting Mycelium FAO experience of their adaptation to submerged body recomm­ end­ cultivation has been satisfactory in the ations authors' experiments. The amino acid com­ Aspartic acid 6.06-10.09 2.66- 9.92 position of the fruiting bodies and the mycelia Threonine 2.07- 5.23 1.83- 5.32 of Leccinum scabrum, Cantharellus cibarius Serine 2.57- 5.10 2.10- 5.24 and Armillariella mellea and the average ami­ Glutamic acid 6.52-15.31 3.41-13.72 no acid composition of the fungi investigated Pyroline 2.76- 5.44 2.69- 5.75 are presented in Table 4 (a corresponding Glycine 3.32- 5.55 1.96- 4.70 comparison of the amino acid contents of the Alanine 2.H3- 6.31 3.82- 5.78 mycelium and fruiting bodies of Boletus Valine 0 0.80 0 1.57 4.2 luteus is presented in the accompanying Cystine 3.47- 5.78 1.66- 5.87 paper; HATTULA & GYLLENBERG, 1969). The Methionine 0 - 0.80 0 - 1.08 2.2 range of variation in the amounts of amino a- e-diamino- acids considering all the fungi investigated pimelic acid 6.93- 21.21 5.05-54.35 as compared to the F AO recommendations Isoleucine 0 6.39 0 - 14.13 4.2 are presented in Table 5. Leucine 0 - 8.82 2.01 - 8.09 4.8 The present results show that the amino Tyrosine 0 - 4.62 1.12- 3.86 acid composition in different species is very Phenylalanine 0 3.64 0 3.86 similar in the mycelium and the fruiting body, y -aminobutyric respectively. The main component is ninhyd­ acid 0.33- 4.69 0.07- 2.67 rinpositive a-e-diaminopimelic acid which is Ammonia 2.18-26.26 5.95- 15 .95 not known to be of nutritional value. All the Ornithine 0 3.93 0 1. 09 essential amino acids are present in most of Tr}'ptophan 1.32- 3.17 1.24- 3.72 1.4 the species but the amounts of valine and Lysine 3.31- 6.14 1.35- 8.39 4.2 methionine are very small. The small amonut H istidine 1.91- 3.44 0.91- 3.44 of methionine in microbial protein is well Arginine 3.31- 6.77 1.61 - 8.39 known but the small amount of valine seems to be characteristic of Finnish fungi. There are, however, very few species of fungi in the amounts of cystine and isoleucine increased which other essential amino acids than and the amount of increase was constant. The methionine and/or valine are lacking. The amount of aumonia atso increase d as a result of the fruiting body of Lactarius necator contains destrunction of labile amino acids. After 72 hours no valine, methionine, isoleucine and phe­ hydrolysis losses of aspartic acid, threonine and nylalanine but in the corresponding mycelium serine occured. In .the final results all the values only methionine is lacking. There occurs in are extrapolated to the zero time. Because of the the fruiting body of this more than complete destruction of tryptophan during the acid 50 % of a- £-diaminopimelic acid as com­ hydrolysis it was determined separately employing pared with the total amount of amino acids. the enzymatic method described by SPIES ( 196 7), If the average contents of the essential amino which is based on the use o.f pronase ( Calbio­ acids are compared to the F AO recommen­ chem) . The selected methods were maintained dations only valine and methionine remain constant throughout all the analyses and every both in the mycelia and in the fruiting bodies determination of amino acids was made immediate­ below the limits defined. The amount of ly after hydrolysis to avoid losses which occur in cystine in the Finnish fungi is unusually high. solutions. Because cystine can substitute for methionine up to 80 50 in the diet the low valine content Because it seems purposeless to describe in seems to constitute the only real amino acid detail the amino acid composition of 18 deficiency of the fungi investigated.

44 SUMMARY

Out of 33 species of fungi only ten were content of the mycelia produced in submerged found to adapt easily to submerged growth. culture was on the average somewhat higher However, these organisms, too, required as a than the corresponding figure for the fruiting rule 14 day or more for a complete utilization bodies of the same fungi. The amino acid of 4 per cent sugar in a synthetic medium. composition of the protein was in general The highest yield coefficient for mycelium the same in corresponding preparations of production (g mycelium per 100 g consumed mycelium and fruiting bodies. Except for sugar) among these fungi was 57 (Cor­ valine and methionine fungal protein seems tinarius hemitrichus) whereas the highest to be a satisfactory source of essential amino protein content in dry mycelium was 49.7 % acids. ( K uehneromyces mutabilis). The protein

REFERENCES : Association of Official Agricultural Chemists, 1960 : LITCHFIELD, J. H ., 1967 b: Submerged culture of Ninth edition A.O .A.C., P.O. Box B, Frank­ Mushroom mycelium, Food T echno!., 21, lin Station, Washington 4 D.C., 158. 55-57. CoNWAY, E. ]., 1957 : Microdiffusion analysis., 4th LITCHFIELD, J. H., R. C. O vERBECK & R . S. DAVID­ edition, Crosby Lockwood and Son ~ td ., soN, 19·6·3 : Factors affecting the growth of London, 465. morel mushroom mycelium in submerged FAO Nutritional Studies No. 16, 195 7: Food and culture, Agr. Food Chern., 11 , 158- 162. Agricultural Organization of the United MoDESS , 0., 19·41: Zur Kenntniss der Mykorrhiza­ Nations, Rome. bildner von Kiefer und Fichte, Sym.bot. GROSSFELD, ] ., 1935 : Kohlenhydrate in Handbuch upsal., V 1:1 16. der Lebensrnittelchemie, ed. A. Bohmer, MosER, ·MEINHARD, 196 7: Die Rohrlinge und Blat­ Springer Verlag, Berlin III, 2, 8·66- 868. terpilze () . In: Helmut Gams: HATTULA, M. L. & H. G. GYLLENBERG, 1969: Pro­ Kleine Kryptogamenflora, Band II b/2 tein and fat composition and vitamin con­ (Basidiomyceten II. Teil) . . Gustav Fischer tent o.f Boletus luteus mycelium produced in Verlag, Stuttgart 433 pp., 429 figs. in 13 submerged culture. Karstenia 9, 46- 50. pls. HuMFELD, H., 1948 : The production of mushroom REUSSER, F ., I. F. T . SPENCER & H. R. SALLANS, mycelium () in submer­ 19·58.: Protein and fat content of some ged culture, Science, 107, 373 . grown in submerged culture, HUMFELD, H ., 1952 : Production of mushroom myc­ Appl. Microbial., 6 : 1-4. elium, U .S. Patent 2 618 000. SPIES, J. R., 1967: Determination of tryptophan in HUMFELD, H. & T . F. SuGIHARA, 1949 : Mushroom proteins, Anal. Chern., 39, 1412-1416. mycelium production by submerged propa- SuGIHARA, T . F. & H . HuMFELD, 1954: Submerged gation, Food Techno!., 3, 355-356. . culture of the mycelium of various species HuMFELD, H. & T . F . SuGIHARA, 1952 : The of mushroom, Appl. Microbial., 2, 170- nutrient requirements of Agaricus cam­ 172. pestris grown in submerged culture, Myco­ SzuECs, ]., 1956 : Mushroom culture, U .S. Patent logia, 44, 605-620. 2 761 246. LITCHFIELD, ]. H. : 1967 a : Submerged culture of WILSON , G. L. & D . W . WILSON, 1960: Compre­ mushroom mycelium, Microbial T echnology, hensive Analytical Chemistry, Elsevier Pub­ Reinhold Publishing Corporation, New lishing Company, Amsterdam, London, York, Amsterdam, London, 107- 144. New York, Princeton, 505 .

45