1. INTRODUCTION

1.1. Brief literature review

Submerged propagation of mushroom mycelium in shaken flasks or fermentors is an easy way of producing large quantities for physiological and biochemical studies. Several industrial applications have been investigated:

1.

Production of mycelium of edible mushrooms for human and animal consumption. This subject has most recently been reviewed by Worgan [160] and Gray [56].

2.

Production of spawn for the cultivation of mushroom fruit bodies as suggested by Humfeld [74].

3.

Production of particular chemical substances like glucan [25], dehydroacetic acid [102], nucleotides [66], antibiotics [17, 117, 127, 167], tumour inhibiting substances [36] and enzymes [11, 82, 85, 104].

This thesis only deals with the cultivation of mushroom mycelium for food purposes. To this end several aspects are important, e.g. the nutritional requirements of the mycelium, its growth rate and maximum yield, the nutritional value of the product, its toxicity and flavour.

Humfeld and Sugihara [72, 73, 74, 75] reported the isolation of several strains from cultivated mushrooms. They selected three fast growing strains (NRRL 2334, 2335 and 2336) giving excellent yields in a defined medium containing glucose, urea and minerals. The nutrient value of the mycelium was satisfactory, but the flavour was weak and unlike mushroom flavour. Neither was the mycelium suitable as spawn [74]. Because of many physiological and morphological differences between the NRRL-strains and other strains of the cultivated mushroom, Molitoris [103] studied the NRRL-strains taxonomically and identified them as Beauveria tenella, an imperfect fungus which may occur on the cap of Agaricus bisporus. Hence it is not surprising that Humfeld was unable to use the mycelium as spawn and that the flavour was unsatisfactory. Our studies [26] have shown that mycelium grown in submerged culture can be used as spawn for the production of fruit bodies. Several other studies of the production of mushroom mycelium by the NRRL-strains mentioned [38, 110, 126, 140] have, of course, become inconclusive. These disappointing results illustrate the problem of isolating reliably pure cultures of mushroom mycelium.

A survey of other higher fungi that have been grown in submerged cultures (including those reported in this thesis) is given in Table 1.

Table 1. Species of higher fungi grown in submerged culture.

Genus

Species 

References

Genus

 Species

References

Agaricus

 

 

 

 

 

 

arvensis

33

Marasmius

 

 

oreades

33

bernardii

33

putillius

157

bisporus

33, 89, 130, 144, 150, this thesis

scorodonius

157

bitorquis

137, this thesis

Morchella

 

 

 

 

 

 

 

 

augusticeps

52

blazei

137

conica

52

campestris

21, 33, 38, .39, 59, 60, 126, 150, 162

crassipes

21, 52, 79, 88, 93, 137

placomyces

137

elata

53

Amanita

caesarea

144

esculenta

33, 52, 79, 88, 93, 130, this thesis

Armillaria

mellea

33, 53, 137, 157, this thesis

hortensis

52, 93, this thesis

Boletus

 

 

 

 

edulis

33, 37, 125

hybrida

39, 126

granulatus

157

semilibera

52

grevillei

157

vulgaris

52

indecisus

39, 126

Myceliumradicus

atrovirens

157, 159

variegatus

120

Oudemansiella

mucida

162, 167

Calvatia

gigantea

130, this thesis

Paxillus

prunulus

157, 159

Cantharellus

 

cibarius

21, 39, 137, 144, 150

Pholiota

mutabilis

157

clavatus

150

Pleurotus

 

 

 

 

cornucopias

33

Cenococcum

graniforme

157

flabellatus

132

Clitocybe

nebulas

33

japonicus

162

Collybia

 

 

butyracea

157

ostreatus

21, 33, 53, 54, 130, 137, 138, this thesis

umbudata

137

velutipes

21, 39, 53, 66, 126, 137, 162

Polyporus

 

borealis

157

Coprinus

comatus

33, 137, 144, 164, this thesis

suphureus

137

Hebeloma

sinapizans

137

Torichoroma

 

matsutake

162

Kuhneromyces

mutabilis

53

robustum

162

Lactarius

piperatus

144

Trametes

gibbosa

157

Lentinus

edodes

138, 141, 162, this thesis

Tricholoma

 

 

 

flavobrunneum

157

Lepiota

 

 

naucina

137

nudum

21, 33, 39, 53, 63, 90, 126, 137, 150

procera

33, 137

personatum

33

rhaeodes

33, 137

pessundatum

157

Limacium

eburneum

144

Schizophyllum

commune

137

Lycoperdon

umbrinum

137

Volvariella

volvacea

6, 33, this thesis

Marasmius

foetidus

157

Xylaria

polymorpha

39, 126

Table 1 is not complete, by way of example a number of woodrotting basidiomycetes of doubtful edibility [80] and a number of species mentioned by Espenshade [35] without any specification of culture conditions and yields are not included. In most papers fructification experiments, which are only possible with a limited number of fungi, were not reported; thus the identity of the isolates is not absolutely sure. The serious difficulties experienced in isolating pure cultures of Cantharellus species in the Netherlands in recent years [12] create doubts about the validity of results reported on submerged cultures of members of this genus.

It is not necessary to describe in this introduction all the experiments carried out with the species listed in Table 1 because there is already an extensive review by Worgan [160], who also considered the production of other metabolic products than cell substance by mushroom mycelium. Only some recent publications have to be mentioned here.

Volz [150] studied submerged growth of strains of Agaricus bisporus, Agaricus campestris, Cantharellus clavatus, Cantharellus cibarius, Pleurotus ostreatus, Tricholoma nudum and Volvariella volvacea, as well as two mutants of Agaricus bisporus and one mutant of Volvariella volvacea. He compared growth in media with 44 carbon and energy sources and 31 nitrogen sources. In addition, he examined the influence of vitamins and growth hormones.

Atacador-Ramos et al. [6] developed an optimal medium for submerged growth of Volvariella volvacea. In this medium, which contained saccharose as source of carbon and energy, urea as source of nitrogen and coconut milk for providing growth factors, they observed maximum mycelial growth (18 g/1) after 3 days. They analysed the amino acid and vitamin content of the mycelium.

Janardhanan et al. [79] used vegetable wastes (turnip and cabbage; cauliflower leaves) as the source of nitrogen for submerged growth of Morchella species. With an extract of cauliflower leaves, supplemented with 5% glucose, they observed yields up to 12.7 g/1 after 7 days.

Srivastava and Bano [132] investigated the nutritional requirements of Pleurotus flabellatus, a mushroom which is eaten by the people in Mysore State (India). The fungus grew well in shake flasks containing a synthetic medium with ammonium citrate as nitrogen source.

Guha and Banerjee [59, 60, 61] examined submerged growth of Agaricus campestris, using 14 sources of carbon and energy and 16 nitrogen sources. The basal medium contained yeast extract and minerals. The highest yield was 7.2 g/1 after 7 days in a medium containing peptone and glucose. They also published results of chemical analyses of the mycelium.

Zarudnaya [164] studied the growth of 7 species of the genus Coprinus, including Coprinus comatus. Growth after 15 - 25 days was compared using 10 carbon and energy sources and 5 nitrogen sources.

Sugimori et al. [138] screened several non-carbohydrate carbon sources (hydrocarbons, alcohols, organic acids) and found good growth of Lentinus edodes and a Schizophyllum species on ethanol. The optimum concentration of ethanol was 2% giving a yield of 10 g/1 after 3 days. They analysed the amino acid composition of the mycelium and studied the digestibility.

Hamid et al. [63] produced mycelium of Tricholoma nudum from industrial wastes (cane and beet molasses, sulphite waste liquor, spent wash, corn steep liquor), supplemented with a simple nitrogen source (ammonium tartrate, ammonium sulphate or urea), and determined the crude protein and lipid content of the mycelium. Cane molasses was the best carbon and energy source; sulphite liquor was less suitable, possibly because of the presence of toxic substances. The best yield was 17 g/1 after 3 days.

Kosaric et al. [83] also used sulphite liquor for submerged growth of Morchella species. The sulphite liquor, containing 40 to 70 g/1 of carbohydrate, was diluted 1: 5 and supplemented with ammonium phosphate and corn steep liquor. They obtained 5 g/1 of dry mycelium after 9 days. The same workers [88] reported results of amino acid and fatty acid analyses of Morchella mycelium grown in this way. They also examined the flavour by gas chromatography and by ultraviolet and infrared spectroscopy, but did not identify any flavour compound.

Ginterová [53, 54] studied submerged cultures of 11 strains of Pleurotus ostreatus. She observed that agitation of the cultures favoured the formation of monokaryotic mycelium, which could be detected microscopically due to the absence of clamp connections. Three strains of Pleurotus ostreatus and five other mushroom species were investigated for their ability to fix molecular nitrogen. Two strains of Pleurotus ostreatus and one strains of Morchella elata and Kuhneromyces mutabilis were able to fix nitrogen up to 7 mg per gram of sugar consumed in a medium of malt extract. Ginterová and Maxianová [55] confirmed the nitrogen fixation by making up the balance of nitrogen in cultures of Pleurotus ostreatus, growing and fructifying on natural substrates. Starting with 530 g of bound nitrogen, the substrate, mycelium and fruit bodies contained 714 g of nitrogen after growth. Ginterová suggested that nitrogen fixation might be quite commonly found among the higher fungi. Nitrogen fixation was also observed in cultures of Pleurotus sajorcaju by Rangaswami et al. [123].

Shannon and Stevenson [130] used brewery wastes for growth of four yeasts and four mushroom species (Agaricus bisporus, Calvatia gigantea, Morchella esculenta and Pleurotus ostreatus). High yields of Calvatia gigantea (39.7 g/1) were obtained in cultures on "trub press liquor" supplemented with ammonium sulphate, after shaking during 8 days. These authors also considered this process as a method of reducing the BOD of industrial waste. The highest BOD reduction was observed with Calvatia gigantea (75%) in "grain press liquor" supplemented with ammonium sulphate.

Yahagi [161] used an unidentified basidiomycete for the production of protein from powdered wood supplemented with a small amount of glucose and dried yeast. About 2% of the weight of the wood was converted into protein after shaking for 7 days at 20 C.

Lee et al. [89] cultured Agaricus bisporus in tryptone-yeast extract media. The highest yield was observed after 12 days. They also used (NH4)2HPO4 as nitrogen source and replaced glucose with other carbohydrates.

Only a few mushroom species have been grown in submerged culture on a pilot plant scale. In the USA Morchella mycelium grown in submerged culture has been produced with the trade name "morel mushroom flavouring" [94].

Torev [144] observed rapid growth of some mushroom species in fermentors with a volume up to 50 m3. It is not clear from his article which species were used for these cultures, but they did not include Agaricus bisporus [145]. According to a recent note [3] 100 tons of mushroom mycelium were produced in the Bulgarian industry in 1974. The production will be increased to 20,000 tons per year. This is possibly the same process as that reported by Torev [144].

Obstacles to the economic production of mushroom mycelium which have to be overcome include the slow growth of most species, the rich media that are needed and a lack of delicious flavour of the product. Many species do not grow in media with simple nitrogen sources such as ammonium salts, but need a complex source of nitrogen or mixtures of amino acids. The rich medium, the slow growth and the required pH-value (often between 6 and 7) make the cultures extremely vulnerable to contamination, so that absolute sterility is required; but these disadvantages need not necessarily be prohibitive if the mycelial cultures at least develop the characteristic flavour of fruit bodies.

Previously the flavour of mushroom mycelium was mainly studied by organoleptic means and results were mostly based on a limited number of judgements. The chemical nature of the flavour of mushrooms was unknown. In recent years the knowledge of the constituents of the cultivated mushroom and of Boletus edulis has increased considerably, because of the application of modern methods such as combined gas chromatography and mass spectrometry. Hence, it is possible now to study the production of flavour compounds in a mycelial culture by physical and chemical means.

1.2. The purpose of the present investigation

Because much work had already been done on the production of protein by higher fungi in submerged culture, the main purpose of the present investigations was to study the production of flavour compounds. To be able to grow sufficient amounts of mushroom mycelium required a detailed knowledge of the nutritional needs of the strains to be used. Because many differences may exist in the behaviour of the strains of the same fungal species, it was impossible to use data from the literature. In Chapter 2 the nutritional requirements, established experimentally for our particular strains of Agaricus bisporus and Coprinus comatus, will be reported.

On the basis of these results, the possibility of utilising the mycelium of these strains as protein sources will be discussed in Chapter 3.

The chemical nature of the flavour of Agaricus bisporus and Coprinus comatus will be dealt with in Chapter 4. The occurrence of some important flavour compounds in other edible mushrooms will also be considered.

After these investigations it was possible to study the production of flavour compounds in submerged cultures by various strains of edible mushrooms, particularly Agaricus bisporus and Coprinus comatus. The results will be reported in Chapter 5.

Some results of the submerged growth of Agaricus bisporus and Coprinus comatus have already been published [26, 27] and will in part be included in Chapters 2 and 5. Several other results of our investigations of the submerged growth of Agaricus bisporus and Coprinus comatus are in press [28] these results are included in Chapter 2. In addition, some articles on our studies of the flavour of mushrooms, described in Chapter 4, have been accepted for publication [29, 30, 31].

REFERENCES TO CHAPTER 1

3.

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6.

Atacador-Ratnos, M., M.A. Pale, D.V. Villadolid, D.S. Cruz. 1967. A study on submerged culture production of banana mushroom (Volvariella volvacea) mycelium as a source of protein, B-Vitamins and food flavor. Philippine J. Sci. 96, 191-216.

11.

Bastide, J.M., M. Bastide, M. Bonte, P. Trave. 1972. Etude de la culture du basidiomyète QM 806 en fermenteur. Production d'une exo-β-(1, 3)-D-glucanase. Trav. Soc. Pharm. Montpellier 32, 93-106.

12.

Bels, P.J. 1975. Domesticatie onderzoek en sortimentsuitbreiding van paddestoelen in Nederland. Mushroom Experiment Station, Horst-America, The Netherlands.

17.

Brian, P.W. 1951. Antibiotics produced by fungi. Bot. Rev. 17, 357-430.

21.

Cirillo, V.P., W.A. Hardwick, R.D. Seeley. 1960. Fermentation process for producing edible mushroom mycelium. U.S.A. Patent 2,928,210.

25.

Davis, E.N., R.A. Rhodes, H. Russell Shulke. 1965. Fermentative production of exocellular glucans by fleshy fungi. Appl. Microbiol. 13, 267-271.

26.

Dijkstra, F.IJ., W.A. Scheffers, T.O. Wikén. 1972. Submerged growth of the cultivated mushroom, Agaricus bisporus. Antonie van Leeuwenhoek; J. Microbiol. Serol. 38, 329-340.

27.

Dijkstra, F.IJ. 1975. Influence of carbon sources on the growth of mushroom mycelium in submerged culture. Delft Progr. Rep., Series A: Chemistry and Physics, Chemical and Physical Engineering 1, 120-124

28.

Dijkstra, F.IJ. Influence of nitrogen sources and vitamins on the growth of mushroom mycelium in submerged culture. Delft Progr. Rep., Series A: Chemistry and Physics, Chemical and Physical Engineering (in press).

29.

Dijkstra, F.IJ., T.O. Wikén. 1976. Studies on mushroom flavours. 1. Organoleptic significance of constituents of the cultivated mushroom, Agaricus bisporus. Z. Lebensm. Unters. Forseh. 160, 255-262.

30.

Dijkstra, F.IJ., T.O. Wikén. 1976. Studies on mushroom flavours. 2. Flavour compounds in Coprinus comatus. Z. Lebensm. Unters. Forseh. 160, 263-269.

31.

Dijkstra, F.IJ. Studies on mushroom flavours. 3. Some flavour compounds in fresh, canned and dried edible mushrooms. Z. Lebensm. Unters. Forseh. 160 (in press).

33.

Eddy, B.P. 1958. Production of mushroom mycelium by submerged cultivation. J. Sci. Food Agr. 9, 644-649.

35.

Espenshade, M.A. 1962. Mushrooms and toadstools. Their growth in liquid media using deep culture techniques. Mushroom Sci. 5, 213-217.

36.

Espenshade, M.A., E.W. Griffith. 1966. Tumor-inhibiting basidiomycetes. Isolation and cultivation in the laboratory. Mycologia 58, 511-517.

37.

Eybergen G.C. van, W.A. Scheffers. 1972. Growth of the mycelium of Boletus edulis on agar media and in submerged liquid cultures. Antonie van Leeuwenhoek; J. Microbiol. Serol. 38, 448-450.

38.

Falanghe, H. 1962. Production of mushroom mycelium as a protein and fat source in submerged culture in medium of vinasse. Appl. Microbiol. 10, 572-576.

39.

Falanghe, H., A.K. Smith, J.J. Rackis. 1964. Production of fungal mycelial protein in submerged culture.of soybean whey. Appl. Microbiol. 12, 330-334.

52.

Gilbert, F.A. 1960. The submerged culture of Morchella. Mycologia 52, 201-209.

53.

Ginterová, A. 1973. Nitrogen fixation by higher fungi. Biologia (Bratislava) 28, 199-202.

54.

Ginterová, A. 1973. Dedikaryotization of higher fungi in submerged culture. Folia Microbiol. (Prague) 18, 277-280.

55.

Ginterová, A., A. Maxianová. 1975. The balance of nitrogen and composition of proteins in Pleurotus ostreatus grown on natural substrates. Folia Microbiol. (Prague) 20, 246-250.

56.

Gray, W.D. 1970. The use of fungi as food and in food processing. Butterworths, London.

59.

Guha, A.K., A.B. Banerjee. 1970. Effect of different nitrogenous compounds on the submerged production of Agaricus campestris mycelium. J. Food Sci. Technol. 7, 23-25.

60.

Guha, A.K., A.B. Banerjee. 1971. Effect of different carbon compounds on the submerged production of Agaricus campestris mycelium. J. Food Sci. Technol. 8, 82-83.

61.

Guha, A.K., A.B. Banerjee. 1973. Nutritive value of mycelia and mycelial protein of Agaricus campestris grown under submerged culture. Indian Agr. 17, 45-53.

63.

Hamid, A., F.H. Shah, M.A. Qadeer. 1972. Production of mushroom mycelium from industrial wastes. Pak. J. Biochem. 5, 57-60.

66.

Hashida, W., T. Mouri, 1. Shiga, S. Teramoto. 1967. Fermentation of nucleic acid related substances by basidiomycetes. 1. Changes of nucleic acid related substances in submerged culture of Collybia velutipes. J. Ferment. Technol. 45, 1108-1118.

72.

Humfeld, H., T.F. Sugihara. 1949. Mushroom mycelium production by submerged propagation. Food Technol. (Chicago) 3, 355-356.

73.

Humfeld, H. 1948. The production of mushroom mycelium (Agaricus campestris) in submerged culture. Science 107, 373.

74.

Humfeld, H. 1950-51. Production of mushroom mycelium. U.S. Dep. Agr. Year Book, p.242.

75.

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79.

Janardhanan, K.K., T.N. Kaul, A. Husan. 1970. Use of vegetable wastes for the production of fungal protein from Morchella species. J. Food Sci. Technol. 7, 197-199.

80.

Jennison, M.W., M.D. Newcomb, R. Henderson. 1955. Physiology of the wood-rotting basidiomycetes. 1. Growth and nutrition in submerged culture in synthetic media. Mycologia 47, 275-304.

82.

Kawai, M., N. Mukai. 1970. Studies on milk clotting enzymes produced by basidiomycetes. 1. Screening tests of basidiomycetes for the production of milk clotting enzymes. Agr. Biol. Chem. 34, 159-163.

83.

Kosaric, N., A. LeDuy, J.E. Zajic. 1973. Submerged culture growth of edible mushrooms on waste sulphite liquors. Can. J. Chem. Eng. 5 1, 186-190.

85.

Kubacková, M., S. Karácsonyi, J. Váradi. 1975. Studies on xylanase from Basidiomycetes. Selection of strains for the production of xylanase. Folia Microbiol. (Prague) 20, 29-37.

88.

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89.

Lee, J.S., S.R. Lee, T.J. Yu. 1975. Production of mushroom mycelium (Agaricus campestris) in shaking culture. Hanguk Sikp'um Kwahakhoe Chi 7, 22-29; Chem. Abstr. 83, ref 94878f.

90.

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93.

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94.

Litchfield, J.H. 1967. Submerged culture of mushroom mycelium. In: H.J. Peppler (ed.): Microbial Technology. Reinhold Publishing Corp., New York, p. 107-144.

102.

Miyazaki, K., M. Sakaguchi, T. Shibamoto, Y. Sacki, N. Hashimoto. 1971. Production of dehydroacctic acid by basidiomycetes. Nippon Nogei Kagaku Kaishi 45, 317-320.

103.

Molitoris, H.P. 1963. Untersuchungen an Beauveria tenella (NRRL 2334, 2335, 2336; bisher Agaricus campestris). 1. Systematik. Arch. Mikrobiol. 47, 57-71.

104.

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110.

Moustafa, A.M. 1960. Nutrition and the development of mushroom flavor in Agaricus campestris mycelium. Appl. Microbiol. 8, 63-67.

117.

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120.

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123.

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125.

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126.

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130.

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132.

Srivastava, H.C., Z. Bane. 1970. Nutrition requirements of Pleurotus flabellatus. Appl. Microbiol. 19, 166-169.

137.

Sugihara, T.F., H. Humfeld. 1954. Submerged culture of mycelium of various species of mushroom. Appl. Microbiol. 2, 170-172.

138.

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160.

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161.

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162.

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164.

Zarudnaya, G.I. 1971. Influence of different sources of carbon and nitrogen on growth of some species of the genus Coprinus. Mikol. Fitopatol. 5, 133-136.

167.

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