MYCELIUM AS SOURCES OF
PROTEIN AND FLAVOUR COMPOUNDS
PROEFSCHRIFT
TER VERKRIJGING VAN DE GRAAD VAN DOCTOR IN DE TECHNISCHE WETENSCHAPPEN AAN DE TECH-NISCHE HOGESCHOOL DELFT, OP GEZAG VAN DE RECTOR MAGNIFICUS PROF. DR. IR. H. VAN BEKKUM, VOOR EEN COMMISSIE AANGEWEZEN DOOR HET
COLLEGE VAN DEKANEN TE VERDEDIGEN OP WOENSDAG 23 JUNI 1976 TE 16.00 UUR
DOOR FRANS I J J E DIJKSTRA SCHEIKUNDIG INGENIEUR GEBOREN TE DOKKUM • ' o •J
M
DRUKK ERIJ J.H 1976 PASMANS, 'S-GRAVENHAGEDIT PROEFSCHRIFT WERD BEWERKT IN HET LABORATORIUM VOOR ALGEMENE EN TOEGEPASTE MICROBIOLOGIE VAN DE TECHNISCHE HOGESCHOOL TE DELFT
op, dan zit hij in verlegenheid.
(Sirach 18 : 6 - 7 )
Voor mijn moeder Voor Lineke en Afke
CONTENTS
Voorwoord 7
1. Introduction 9 1.1. Brief literature review 9
1.2. The purpose of the present investigation 14 2. The nutritional requirements of Agaricus bisporus and
Copri-nus comatus 16
2.1. Choice and identity of the organisms 16 2.2. Influence of temperature and pH 16 2.3. Influence of the carbon and energy source 18
2.4. Influence of the nitrogen source 26 2.5. Influence of growth substances 32 2.6. Influence of Maillard compounds 33 2.7. The use of complex media 36
2.8. Discussion 38
3. Mushroom mycelium as a source of protein 40
4. The chemical nature of the flavour of fruit bodies of
mush-rooms 45 4.1. Brief literature review 45
4.2. Organoleptic significance of constituents of Agaricus bisporus 48
4.3. Flavour compounds in Coprinus comatus 52 4.4. Flavour compounds in some other mushrooms 56
4.5. Discussion 59
5. Production of flavour compounds by mushroom mycelium in
submerged culture 60 5.1. Brief literature review 60 5.2. Production of some flavour compounds by higher fungi in
sub-merged culture 60 5.3. Some factors influencing the production of flavour compounds
by submerged mycelium oi Agaricus bisporus (isolate Al) and
Coprinus comatus (isolate L) 65
5.4. The cultivation of Agaricus bisporus and Coprinus comatus in
a 10 litre fermentor 70 5.5. Comparison of the flavour compounds in mushroom mycelium
grown in submerged culture with those present in fruit bodies 76
6. Mat€ 6.1. 6.2. 6.3. 6.4. 6.5. 6.6. jrials an d methods Organisms Media Chemicals Culture conditions
Preparation of mushroom extracts Analytical procedures 6.6.1. 6.6.2. 6.6.3. 6.6.4. 6.6.5. 7. Summary Samenvatting References
Chemical and physical methods Organoleptic methods
Statistical interpretation of results
Temperature sensitivity of the Maillard reaction Fractionation of laccase 80 80 80 82 83 84 85 85 88 88 89 89 91 95 99 Abbreviations 106
VOORWOORD
Bij de voltooiing van mijn promotieonderzoek gaan mijn gedachten in de eerste plaats terug naar mijn vader, wiens warme belangstelling steeds een belangrijke stimulans is geweest bij mijn studie, waarvan hij het eindresul-taat niet heeft mogen zien.
Het heeft mij verheugd, dat Prof. Dr. T.O. Wiken mij de kans bood in zijn laboratorium te werken. Zijn adviezen en zijn inbreng in de ma-nuscripten, ook als co-auteur, heb ik op hoge prijs gesteld.
De bijdrage van de heer W.J. Soeting en medewerkers van het Uni-lever Research Laboratorium te Vlaardingen heeft mijn verwachtingen overtroffen. Dankzij hun ervaring in het identificeren van kleine hoe-veelheden stof heeft het onderzoek een belangrijke vordering kunnen maken.
Verschillende leden van de vakgroepen Organische Chemie en Ana-lytische Chemie hebben mij door raad en daad bijgestaan bij het betre-den van hun vakgebied. Dit geldt in het bijzonder voor Dr. P.J.W. Schuijl en de heer A. van Estrik.
Binnen de vakgroep Algemene en Toegepaste Microbiologic is van groot belang geweest de bijdragen van Ir. P.M. van den Berg, Mej. M.J.A. van Driel, de heren A.K.S. Polderman en F.G.M. Snijdewint, Ir. J.F.M. van Vliet, de heer F. Wielaard en Ir. G. van Woggelum, die zich als stu-dent met enthousiasme en geduld hebben ingezet voor de dikwijls lang-durige experimenten. Aan de heer S. de Vries dank ik het welslagen van talloze fermentor proeven. De heer J.A. Schuur heeft het fotowerk ver-richt en bij verschillende publicaties de figuren verzorgd. Zonder de medewerking van de leden van het proefpanel zouden veel van mijn ver-moedens onbewezen hebben moeten blijven.
Tenslotte dank ik de heer B.P. May, Dr. J.G. Kleyn en Mej. L.A. Robertson voor het in goede banen leiden van mijn gebruik van hun moedertaal.
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 inves-tigated:
1) Production of mycelium of edible mushrooms for human and animal consumption. This subject has most recently been re-viewed 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 Hke glucan [25], dehydroacetic acid [102], nucleotides [66], antibiotics [17, 117,127,167], tumor 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 nutri-tional 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 sev-eral strains from cultivated mushrooms. They selected three fasc 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 unhke mush-room 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 men-tioned [38,110,126,140] have, of course, become inconclusive. These disappointing results illustrate the problem of isolating reliably pure cul-tures of mushroom mycelium.
Agaricus arvensis bernardn bisporus bitorquis blazei campestns placomyces Amanita caesarea Armillana mellea Boletus edulis granulatus grevtllei indecisus variegatus Calvatia gigantea Cantharellus cibanus clavatus Cenococcum graniforme Clttocybe nebulans Collybia butyracea umbudata velutipes Coprinus comatus Hebeloma smapizans Kuhneromyces mutabilis Lactanus piperatus Lentinus edodes Lepiota naucina procera rhacodes Limacium eburneum Lycoperdon umbrinum Marasmms foetidus 33 33 33,89,130,144,150, this thesis 137, this thesis 137 21,33,38,39,59,60,126, 150,162 137 144 33,53,137,157, this thesis 33,37,125 157 157 39,126 120 130, this thesis 21,39,137,144,150 150 157 33 157 137 21,39,53,66,126,137,162 33,137,144,164, this thesis 137 53 144 138,141,162, this thesis 137 33,137 33,137 144 137 15" Marasmms Morchella oreades putillus scorodonius augusticeps cornea crassipes elata esculenta hortensis hybrida semilibera vulgaris Mycelium radicus atrovirens Oudemansiella mucida Paxillus prunulus Pholiota mutabilis Pleurotus Polyporus Torichoroma cornucopiae flabellatus japonicus ostreatus borealis suphureus matsutake robustum Trametes gibbosa Tricholoma flavobrunneum nudum personatum pessundatum Schizophyllum commune Volvariella volvacea Xylaria polymorpha 33 157 157 52 52 21,52,79,88,93,137 53 33,52,79,88,93,130, this thesis 52,93, this thesis 39,126 52 52 157,159 162,167 157,159 157 33 132 162 21,33,53,54,130,137,138, this thesis 157 137 162 162 157 157 21,33,39,53,63,90,126,137,150 33 157 137 6,33, this thesis 39,126
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 is not complete, by way of example a number of wood-rotting basidiomycetes of doubtful edibiUty [80] and a number of species mentioned by Espenshade [35] without any specification of culture con-ditions and yields are not included. In most papers fructification experi-ments, 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 o^ 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 experi-ments carried out with the species listed in Table 1, because there is al-ready 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 oi Agaricus
bis-porus, 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
volva-cea. He compared growth in media with 44 carbon and energy sources
and 31 nitrogen sources. In addition, he examined the influence of vita-mins and growth hormones.
Atacador-Ramos et al. [6] developed an optimal medium for sub-merged growth of Volvariella volvacea. In this medium which contained saccharose as source of carbon and energy, urea as source of nitrogen and coco-nut 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 require-ments 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
ni-trogen sources. The basal medium contained yeast extract and minerals. The highest yield was 7.2 g/1 after 7 days in a medium containing pep-tone 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 1 5 - 2 5 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 digestibihty.
Hamid et al. [63] produced mycehum of Tricholoma nudum from industrial wastes (cane and beet molasses, sulphite waste liquor, spent wash*, com steep hquor), 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 hquor for submerged growth of Morchella species. The sulphite hquor, containing 40 to 70 g/1 of carbohydrate, was diluted 1: 5 and supplemented with ammonium phos-phate and corn steep hquor. 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.
Ginterova [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 abihty 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. Ginterovd and Maxianov^ [55] confirmed the * A by-product of the alcohol industries.
nitrogen fixation by making up the balance of nitrogen in cultures of
Pleurotus ostreatus, growing and fructifying on natural substrates.
Start-ing with 530 g of bound nitrogen, the substrate, mycelium and fruit bodies contained 714 g of nitrogen after growth. Ginterov^ suggested that nitrogen fixation might be quite commonly found among the higher fungi. Nitrogen fixation was also observed in cultures of Pleurotus
sajor-caju 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 produc-tion of protein from powdered wood supplemented with a small amount of glucose and dried yeast. About 2% of the weight of the wood was con-verted 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 )2 HPO4 as nitrogen source and replaced glucose with other carbo-hydrates.
Only a few mushroom species have been grown in submerged cul-ture on a pilot plant scale. In the U.S.A. Morchella mycelium grown in submerged culture has been produced with the trade name "morel mush-room flavouring" [94].
Torev [144] observed rapid growth of some mushroom species in fermentors with a volume up to 50 m^. 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 possi-bly 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-val-ue (often between 6 and 7) make the cultures extremely vulnerable to contamination, so that absolute steriUty is required; but these disadvan-tages 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 num-ber of judgements. The chemical nature of the flavour of mushrooms was unknown. In recent years the knowledge of the constituents of the cul-tivated mushroom and of Boletus edulis has increased considerably, be-cause 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 de-tailed knowledge of the nutritional needs of the strains to be used. Be-cause many differences may exist in the behaviour of the strains of the same fungal species, it was impossible to use data from the hterature. 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 possibihty of utihzing the myce-lium of these strains as protein sources will be discussed in Chapter 3.
The chemical nature of the flavour of Agaricus bisporus and
Copri-nus comatus will be dealt with in Chapter 4. The occurrence of some
im-portant flavour compounds in other edible mushrooms will also be con-sidered.
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
investiga-tions of the submerged growth of Agaricus bisporus and Coprinus
coma-tus are in press [ 2 8 ] ; 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 pubhcation [29,30,31 ] .
2. THE NUTRITIONAL REQUIREMENTS OF AGARICUS BISPORUS AND COPRINUS COMA TUS
2.1. Choice and identity of the organisms
During the investigations described in this thesis, submerged growth and flavour production of several mushroom species were compared. The choice of the organisms for the more detailed studies was rather arbitrary.
Agaricus bisporus and Coprinus comatus were chosen because sufficient
amounts of fruit bodies for investigation of the flavour could be ob-tained easily; the origin of the strains is described in Chapter 6. Unless stated otherwise, our experiments with Agaricus bisporus and Coprinus
comatus were done with strains Al and L, respectively. The identity of Agaricus bisporus, strain A l , was verified by fructification experiments
[26]. Coprinus comatus, strain L, did not produce fruit bodies; fructif-ication experiments with this organism have always been difficult. Mounce [109] observed fruit bodies after nine months mycelial growth on a mixture of horse manure and sawdust. The positive results of Lam-bert (cited by Stoller [135]) were not reproducible. Nevertheless there is no reason to question the identity of our isolate L, especially because the similarity of the odour of this mycelium to that of fruit bodies was confirmed by chemical analyses (Chapters 4 and 5).
Agaricus bisporus, strain Al, needed 20 to 25 days to give the
maximum yield in shaken flasks (see Fig. 6). With Coprinus comatus, strain L, the maximum was reached earlier and the yield was three to four times as high as with Agaricus bisporus. Unless stated otherwise, our shaken cultures of Agaricus bisporus were harvested after 21 days, and those of Coprinus comatus after 14 days. Both organisms were thus harvested near the end of the phase of rapid growth.
2.2. Influence of temperature and pH
According to the hterature [5,43,146] the optimum temperature for mycelial growth of Agaricus bisporus is 23° to 25° C, while for produc-tion of fruit bodies it is 15° to 20° C. The growth rate of the mycelium quickly decreases above 25° C. The heat tolerance differs from strain to strain, but most strains hardly grow above 32° C and die at 34° C [51]. Nothing is known from the literature about the influence of temperature on the growth of Coprinus comatus.
Table 2. Influence of temperature on the growth of Agaricus bisporus and Coprinus comatus Medium: CME(A). Temperature "C 30 25 20
Growth of Agaricus bisporus^ Agar culture'^ Submerged culture**
35 3.3 50 5.1 41 3.0
Growth of Coprinus comatus^ Agar culture*^ 44 78 60 Submerged culture'' 9.0 13.1
^After 21 days. "After 7 days. '^Colony diameter in mm. ''Dry weight of mycelium in g/1.
Table 2 shows that for our strains the optimum temperature for the rate of growth on sohd as well as in liquid media is 25° C.
The optimum pH for both organisms lies between 6.5 and 7.5 (Fig. 1), which agrees with observations made by Treschov [146] and Volz [ 150] of other Agaricus bisporus isolates.
Fig. 1. The influence of the initial pH of the medium on the submerged growth of Agaricus bisporus and Coprinus comatus. Basal medium with glucose as carbon and energy source and an amino acid mixture as nitrogen source.
o - o - o - o Dry weight of mycelium. • - • - • - • Final pH of the medium.
In all further experiments the temperature was kept at 25° C and the initial pH was set at 7. In media with suitable carbon and nitrogen sources the pH decreased during growth. When a carbon source unsuitable for mycelial growth was tested a slight increase of the pH was observed, possibly because the organic nitrogen source was also used slowly as car-bon source, resulting in the formation of ammonia. In other experiments no increase of the pH was observed.
2.3. Influence of the carbon and energy source
With casein as nitrogen source for submerged growth in shaken flasks,
several carbon compounds were tested as carbon and energy sources. The composition of the basal medium is described in Chapter 6.2. When varying the initial concentration of glucose the optimum concentration was found to be between 20 and 30 g/1 for Agaricus bisporus and be-tween 60 and 70 g/1 for Coprinus comatus (Fig. 2).
g / l Yield
30 60 90 120 g/1
Fig. 2. The influence of the initial concentration of glucose on the growth of Agaricus bisporus and Coprinus comatus m shaken tlasks.
The yield of Agaricus bisporus could not be improved by gradually adding more glucose during the growth period. This speaks in favour of the view that many other factors than the amount of glucose present Umit mycelial growth.
The consumption of other carbohydrates is shown in Table 3. When no carbohydrate was added, growth was not zero as the nitrogen source (casein) could slightly serve as a carbon and energy source. The weight of the inoculum was, at most, 0.006 g/1. Our results are compared in Table 4 with those of investigators who used other strains. As far as they were specified by these investigators, not all the culture conditions were simi-lar. Volz [150], for example, used media with a pH of 4.5 while the other workers used pH values between 6.6 and 7. This again shows that different strains may grow in different conditions. Treschov [ 146] used static cultures, while the other experiments were carried out in shake flasks. The incubation times varied from 10 to 30 days. The temperatures were between 23° and 30° C. Treschov [ 146], Volz [ 1 50] and Zarudnaya
Table 3 Growth of Agaricus bisporus and Coprinus comatus m the basal medium on various carbohydrates m shaken flasks
Carbohydrate^ glucose galactose fructose maltose saccharose hydrolysed saccharose lactose trehalose xylose soluble starch mannitol no carbohydrate LSD«
Yield'' of Agaricus bisporus Compl <= Sep.^ 5 8 4 4 2 3 1 5 7 8 6.5 4 1 3 7 1 7 2 2 5 8 3 1 2 5 4 8 3.6 4 2 4.3 3 6 3 8 2 2 2.1 1.6 30%
Yield'' of Coprinus comatus Compl. 21 3 2.9 19 7 20.1 2.5 20.0 3.8 19.2 3.0 23.8 3 1 2 1 23% Sep.d 18 0 1 1 20.3 18.3 1.6 2.0 18.8 2 0 20.5 3 1
^Concentration 30 g/1 for Agancus bisporus and 60 g/1 for Coprinus comatus ''Dry weight of mycelium (g/1)
'^Compl = complete medium heat sterilized ''Sep. = carbohydrate sterilized separately ® Least significant difference
Table 4. Growth of Agaricus bisporus and Coprinus comatus on various carbohydrates Com-parison of results from different strains
Carbohydrate glucose galactose fructose maltose saccharose lactose trehalose xylose soluble starch mannitol + + = Good growth. Ref. 89 + + + + + + + + + + Agaricus bisporus 146 + + + + + + + + + + + -+ = Moderate growth. 150 + + + + + + + + + + + + This thesis + + + + + + -+ + + + + + -- = No growth. Coprinus Ref. 164 + + + + + -+ comatus This thesis + + + + + + -— + + + +
-[164] used synthetic nitrogen sources (asparagine, urea) while Lee [89] used tryptone and we used casein. We may, however, assume that all workers used standardized conditions throughout their experiments, so that their conclusions on the influence of different carbohydrates can be compared. It is clear that many important differences occur between strains of the same species.
Differences in growth occurring when the carbohydrate was steril-ized separately or, alternatively, together with the rest of the medium (Table 3) were caused by Maillard compounds; we shall return to this subject in Chapter 2.6.
Some of the sugars that were not used by our strains could be made suitable by simple means. Acid hydrolysis of saccharose followed by neu-tralization of the acid made it effective for the growth of both organisms (Table 3). The same procedure did not improve the efficacy of lactose. The consumption of lactose by Coprinus comatus was increased by addi-tion of a relatively small amount of glucose (Table 5 and Fig. 3), as had been found in tests with Coprinus lagopus [105]. Galactose was partly consumed in the presence of glucose, but growth on mixtures of glucose and galactose was hardly more abundant than when galactose wasomitted.
Table 5.Consumption of glucose, lactose and galactose by Coprinus comatus during growth in shaken flasks.
Initial concentration (g/l) Glucose Galactose Lactose
Final concentration (g/1)
Glucose Galactose Lactose Mycelial dry weight 0.4 13.5 52.5 0.3 13.7 0.0 0.0 0.0 49.0 42.0 54.0 47.3 0.0 0.0 0.0 0.4 0.3 12.1 0.0 0.8 0.5 0.0 0.0 49.5 31.0 50.0 20.0 0.0 0.0 0.0 2.7 14.6 19.1 1.4 8.0 g/l 1 6 ' 12-e 4
Yield of Coprinus comatus
30 ) 1 1 V ~--^^ • • \ Lactose 1 \ i > 60 g/l 60 30 Glucose 0 g/l
Fig. 3. Growth of Coprinus comatus in shaken flasks on mixtures of lactose and glucose. Total sugars initially 60 g/1.
No galactose was found in the culture medium after lactose had been consumed If we assume that lactose was hydrolysed in the culture me-dium, this would imply that galactose could be used completely when it was liberated gradually Another possibility is that there was no initial splitting of lactose Glucose might have induced the uptake of lactose from the culture medium A stimulating effect of glucose on the con-sumption of saccharose was not observed, nor did glucose appear to have any influence on the consumption of other sugars by Agaricus bisporus Metz [168] studied the growth of Penicilhum chrysogenum using a carbon source consisting of a mixture of lactose and glucose (25 g and
10 g per litre, respectively) In the initial stage of the fermentation, glu-cose was used causing a high growth rate When thegluglu-cose was exhausted, lactose was consumed at a much lower rate, due to the rate-limiting hy-drolysis of lactose The concentrations of glucose and galactose remained very low during this hydrolysis
Table 6 Growth of Agaricus bisporus and Coprinus comatus m shaken cultures on non-carbohy-drate carbon and energy sources m the basal medium
Carbon source ethanol methanol decane tetradecane hexadecane olive oil potassium dl lactate potassium citrate potassium 1-malate potassium 1 tartrate potassium oxalate potassium succmate potassium fumarate potassium maleate potassium acetate no carbon source added
Concentration (g/1) 4 8 24 4 8 24 3 7 22 7 8 3 9 23 17 6 7 6 7 7 5 6 3 5 7 5 8 5 8 6 0
Dry weight of mycehum (g/1) Agaricus bisporus 1 3 1.3 0.4 1.2 1.3 0.2 1.2 0.0 0.0 1.5 0 0 12 4 0.1 0.1 0.1 0.1 0.1 0.2 0.4 0.1 0.2 1 2 Coprinus comatus 1 3 1 7 0 5 0 9 1 0 0 2 1 3 1 3 1 7 1 3 2 1 16 4 0 3 0 2 0 2 0 2 0 2 1 0 0 6 0 6 0 9 0 9
Table 7. Growth of Agaricus bisporus and Coprinus comatus in shaken cultures on fatty acid esters as carbon and energy source in the basal medium.
Carbon source* methyl stearate methyl oleate ethyl oleate methyl linoleate methyl linolenate methyl palmitate methyl palmitoleate no carbon source added
LSD'' Dry weight of Agaricus bisporus 3.3 11.0 7.5 0.0 0.0 11.6 5.9 1.2 30% mycelium Cof (g/1) rinus comatus 6.5 11.3 13.6 0.0 0.0 9.0 9.7 1.0 23%
^Concentration: 18 g/1 for Agaricus bisporus and 36 g/1 for Coprinus comatus. ''Least significant difference.
Several non-carbohydrate compounds were tested as carbon and energy sources (Tables 6 and 7). Methanol, ethanol, decane, tetradecane, hexadecane and several salts of organic acids hardly stimulated growth of either organism but often even inhibited it. Sugimori etal. [138] ob-served good growth of Lentinus edodes on ethanol, but not on salts of organic acids (see Chapter I). Some fatty acid esters were suitable as carbon and energy sources: methyl oleate, ethyl oleate, methyl palmi-tate, methyl palmitoleate and, with lower yield, methyl stearate (Table 7). The concentration of ethyl oleate, giving the highest yield observed, was 18 g/1 for Agaricus bisporus and 52 g/1 for Coprinus comatus (Fig.4).
g/l 16 • 12 6 I.-Yield . /
A
^^^^ ^^^^^ comatus ^ \ 4 bisporus Ethyl oleate 10 30 50 g/lFig. 4. Influence of the concentration of ethyl oleate on the submerged growth of Agaricus
Table 8. Growth of Agaricus bisporus and Coprinus comatus in shaken cultures on some carbon and energy sources in the basal medium.
Carbon source''
methanol oleic acid
methanol + oleic acid methyl oleate potassium oleate no carbon source added
Dry weight of mycelium (g/1) Agaricus bisporus 1.6 0.7 0.9 11.0 1.4 1.3 Coprinus comatus 1.2 1.1 1.6 11.3 0.9 0.9 *AI1 carbon sources 0.06 M.
The effect of higher concentrations of ethyl oleate on the yield of
Copri-nus comatus was not investigated. From the experimental values listed in
Table 8 it may be concluded that oleic acid should be esterified to be suitable as a carbon and energy source, probably because the free fatty acid and even its anions are toxic to the fungi studied.
Apart from being used as the sole source of carbon and energy, the lipids mentioned may play another role in the nutrition of Agaricus
bisporus and Coprinus comatus. When added in small amounts to a
medi-um, containing a sugar as the principal carbon source, they often stimul-ate the growth of Agaricus bisporus, as found by Wardle and Schisler
[ 152]. Such effects were also observed in our shaken cultures of Agaricus
bisporus and Coprinus comatus (Table 9). It is interesting that methyl
linoleate, which is not suitable as the sole source of carbon and energy, may be used for stimulating the growth of Coprinus comatus in the CME-medium. It is evident that the higher amounts of methyl linoleate added when it was the sole carbon and energy source are toxic for the organism.
Stimulation by lipids varies from strain to strain. Wardle and Schisler [152] reported that some strains of Agaricus bisporus were stimulated by oleates as well as by linoleates, whereas one was not stimulated by lipids at all. 0\xr Agaricus bisporus strain was stimulated by oleic, but not linoleic acid esters.
The stimulation by small amounts of ethyl oleate significantly ex-ceeded the weight of the lipid (Fig. 5). Higher amounts (more than 0.5 ml per litre of culture medium) were also taken up, but the increase in yield could be attributed solely to an uptake of lipids from the culture
Table 9 Effect of fatty acids and their esters on the growth of Agaricus bisporus and Coprinus comatus m shaken cultures Medium CME
Unsaturated fatty acids and esters, 2 5 ml/1 oleic acid hnoleic acid Imolenic acid methyl oleate ethyl oleate methyl hnoleate ethyl hnoleate methyl hnolenate Agaricus bisporus Suitabihty of hpid as sole carbon source + + -Yield after 18 days (g/1) 0 1 0 0 0 2 7 2 7 9 3 2 3 9 0 0 Coprinus Suitabihty of lipid as sole carbon source + + -comatus Yield after 10 days (g/1) 8 2 0 1 0 1 17 2 18 4 19 6 15 7 0 2 Saturated fatty acids
and esters, 2 5 g/1 stearic acid palmitic acid methyl stearate methyl palmitate control (no bpid added)
+ + 1 2 0 9 3 8 5 9 4 1 + + 19 4 16 5 19 6 19 7 14 3 Yeld of Agaricus bisporus offer 18 doys
Fig 5 Effect of ethyl oleate on the yield of Agancus bisporus when grown m submerged culture on the CME-medium
o - o - o - o Dry weight of mycehum Weight of ethyl oleate
medium. Van den Berg [14] found a curve similar to that in Fig. 5 in tests with Coprinus comatus (strain L).
From Fig. 6 it is evident that with olive oil the yield is iiigher at all stages of growth. The lipid content of the mycelium is maximal after about two weeks, when the yield is approximately half the final yield or higher. Perhaps the hpids are partially incorporated in the cell membrane, thereby facilitating the uptake of nutrients from the culture medium. In later stages of growth the hpids seem to be converted into non-hpid com-pounds or at least into non-extractable Hpid complexes.
The use of hpids in the culture medium changed the composition of the mycelium of Agaricus bisporus. The lipid content of the mycelium increased (Fig. 6) while the nitrogen content descreased, as will be shown in Chapter 2.7.
10 20 30 Days
Fig. 6. Effect of olive oil on the growth curve and lipid content of Agaricus bisporus in shaken flasks on the CME medium supplemented with olive oil.
Dry weight of mycelium: o-o—o 5 ml/I olive oil, • - • - • 2.5 mI/1 olive oil, n—a-a control. Lipid content:
o—o—o 5 ml/1 olive oil, •—•—• 2,5 ml/I olive oil, n—•—• control.
2 4 Influence of the nitrogen source
With glucose as the carbon and energy source, several nitrogenous com-pounds were tested as nitrogen sources Inorganic nitrogen sources, amines, urea and asparagine provided little or no growth (Table 10) Casein was a good nitrogen source, as was a mixture of 17 amino acids having a similar composition as casein With Coprinus comatus practically the same growth was obtained with the mixture of 17 amino acids as with a mixture of asparagine and phenylalanine, thus this strain does not re-quire any more amino acids The same conclusion was drawn earlier [26] for Agaricus bisporus (strain Al) from results obtained when its growth on a mixture of asparagine and phenylalanine was compared with growth on a mixture of 20 amino acids, all of which were in the same concen-tration, while the basal medium contained less glucose (10 g/1 as com-pared to 30 g/1) From the results presented m Table 10 it is clear, that in more favourable conditions additional amino acids are required for maxi-mal growth of this strain
Table 10 Growth of Agaricus bisporus and Coprinus comatus on various nitrogen sources m shaken cultures m the basal medium
Nitrogen source^ \is\i^ of Agaricus bisporus Yield^ of Coprinus comatus ammonium chloride 0.2 1 5 ammonium tartrate 0.3 1 4 potassium nitrate 0.2 1 4 methylamme 0.1 0 6 dimethylamine 0.1 0 3 tnmethylamme 0.2 0 7 urea 0.3 4 1 urea + phenylalanine'^ 0.2 3 9 asparagine 0.6 2 4 asparagine + phenylalanine'^ 2.5 9 1 mixture of 17 ammo acids'^ 3.6 10 4 casern 5.0 16 3 no nitrogen source 0 3 0 9 LSD^ 30 % 23 % ^Concentration of nitrogen 0 06 M (Agaricus bisporus) and 0 12 M (Coprinus comatus) ''Dry weight of mycelium (g/1)
'^3mM (Agancus bisporus) and 6 mM (Coprinus comatus) ''composition, see Table 11
Table 11 Uptake and release of ammo acids by Agaricus bisporus and Coprinus comatus The basal medium without casein was supplemented with amino acids m the concentra-tions indicated m the column "Initial"
Agancus bisporus Coprinus comatus
Amino acid — Asn Asp Thr Ser Glu Pro Ala Gly Val Met He Leu Tyr Phe HIS Lys Arg Yield of mycehum Initial (g/1) 0 00 0 42 0 25 0 32 1.19 0.55 0.24 0 13 0 44 0.15 0.45 0 55 0 00 0 62 0 1 3 0.27 0.13 After 24 days (g/1) 0 00 0 44 0 21 0 24 1 0 4 0 54 0 18 0 14 0.21 0.02 0 30 0 09 0 0 1 0 24 0 03 0 12 0 03 3.6 % Taken up (+) or released ( - ) - 5 +16 +25 +13 + 2 +25 - 8 +52 +87 +33 +84 +61 +77 +56 +77 Initial (g/1) 20 10 0 53 0 00 0 00 0 09 0 00 0 04 0 08 0 00 0 00 0 00 0 00 0 00 1 22 0 00 0 00 0 00 After 24 (g/1) 4 65 1 30 0 00 0 00 3 03 0 00 0 13 0 10 0 07 0 00 0 00 0 00 0 00 0 00 0 17 0 12 0 20 13 5
The amino acid requirements of Agaricus bisporus were investigated by analysing uptake and release from the mixture (Table 11). A medium with the same mixture of amino acids as reported in Table 10 was used. Most of the amino acids were taken up from the medium and there was a particularly high uptake of valine, methionine, leucine, phenylalanine, histidine, lysine and arginine. Probably some of these amino acids cannot be synthesized in sufficient amounts by the strain concerned. This phenomenon was investigated further by using a nitrogen source consis-ting of mixtures of the same initial concentrations of the amino acids and tryptophane. The initial concentration of tryptophane, which was not determined in the experiments reported in Table 11, was 0.15 g/1. Every amino acid was in turn omitted from the mixture and replaced with an equal amount of leucine. When leucine was omitted, it was replaced with an equal amount of vahne. The results are shown in Table
12A. Omission of phenylalanine, histidine, valine and methionine resulted m a significant decrease m growth, while the need for lysine was doubt-ful. Leucine, arginine and tryptophane were not necessary. These conclu-sions were confirmed by the results of another experiment, in which
Table 12. Growth of Agaricus bisporus in shaken cultures on mixtures of amino acids in the basal medium. Values not followed by the same letter are significantly different.
Amino acids Dry weight of mycelium (g/1)
A. Phe, His, Val, Met, Lys, Leu, Arg, Trp His, Val, Met, Lys, Leu, Arg, Trp Phe, Val, Met, Lys, Leu, Arg, Trp Phe, His, Met, Lys, Leu, Arg, Trp Phe, His, Val, Lys, Leu, Arg, Trp Phe, His, Val, Met, Leu, Arg, Trp Phe, His, Val, Met, Lys, Arg, Trp Phe, His, Val, Met, Lys, Leu, Trp Phe, His, Val, Met, Lys, Leu, Arg
2.7 0.6 1.7 1.5 1.7 2.1 2.5 2.5 3.0 0.5 2.1 2.5 3.4 4.3 4.6 3.7 3.3 DEF A BC B BC CD DE DE EFG A CD DE FGH HK K GHK EFGH B. Asn, Asn, Phe Asn, Phe, His Asn, Phe, His, Val Asn, Phe, His, Val, Met Asn, Phe, His, Val, Met, Lys Asn, Phe, His, Val, Met, Lys, Leu 17 amino acids (see Table 11)
phenylalanine, histidine, valine, methionine, lysine and leucine (concen-trations, see Table 11) were added successively to asparagine as the major nitrogen source. The concentrations of asparagine were calculated, so that the total initial concentration of nitrogen was the same within this series of media. The results (Table 12B) showed again the need for phenylalanine, histidine, valine and methionine while the effect of lysine was not significant. Because the effect of phenylalanine was much greater than that of the other amino acids, we may say that the strain concerned had an absolute need for phenylalanine and a relative need for histidine, valine and methionine. Growth with the mixture of asparagine, phenyl-alanine, histidine, valine, methionine and lysine was significantly better than with the mixture of 17 amino acids.
Frazer and Fujikawa [45] also came to different conclusions about the amino acid requirements of an Agaricus bisporus strain in media with different ratios of glucose and asparagine. In Table 13 the requirements of some strains are compared.
Table 11 also shows uptake and release of amino acids by Coprinus
comatus in a medium containing asparagine and phenylalanine. The
other amino acids initially present were either impurities in the chemicals or they were introduced with the inoculum. After 24 days the medium
Table 13. The amino acid requirements of Agaricus bisporus. Comparison of different strains.
Amino Ref. 45 124 146 This thesis acid
Wild type Mutant Mutant Phe* + _
-His* - - + Val*
Met* + Pro + -+ -+ = Absolute need for the amino acid. + = Growth is promoted by the amino acid. - = Good growth without the amino acid. * Essential amino acid for human nutrition.
contained traces of glycine, alanine, valine, histidine, lysine and arginine in addition to larger amounts of aspartic acid and glutamic acid. The con-centration of glutamic acid was so high, that this substance may be an important flavour compound in the culture broth, especially if 5'-GMP has also been formed. The high excretion of glutamic acid was possibly caused by the high concentration of asparagine in the medium, which had shown to result in high yields of mycelium.
Our strains of Agaricus bisporus and Coprinus comatus cannot grow with molecular nitrogen as the only nitrogen source (Table 10). The abil-ity of these strains to fix gaseous nitrogen in a complex medium, as found for some mushroom species [53,55,123], was investigated by determination of the total amount of bound nitrogen in a shaken fiask containing the CME medium before and after growth; no nitrogen fixa-tion could be demonstrated within the limits of experimental error.
Casein was a better nitrogen source for both organisms than the amino acids, indicating that a complex nitrogen source is better than a defined one, as stated earher by Styer [136]. This might be caused by slow hydrolysis of casein by extracellular enzymes of the fungi, so that the concentration of free amino acids never becomes as high as in the synthetic mixtures. This theory was supported by the results x)btained in growing the fungi in media with lower initial concentrations of amino acids. More amino acids were added after one or two weeks, so that the amount of nitrogen totally supplied during the growth period was as high as in the experiments reported in Table 10. In this way the growth could be improved significantly; but it was not as high as with casein, probably because the enzymatic hydrolysis of casein supplies the amino acids
+ + + + +
5 •
Yield of Agaricus bisporus
^A
\^ \ ©x
\K
n. A ^-T^=6
2.5 10 20 iO C/N ratio (at/at) 24 g/l 20Yield of Coprinus comatus
2.5 5 10 20 iO
C/N ratio (at/ati
U g/1 12
Yield of Agaricus bisporus
1/. g/l 12 10 8 6 L 2 Y eld of fl a D Coprinus comatus
y^XZtl
• "~--o-^<x^^7^
-5^-^
> ~ ^ 2.5 5 10 20 iO C/N ratio (at/at) 2.5 5 10 20 iO C/N ratio lat/at)Fig. 7. Yield of Agaricus bisporus and Coprinus comatus in submerged cultures on media with different carbon to nitrogen ratios, calculated as atoms carbon per atoms nitrogen. Total amounts of carbon and nitrogen:
o o C + N 0.5 gramat/l, * * C + N 3 gramat/1, • • C + N 1 gramat/1, • o C + N 4 gramat/l. A A c + N 2 gramat/1,
Media in experiments A and B. Nitrogen and carbon sources: casein and glucose. Minerals and growth substances as in the basal medium (see Chapter 6.2).
Media in experiments C and D. Nitrogen and carbon sources: amino acid mixture (pro-portions as in Table 11) and glucose; 2 g/1 K^HPO^; 250 ml/1 vegetable extract (supplying 0.03 gramat/1 carbohydrate-carbon and 0.01 gramat/1 nitrogen); 2.5 ml/1 olive oil (supplying 0.13 gramat/1 carbon).
more gradually than was done in this experiment. It is also possible, that the casein contained one or more unknown compounds stimulating growth.
The above results also show that the optimum initial concentration is not the same for all nitrogen sources. This can also be concluded from Fig. 7 where the yield is shown as a function of the carbon to nitrogen ratio with different total amounts of carbon and nitrogen. For the calcu-lation of the C/N ratio the carbon content of casein was ignored because it hardly stimulates mycelial growth (see Table 3). With casein as nitrogen source and glucose as carbon source (Fig. 7A and B) the highest yields of
Agaricus bisporus were obtained at low C/N ratios (2.5 to 5); this might
be attributed to slow hydrolysis of casein, resulting in low concentrations of available nitrogen. Coprinus comatus grew well with a higher C/N ratio (5 to 10). This suggests that Coprinus comatus hydrolysed casein faster than Agaricus bisporus so that the concentration of available nitro-gen became high enough with lower concentrations of casein.
The influence of the C/N ratio was also studied with mixtures of amino acids, thereby providing a readily available nitrogen source. In order to get as high yields as possible, vegetable extract and olive oil were also added to the medium. The results (Fig. 7C and D) show that Agaricus
bisporus grew well with C/N ratios between 5 and 20, there being no
dis-tinct optimum. When the total amount of carbon and nitrogen was higher than 1 gramat/1 the yield decreased. We might attribute this to the sensitivity of Agaricus bisporus to high initial sugar concentrations (Fig. 2), while high concentrations of free amino acids are also less favourable. The C/N ratio resulting in maximal yield of Coprinus comatus growing on the mixture of amino acids (Fig. 7D) was the same as found when the organism grew on casein (Fig. 7B); but the yield was lower. There seemed to be a shift of the optimum C/N ratio to lower values when the total amount of carbon and nitrogen decreased.
Our experiments show, that the optimum C/N ratio may depend on the nature of the nitrogen source and on the total amount of carbon and nitrogen. Thus far we, as most previous workers, have only considered the influence of the C/N ratio on the yield; but the nitrogen content of the mycelium and the uptake of carbon and nitrogen from the medium are also important for the efficiency of the production of mycelium, and these may have their optimum with different C/N ratios. Litchfield et al. [93] found a maximum yield of Morchella hortensis, when the C/N ratio was 10. When this ratio was decreased to 5 a slightly higher yield was observed, but the difference was not significant. With Morchella hybrida
Reusser et al. [126] obtained similar results. Shannon and Stevenson [130] demonstrated maximal growth of Calvatia gigantea when the ratio of reducing sugars to nitrogen was 32 to 44 in media based on brewery wastes supplemented with ammonium sulphate. If the non-carbohydrate carbon is ignored, this corresponds to a C/N ratio of 13 to 18, which is higher than in the other observations cited.
2.5. Influence of growth substances
As regards the growth factor requirements of fungi in general we may refer to Fries [47,48] and Schopfer [129]. Most higher fungi need thiamine as growth factor [47,77,80,151]. In addition, some species need adenine (Fomes officinalis and Poria vaillanti), biotin and ribofla-vine (Porta vaillanti) or pyridoxine (an unidentified psychrophiUc basi-diomycete). Only a few species {Morchella conica, Ptychogaster
rubes-cens, Trametes serialis) have been reported to be prototrophic. The
growth of Boletus variegatus and Collybia velutipes can be stimulated by fructose-1,6-diphosphate [113].
Treschov's Agaricus bisporus strain needed thiamine and biotin [146]. All wild-type strains described by Raper etal. [124] only required thiamine and one mutant needed adenine. Ferulic acid promoted growth of another strain of Agaricus bisporus [118]. Coprinus comatus was also shown to need thiamine [33]. In a medium containing yeast extract the strain grew much better, which may be attributed to the need for one or more additional growth substances.
Table 14. Effect of vitamins and nucleobases on the growth of Agaricus bisporus and Coprinus comatus in shaken cultures.
Substances added control 8 vitamins thiamine" 8 vitamins + 4 nucleobases thiamine* + 4 nucleobases thiamme" + adenine'' 4 nucleobases LSD'^ Dry weight of Agaricus bisporus 0.2 2.6 2.8 3.5 2.5 3.0 0.3 3 0 % mycelium (g/1) Coprinus comatus 3.2 12.4 7.8 12.0 10.3 11.8 2.4 23% "400 Aig/l. ''50 mg/1.
The requirements of our strains for vitamins and nucleobases are illustrated in Table 14. Agaricus bisporus only needed thiamine. The growth of Coprinus comatus was further stimulated by the addition of adenine; the same growth as with thiamine plus adenine was obtained with a mixture of 8 vitamins (including thiamine). Fructose-1,6-diphos-phate and ferulic acid did not stimulate the growth of our strains.
In Chapter 2.7 we shall see, that the growth of our strains in various complex media was considerably higher than in the synthetic media studied so far. Possibly there are still unknown factors in complex media which are able to stimulate the mycelial growth.
2.6. Influence of Maillard compounds
If a solution containing a reducing sugar and an amino compound is heated, the development of a brown colour may be observed. This reac-tion, which was first reported in 1912 by Maillard [99], depends on the pH of the solution: it is rapid at high pH values, still occurs at pH 6 and is inhibited at pH 4. On the other hand the Maillard reaction causes a decrease of the pH of the medium. The chemistry of this reaction is not yet fully understood, although the first steps are well known [57].
During autoclaving of a culture medium, the Maillard reaction may also occur, and the resulting products may effect the growth of micro-organisms. The reaction can be avoided by sterihzing the carbohydrate and the nitrogen source separately. If the pH of all media is adjusted to the same value after sterilization, the effect of Maillard compounds can be studied. Impens and Willam [76] cited several workers who had found a stimulation of the growth of yeasts and lactic acid bacteria by Maillard compounds. Miura et al. [101] found that the lag-phase of
Sporobolomyces odorus was shortened by
l-deoxy-l-(L-asparagino)-D-fructose formed during autoclaving of the medium. Impens and Willam [76] also cited some examples of organisms, the growth of which was partly inhibited by Maillard compounds (a Streptococcus species and
Bacillus polymyxa).
Impens and Willam [76,77] investigated the effect of Maillard com-pounds on the growth of some higher fungi, and found stimulation of
Volvariella volvacea and Morchella esculenta, but not of Morchella conica.
Growth of some other fungi {Phycomyces blakesleean'is and Aspergillus
niger) was also stimulated.
In our investigations the influence of Maillard products was tested in media containing 8 g/1 of casein, 30 or 60 g/1 of glucose and the usual
Table 15. Influence of the Maillard reaction on the growth of higher fungi in shaken cultures. Species Agaricus bisporus ( A l ) Agaricus bisporus (18) Agaricus bitorquis Armillana mellea Calvatia gigantea Coprinus comatus (L) Coprinus comatus (CBS 150 39) Lentinus edodes (IFO 7123) Morchella esculenta (CBS 369.68) Pleurotus ostreatus(fiRRL 2366) Volvariella volvacea (NRRL 3723) Incubation time (days) 21 28 28 23 28 14 14 21 14 14 14
Yield as dry weight of mycehum (g/1) Glucose concentration 30 g/1 Compl." 4.2 6.6 6.5 14.8 7.6 9.9 14 8 7.9 9 1 10 2 6.1 Sep." 3.9 6 0 5 3 " 7 1" 7 1 9 9 8.8'' 0 6 ' " -9.8 9.6 5.7 Comp 2.8 5 3 6 3 11 0 8 5 15.9 13.1 8.1 14.7 7 7 8.5 60 g/l " Sep." 1 8'' 4 l ' ' 6.1 6.6" 4.8" 14.1 11.2 0 - 5 " " ^ 15.1 7.8 7.0" "Compl. = complete medium heat sterihzed. Sep. = carbohydrate sterilized separately. ''Significantly lower yield without Maillard products
'^Varying yield
minerals and growth substances (see Chapter 6.2). The pH was adjusted to 7.0 before and after sterilization. The results obtained with 11 strains are shown in Table 15. Most strains were stimulated by Maillard com-pounds, although the result was often just below the significant level. The greatest differences were observed in experiments with Armillaria
mellea and Lentinus edodes at both sugar concentrations. The yield of Lentinus edodes varied considerably in the absence of Maillard
com-pounds. Five more Lentinus edodes strains were received from the CBS (Baarn, the Netherlands). Their growth was more reproducible; Maillard compounds had a smaller stimulating effect on three of them and none at all on two. A great difference was also found with Coprinus comatus, CBS 150.39, at the lower sugar concentration.
With Agaricus bisporus, strain A l , and Coprinus comatus, strain L, the effect of Maillard compounds was studied with larger numbers of flasks (Table 16). Variations resulting from differences in the shakers on which the flasks were incubated had to be eliminated. The yields and standard deviations of all flasks (maximally 36) incubated on a shaker were expressed as percentages of the average yield in the flasks contain-ing Maillard compounds, on that shaker. The results from all shakers, calculated in this way, were combined. The stimulating effect of Maillard
Table 16. Reexamination of the influence of Maillard compounds on the growth of Agaricus bisporus (strain Al) and Coprinus comatus (strain L) in shaken cultures.
Species and glucose concentration Sterilization Number of observations Average yield (%) Standard deviation (%)
Agaricus bisporus (Al) (30 g/1) Coprinus comatus (L) (60 g/1) Compl." Sep. Compl. Sep. 80 80 101 103 lOO.O" 90.9 loo.o" 99.7 18.6 23.5 14.9 17.7 "Compl. = complete medium heat sterilized. Sep. = carbohydrate sterilized separately. "Arbitrarily set to 100 %.
compounds on the growth of Agaricus bisporus, strain Al, was demon-strated conclusively (significance 99%). The growthof Coprinus comatus, strain L, was not stimulated by Maillard compounds, this in contrast to strain CBS 150.39 at the lower glucose concentration (Table 15).
The effect of Maillard compounds was also observed with other carbohydrates (Table 3).
Several explanations of the effect of Maillard compounds on the growth of microorganisms have been suggested. These include activation of the sugar [116] and activation of thiamine [77]. The inhibition of the formation of a toxic product is also possible. For example, when cystine is autoclaved, decomposition may lead to the formation of substances that are toxic for Lactobacillus bifidus; this decomposition is inhibited when cystine is autoclaved together with glucose [20].
150 Doys
Fig. 8. Effect of temperature on the Maillard reaction between glucose and glycine. As regards experimental conditions, see Chapter 6.6.4. No browning was measured at 25° C.
Although the Maillard reaction takes place most quickly at higher temperatures, (60° to 120° C), it also occurs after prolonged incubation (some months) at 37° C and it does not stop at 30° C (Fig. 8). We did not measure any browning at 25° C, although a pale yellowish colour was visible after 170 days. Thus Maillard compounds may also be found in natural conditions. It does not seem unlikely that many microorgan-isms are adapted to grow in the presence of Maillard compounds.
2.7. The use of complex media
Complex sources of nutrients (preferably waste materials) are most often used in large scale cultivation of fungi, since they are cheaper than syn-thetic media or have to be disposed. In addition, growth of many strains is more abundant in complex media, either because unknown growth substances are present, or because high molecular carbon or nitrogen sources are broken down gradually, so that the concentration of sugars and amino acids does not become excessive. The following waste materials have been used as carbon and energy sources for higher fungi: citrus press water [16], sulphite waste liquor [21,63,83,126], corn steep liquor [63,125], vinasse [38], molasses [63,126], soybean whey [39], soy been oil [125], spent wash, a by-product of the alcohol industries
[63], and brewery wastes [130]. As complex sources of nitrogen were used: yeast extract or yeast autolysate [33,138], corn steep liquor [125], vegetable wastes like cauliflower leaves [79] and brewery wastes [130]. Several higher fungi also utilized simple ammonium salts.
The complex carbon and nitrogen sources mentioned also provided trace elements and growth substances. A small amount of corn steep liquor or yeast extract can also be added as source of growth substances to media with simple carbon and nitrogen sources [52,59,60,93]. Coco-nut milk was used as a source of growth substances for Volvariella
volva-cea [ 6 ] .
For our Agaricus bisporus and Coprinus comatus strains it was necessary to find an organic source of nitrogen (amino acids) and growth substances, all of which were needed by each strain. In Table 17 the growth in several natural media is listed. Malt extract contains carbohy-drates, vitamins and a small amount of nitrogenous substance; supple-mentation with a suitable nitrogen source increased the growth. As might be expected from the results of former experiments, casein was the best supplementary nitrogen source; for Coprinus comatus asparagine was also effective. Surprisingly, also urea stimulated the growth of Coprinus
Table 17 Growth of Agaricus bisporus and Coprinus comatus in various supplemented natural media in shaken cultures
No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Components of culture medium (dissolved in deionized water)
malt extract"+ K , H P 0 4 "
mah extract" + K, HPO4 " + asparagine'^ malt extract" + K, HPO^ " + urea'' malt extract" + K^ HPO^ " + casern^ skim milk powder'
skim milk powder'' + glucose^
skim milk powder + maltose (technical)^ skim milk powder + glucose^ + yeast extract skun milk powder*^ + glucose^ + meat extract" skim milk powder*" + glucose^ + vegetable extracfP casern* + vegetable oU''
casem® + vetegable oU'' + meat extract" casein® + vegetable oil*^ + vegetable extractP proflo'
proflo' + glucoseS corn steep hquor*"
corn steep hquor"" + glucose^
smgle ceU protein" + K^ HPO4 " + glucose^ ESDI
Dry weight of mycehum (g/i) Agaricus bisporus 2.5 2.8 2.0 5.3 2.7 4.8 5.1 4.9 4.8 6.9 7.1 10.6 13.0 0.4 0.8 1.1 0.0 1 3 3 0 % Coprinus comatus 11.5 16.6 15.0 22.2 5.6 17.6 23.6 11.2 25.0 23.8 18.2 19.5 18.9 4.0 20.3 3.4 1.0 16.4 2 3 % "250 ml/1 (Agaricus bisporus) and 500 ml/1 (Coprinus comatus). "2 g/1. '^4 g/1 ' ' l g/1. ®5 g/1 (Agancus bisporus) and 10 g/1 (Coprinus comatus) *^60 g/1. ^30 g/1 (Agancus bisporus) and 60 g/I (Coprinus comatus). ''^20 ml/L '20 g/1 ""200 ml/1 "0.4 g N/1. P3 9 g Sohds/1. ''Least significant difference.
comatus in malt extract. Coprinus comatus also used skim milk after the
addition of glucose. Better growth was obtained after addition of technical maltose, a rather impure preparation. The same yield was obtained when glucose and meat or vegetable extract were added to skim milk. Agaricus
bisporus cannot exhaust skim milk media completely because it only
uses lactose moderately, even in the presence of glucose. However, fairly good growth was obtained, especially when technical maltose or vegetable extract were added.
The best growth of Agaricus bisporus was observed when vegetable oil was the carbon source and casein the nitrogen source. Addition of meat extract or vegetable extract stimulated the growth. Coprinus
coma-tus also grew well in these lipid containing media, but not as well as in
the skim milk media. Corn steep hquor was not suitable for growth of the fungi studied. Proflo is a cheap cotton-seed flour containing a high amount of protein, several minerals, vitamins, some lipids and a hmited amount of carbohydrates. When supplemented with more glucose, it
was suitable for growth of Coprinus comatus. WOWQ^QV, Agaricus bisporus
did not grow with Proflo. Single cell protein, prepared from cells of the yeast Pichia pastoris grown on methanol, also promoted the growth of
Coprinus comatus while growth of Agaricus bisporus was not abundant
with this nitrogen source.
Table 18. Conversion of carbon and nitrogen by mycelium of Agaricus bisporus and Coprinus comatus in shaken cultures.
carbon in medium carbon in mycelium lactose in medium glucose" in medium nitrogen in medium nitrogen in mycelium dry weight of myceUum
Agaricus bisporus (medium 13") Initial Fmal (g/1) (g/1) 20.1 9.0 7.9 1.92 1.32 0.34 13.0
Coprinus comatus (medium 9") Initial Final (g/1) (g/1) 50.0 28.0 12.6 18.6 10.9 22.0 10.9 4.08 1.71 2.08 25.0 "Numbers refer to Table 17.
"Expressed in g carbon/1.
Table 18 shows the conversion of carbon and nitrogen by the myce-lium when grown in the media affording the highest yields mentioned in Table 17. Coprinus comatus, grown in medium 9, contained 50% carbon and 8.3% nitrogen. Agaricus bisporus, grown in medium 13, contained 61% carbon and 2.6% nitrogen. This low nitrogen content is related to the high hpid content of the mycelium, grown in a lipid containing medium, as was already mentioned (Chapter 2.3). When grown in a medium based on skim milk (no 7 in Table 17), Agaricus bisporus myce-hum contained 10.8% nitrogen. The fungi did not exhaust the media. On varying the concentrations of components of several media listed in Table 17 higher yields were sometimes observed, but the gain in yield was much less than the additional nutrients supplied. Probably these more concentrated media are utilized less efficiently. For an economically feasible conversion of the carbon and nitrogen sources more dilute media should be used.
2.8. Discussion
Coprinus comatus are rather complex, but it may be possible to find less
expensive sources of suitable nutrients. The results with Coprinus
coma-tus growing on Proflo plus glucose and on malt extract plus urea, and the
ability of Agaricus bisporus and Coprinus comatus to use a vegetable extract (from cauliflower leaves) as a source of growth substances suggest possibilities. Moreover, many other complex media may be investigated. The examples that were given on the utilization of sugars, the need for growth substances and the stimulation by hpids show that much variation exists between different strains of the same mushroom species. So far, little or no variation has been found in the optimum pH and temperature.
3. MUSHROOM MYCELIUM AS A SOURCE OF PROTEIN
The production of protein is an important aspect which has received much attention from a great many workers studying the submerged cul-ture of mushroom mycelium. As a rule protein in the mycelium was determined by the Kjeldahl method in wich the total amount of nitrogen is ascertained and the crude protein content calculated by multiplication of the nitrogen content by a factor of 6.25, based on the assumption that the protein on an average contains 16% nitrogen and other nitroge-nous compounds are present only in negligible amounts. Reports about the crude protein content of mushroom mycelium vary considerably. For Morchella species values ranging from 10 to 55% were found [39,83, 93,126]. Tricholoma nudum contains 21 to 61 % crude protein [39,63],
Pleurotus ostreatus 24 to 35% [53] and Volvariella volvacea 34% [ 6 ] .
Sugimori et al. [138] found that the protein content of Lentinus edodes mycelium varied with the carbon source. Grown on glucose, the myce-lium contained 49% crude protein and on ethanol 58%. Lee et al. [89] found 48% crude protein in submerged grown mycelium of Agaricus
bisporus.
The crude protein content of the mycelium of our strains of
Agari-cus bisporus and Coprinus comatus also depended on the composition
of the medium. With lipids as carbon and energy source, a high dry weight of mycelium was observed, but the crude protein content was low (16% for Agaricus bisporus, see Table 18). In the CME medium the crude protein content of Agaricus bisporus was 38% [26] and in a medium based on skim milk it was 68%. Mycelium of Coprinus comatus grown in a medium based on skim milk contained 52% crude protein (Table 18).
The crude protein content, calculated from the results of the Kjel-dahl method, is higher than the protein content determined by amino acid analysis after hydrolysis of the mycehal protein, because the total nitrogen content also includes non-protein nitrogen, especially from nucleic acids. From the amino acid analyses of LeDuy et al. [88] it can be calculated that in hydrolysates of Morchella mycelium 16 g of nitrogen represented 69 to 83 g of amino acids. This means, that only 62 to 75% of the crude protein (N x 6.25) was true protein.
We analysed amino acids in hydrolysates of the mycelium of
Agaricus bisporus and Coprinus comatus, as well as free and bound
Asp Thr* Ser Glu Pro Gly AU Val* Met* lie* Leu* Tyr Phe* His* Lys* Arg* Initial concentrations Free 0 10 0 06 0 20 0 05 0 08 0 04 0 16 0 09 0 01 0 03 0 06 0 03 0 08 0 03 0 03 0 06 Total amount of nitrogen 1 38 manuno acids Medium Bound 2 7 1 3 1 5 8 3 4 4 8 2 0 9 2 7 0 6 1 8 3 1 1 1 1 3 0 8 1 4 0 7 45 9 Agaricus bisporus Final concentrations Medium Free 0 04 0 1 3 0 25 0 84 0 50 0 29 0 25 0 1 3 0 00 0 16 0 04 0 08 0 04 0 04 0 04 0 04 3 1 1 Bound 1 9 0 8 0 8 4 9 1 9 9 2 1 1 1 0 0 3 0 8 0 8 0 1 0 4 0 4 0 6 0 3 27 6 Myc 0 9 0 4 0 4 1 2 0 5 2 1 0 7 0 6 0 1 0 4 0 6 0 2 0 3 0 2 0 4 0 4 1 1 4 Balance 0 0 0 0 - 0 3 - 1 4 - 1 6 +3 4 +1 0 - 1 1 - 0 2 - 0 5 - 1 7 - 0 8 - 0 6 - 0 2 - 0 4 0 0 - 5 2 Coprinus Initial concentrations Medium Free 0 04 0 03 0 16 0 16 0 05 0 09 0 16 0 10 0 03 0 08 0 1 3 0 05 0 10 0 04 0 64 0 27 3 66 Bound 7 7 3 1 2 2 15 8 13 7 6 0 7 6 8 2 1 9 5 8 10 0 2 6 3 6 2 2 4 7 2 8 115 4 comatus Fmal concentrations Medium Free 0 12 O i l 0 34 0 34 0 4 1 0 13 0 56 0 18 0 05 0 19 0 27 0 01 0 15 0 05 0 1 3 0 21 4 1 1 Bound 5 0 2 1 1 4 9 7 8 6 4 8 4 5 4 3 0 7 3 3 4 8 0 8 1 6 0 9 1 3 1 3 62 1 Myc 3 2 1 4 1 0 3 4 3 0 2 4 3 1 2 6 0 5 2 0 3 2 0 8 1 5 0 7 1 4 1 5 39 0 Balance +0 6 +0 5 +0 3 - 2 5 - 1 7 + 1 2 +0 4 - 1 2 - 0 7 - 0 4 - 1 9 - 1 0 - 0 5 - 0 6 - 2 5 - 0 1 - 1 3 8
