Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN968-878-105-3
STUDIES OF MEDICINAL MUSHROOMS IN SUBMERGED CULTURES
M. L. Lomberh1, E. F. Solomko1, A. S. Buchalo1, B. Kirchhoff12
1M.G. Kholodny Institute of Botany, National Academy of Sciences of Ukraine, 2 Tereschenkivska St., Kiev 01601, Ukraine.
2WESER-CHAMPIGNON, Neue Heerstr. 35, Hessisch-Oldendorf 31840, Germany
ABSTRACT:
The morphology and growth rate of over 70 strains of 20 species of edible and medicinal mushrooms from the culture collection of N.G. Kholodny Institute of Botany, National Academy of Sciences of Ukraine were studied on 5 agar media. The optimal incubation time, temperature, and suitable media were determined for each culture. One natural and three synthetic liquid media with mineral and organic nitrogen sources were also tested. The effect of medium pH on the mycelial growth was determined by using phosphate buffers with pH values between 3.0 - 8.0. In each stage of inoculum preparation we used physiologically active mycelia. Factors affecting mycelial growth of some medicinal mushrooms in submerged culture were investigated. Inoculum size and composition and nutrient medium pH were important for maximising growth rate and biomass yield and minimising the duration of the lag phase of growth. Techniques for laboratory scale submerged culture of some medicinal mushrooms including Agrocybe aegerita (Brit.) Sing., Auricularia auricula (Bull.) Wettst., Flammulina velutipes (Curt.: Fr.) P.Karst., Ganoderma lucidum (Curt.: Fr.) P.Karst., Grifola frondosa (Dicks.: Fr.) S.F. Gray, Hypsizygus marmoreus (Bull.: Fries) Singer, Lentinula edodes (Berk.) Sing., Piptoporus betulinus (Bull.: Fr.) Karst., Pleurotus ostreatus (Jacq.: Fr.) Kumm. and others were determined.
INTRODUCTION
Although fungi have been valued throughout the world as both food and medicine for thousands of years, their role in human civilisation is expanding every day. Mushrooms are promising sources of physiologically functional food and as materials for the development of medicines, pharmaceutical products such as new drugs (protein-bound polysaccharides, terpenoids, steroids, etc.), dietary supplements and healthy beverages, cosmetic products, etc. Numerous articles and monographs contain detailed information on the physiology, chemistry, pharmacology, and medicinal value of mushrooms (Chang 1999, Hobbs 1996, Mizuno 1999, Royse 1996, Stamets 1993, Wasser and Weis 1999a,b). However, the mushroom industry is growing and new techniques are being devised to increase the number of cultivated species. Submerged pure culture techniques are used in the propagation of mushrooms mycelia in liquid media and have three main applications. It is possible to obtain in a short period of time: 1 - liquid spawn for fruit body and spawn production, 2 – biomass that can be used for food and dietary supplements, 3 – biomass and/or exometabolites of medicinal mushrooms as a raw material for medicine. This method has not been broadly applied, if at all, in cultivation of some species of medicinal mushrooms. Therefore the investigation of the cultivation conditions on solid agar and liquid media of different species and strains of medicinal mushroom was the aim of this work.
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Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN968-878-105-3
MATERIALS AND METHODS
Strains
The strains (Table 2) are deposited in the culture collection of mushrooms in the N.G. Kholodny Institute of Botany, National Academy of Sciences of Ukraine, Kiev (Buchalo and Mitropolska, 2001). Stock cultures were maintained in test tubes on beer wort agar (see below). The master cultures were incubated in 90 mm diameter plastic petri dishes at 25°C on 20 ml of the medium determined to be optimal for that fungal species.
Culture media and performed assays
To investigate radial growth rate and morphological and physiological characteristics of colonies, the following solid agar media were used:
1)Beer wort agar: liquid beer wort extract diluted by distilled water, to give a carbohydrate of 8.00.2, 1 liter, agar-agar, 20.0 g (Roth, Germany), pH 4.70.1 (data not shown).
2)Czapek’s medium (CzA) (g/l): sucrose, 30.0; NaNO3, 2.0; KH2PO4, 1.0; MgSO47H2O, 0.5; FeSO47H2O, 0.01; KCL, 0.5; agar-agar, 20.0 (Roth, Germany); pH, 5.80.2 (data not shown).
3)Malt extract agar (MEA): 48 g/l (Oxoid), pH 5.40.2.
4)Oat agar (OA): liquid decoction of oat, 1 liter, the preparation by A.S. Buchalo (1988), agar-agar, 20.0 g (Roth, Germany), pH 6.50.2
5)Potato dextrose agar (PDA): 39 g/l (Oxoid), pH 5.60.1.
6)Wheat agar (WA): liquid decoction of wheat, 1 liter, the preparation by A.S. Buchalo (1988), agar-agar, 20.0 g (Roth, Germany), pH 6.30.2
All agar media were sterilised for 30 min. at 121°C then cooled and poured into sterile petri dishes. In compliance with the current methodology of Weis et al. (1999), one agar disk (5 mm diameter) colonised by mycelium in the active physiological state was used to inoculate each dish.
To investigate the influence of pH and growth of mycelia of mushrooms in submerged culture, the following liquid media were used (Table 1):
Table 1. Composition of liquid media.
Nutrient / Concentration of nutrients in medium (g/l)A / B / C / D
Dextrose / 20.0 / 50.0 / 50.0 / 25.0
Peptone / 2.5 / 5.0 / 2.0 / 10.0
Yeast extract / 1,0 / 2.0 / 2.0 / 1.0
(NH4)2SO4 / 0 / 0 / 3.0 / 0
MgSO4*7H20 / 0.3 / 0.5 / 0.5 / 0.25
CaCl2*2H20 / 0 / 0.3 / 0 / 0.15
K2HPO4 / 0 / 0 / 0-7.5 / 0
KH2PO4 / 0.3 / 1.0 / 1.0-7.5 / 0.44
Corn steep liquor / 2 ml / 5 ml / 0 / 2.5 ml
Trace element solution
Concentrate / 10 ml / 10 ml / 10 ml / 10 ml
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Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN968-878-105-3
A liquid decoction of wheat (medium I) was also used. It was prepared as follows: 1 kg of wheat grain was added to 1.5 litres of distilled water and boiled for 20 minutes. After filtration the decoction was adjusted to pH 7.5 and it was sterilized. Liquid beer wort medium (medium J) was diluted with distilled water to give a carbohydrate content of 4.0 0.2%
The media were sterilised in an autoclave at 121C for 20 min. The different concentrations of the potassium dehydro- and hydrogen phosphate for medium C were sterilised separately. After cooling to 40C trace metal solution concentrate (10 ml) was added to each litre of media. The trace metal solution consisted (g per litre): 0.4 MnCl24H2O, 0.2 ZnCl2; 0.8 FeCl36H2O, 0.1 CuSO45H2O. The original media A, B, and D had a pH of 4.80.1 The pH of this media was adjusted with 20% KOH and 1N HCl to the optimal pH for every species tested. The medium C was adjusted to different levels of pH by changing proportions of phosphates.
Determinations of pH optima were performed with D medium in stationary liquid cultures. The pH was adjusted to different levels with 20% KOH and 1N HCl and dispensed in 20 ml aliquots into 100 ml flasks.
We have investigated growth of some species of medicinal mushrooms in submerged cultures by methods described previously (Solomko and Sasek 1984, Solomko 1992, Weis et al. 1999). Investigations of the growth of basidiomycetes were done after the optimal pH for each species were determined. Submerged cultivation was done on 75 ml of media in 500 ml Erlenmeyer flasks and on one litre in 2.5 Fernbach flasks incubated at 26°C on a rotary shaker at 120 rpm. 10 ml of homogenised mycelium representing a biomass of 0.9 g/l dry matter, were used as inoculum. At the end of the assays, the mycelium was collected in porous glass funnels, washed with distilled water, and dried at 105°C to a constant weight.
Repetitions and statistical analyses
Three petri dishes were used for each variant of agar medium studied. Radial growth of the colonies was monitored every 2 to 3 days in four directions, so that values represent the mean data of 12 measurements. When the increase of the colony radius was linear, the mean of the radial growth rate (VR, mm/day) of each colony was calculated.
Determination of optimal pH level for the growth of different strains in stationary liquid cultures was done in four repetitions on 100 ml flasks. Submerged cultivation was done also in four repetitions in 500 ml and 1 litre flasks on a rotary shaker. The tables and figures consist of the average data of four replications.
Statistical analyses were performed and correlation coefficients were calculated using the ORIGIN (v5.0) statistics package.
RESULTSAND DISCUSSION
The rates of radial mycelial growth of 30 species of edible and medicinal mushrooms on different natural and commercial agar media have been studied and the results published (Bilay etal. 2000, Solomko et al. 2000). Analysis of data given in Table 2 shows that the composition of agar media and its pH strongly influences the time it takes to obtain physiologically active inoculum.
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Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN968-878-105-3
Table 2. The optimal time to obtain inoculum of some species of medicinal mushrooms on different agar media.
Time of cultivation, days
Species strains / MEA / WA / OA / PDAAgrocybe aegerita 1503 / 10 / 10 / — / 12
Auricularia auricula 961 / 14 / 10 / — / 10
Ganoderma applanatum 920 / 8 / — / 7 / 14
G. lucidum 921 / 9 / 12 / 6 / 20
G. lucidum 1621 / 7 / 7 / 6 / 6
Grifola frondosa 923 / 21 / 19 / — / 14
Hypsizygus marmoreus 1611 / 14 / 15 / 14 / 14
H. marmoreus 1612 / 14 / 14 / 12 / 13
Piptoporus betulinus 1649 / 6 / 6 / 19 / 10
P. betulinus 1653 / 9 / * / 10 / 9
Pleurotus calyptratus 189 / 8 / 5 / 7 / 7
P. dryinus 205 / 12 / 6 / 9 / 11
P. eryngii 10 / 10 / 8 / 10 / 9
P. ostreatus 1016 / 10 / 6 / 6 / 6
1017 / 9 / 6 / 6 / 7
274 / 10 / 5 / 5 / 7
295 / 7 / 7 / 6 / 6
1637 / 7 / 9 / 6 / 7
1638 / 7 / 6 / 6 / 6
133 / 7 / 6 / 6 / 7
Lentinula edodes 57 / 13 / 14 / * / 11
704 / 12 / 12 / * / 10
711 / 15 / 11 / * / 10
712 / 12 / 12 / * / 9
717 / 14 / 12 / * / 9
Flammulina velutipes 47 / 10 / 10 / 8 / 9
50 / 12 / 9 / 15 / 8
Comment: «—» no studies were done, «*» - time of cultivation was more as 30 days.
The experiment on influence of pH with liquid medium D could be interpreted, as the adjusted pH values were maintained both after autoclaving and roughly during 13 days of cultivation. At this time, a mycelial mat had covered the surface of the liquid medium and the dry weight was measured.
The result obtained indicates that optimum pH for A. auricula 975 is between 5.5 and 6.7 (Figure 1). Our attempts to determine more precisely the pH optimum for this species showed higher optimum from 6.7 to 7.4 for strain 961. Under these conditions strain 975 was the best one and produced up to 2.5 and 4 g/l, on media I and D respectively (Figure 6, 7). In contrast, strain 961 grew very slowly in both submerged media.
The result of the present study demonstrates that in contrast to A. auricula the pH optimim of P. betulinus was on the acidic side of about 3.2 (Figure 2).
Optimum pH for the growth of H. erinaceus strains investigated appeared to be between 5.8 and 6.2 (Figure 2). No growth occurred in medium C below a pH of 2.8 and above 7.0. In comparison with our results the same data was obtained by Grigansky et al. (1999). The lag-phase in submerged
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Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN968-878-105-3
culture was shorter in liquid medium A (2 days) then in medium B and C (3-4 days). However after 10 days of incubation period at 26C on a rotary shaker, maximum yield of dry mycelium was obtained in medium B (Table 3). These results show that this composition of liquid media provided more biomass growth as than was shown in earlier work by Kirchhoff (1996a) on molasses, biomalt and with malt extract and peptone. The growth of strain 965 on liquid media I and J is shown in Figures 7 and 8.
Table 3. Dry weight of mycelium of some medicinal mushrooms after ten days
incubation on different liquid media in submerged conditions.
Strains
/ Dry weight of mycelium, g/lА / В /
С
Agrocybe aegerita 197
/ 6.4 / 17.0 / 4.2A. aegerita 168
/ 4.5 / 12.5 / 7.5A. aegerita 960
/ 7.2 / 14.0 / -Hericium erinaceus 991 / 2.7 / 5.8 / 6.4
H.erinaceus 965 / 3.2 / 8.9 / 6.0
H.erinaceus 963 / 3.0 / 9.2 / 6.8
Ganoderma applanatum 920*
/ 3.4 / 2.5 / 4.4G.lucidum 921* / 2.7 / 5.7 / 6.0
G.lucidum 922* / 1.0 / 5.6 / 5.2
G.lucidum 1621* / 4.4 / 8.0 / 8.2
Grifola frondosa 976*
/ - / 2.7 / -Lentinula edodes 713 / - / 4.2 / 6.1
L. edodes 374 / - / 4.0 / 3.2
* after 12 days of incubation
Two strains of Grifola frondosa were investigated to determine their pH optimum. It was found to be between 5.2-5.5 for strain 923 (Figure 3). Strain 976 achieved maximum biomass of 2.7 g/litre after two weeks incubation in medium B with an initial pH of 6.0. All strains were tested in fruiting trials (Kirchhoff 1996b) and identified as belonging to G. frondosa.
Strains of A.aegerita (168, 197, 960) grew very well under our environmental conditions. The optimum pH for strain 197 is 6.3 (Figure 3). The production of biomass in 10 days incubation in shake-flask culture is shown in Table 3. The best biomass production was on medium B (pH 6.0) for all investigated A. aegerita strains while the least was produced on medium C (pH7.4). Strain 960 also showed very good results on Medium D – up to 13 grams/litre (Figure 6). In some cases after submerged cultivation was completed, the cultures were kept in a static condition as surface cultures. After two weeks in static culture fruit body formation was observed, with cap and basidiospore formation characteristic of this mushroom.
Several studies have been made about submerged cultivation of another significant and very popular medicinal mushroom, L. edodes, and some liquid nutritional media have been proposed. Among these there is a synthetic medium for the production of submerged cultures of L. edodes and some complex media (Solomko and Mitropolskaja 1994). Our current investigation is in accord with data obtained earlier for other strains. Optimum pH for growth appeared to be between 4.5 and 5.2. Maximum dry weight of mycelium was observed on medium C for different strains of L. edodes (Table 3). PH dropped from 4.7 to 3.5 during mycelial growth, which limited further growth. The results of the growth on J medium are presented on Figure 8.
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Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN968-878-105-3
Investigated strains of G. lucidum had very precise pH optima. They were very different for each strain (Figure 4) and depended on their origin. Investigated strains of G. lucidum and G.applanatum grew on liquid media A, B and C with a pH of 4.75 to 6.0. These studies have shown that the best growth of strain 1621 was on media B and C (Table 3). After incubation for 12 days, maximum biomass was 8.2 g/l. During that same incubation period on medium D, these strains showed biomass production of 20-25 g/l (Figure 9).
The optimum pH of H. marmoreus was 7.2 (Figure 5) which resulted in biomass production of 8.1 g/l. No growth occurred at a pH lower than 3.1. The growth of this mushroom on D medium is shown on Figure 9. Three strains were tested in fruiting trials (Lomberh et al. 2000) and identified as belonging to H. marmoreus.
Submerged cultivation in liquid media of mushroom mycelium is a promising method which can be used in novel biotechnological processes for obtaining pharmaceutical substances of anticancer, antiviral, immunomodulating, and antisclerotic action from fungal biomass and cultural liquids and also for the production of liquid spawn. As a result of our recent studies new data was obtained on the cultivation conditions which provide for fast growth and high productivity of the investigated strains in liquid media. The culture media including glucose, peptone, yeast and corn steep extract was proposed to be the best for most of the species investigated. For each species the optimal pH of the medium was determined. The yield of dry biomass was up to 6-25 g/l after 7-10 days of submerged cultivation for L. edodes, A. aegerita, A. auricula, G. lucidum, H. erinaceus, H. marmoreus, (depending on taxonomic status of cultures). G. frondosa was comparatively less productive (up to 4 g/l of dry biomass after 9 days of growth). In all investigated species the period of active growth in our experiments was 4-7 times shorter and the yield of biomass 4-7 times higher compared with the data reported earlier for these species (Kirchhoff 1996a,b).
Figure 1. Effect of medium pH on the growth of Auricularia auricula strains.
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Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN968-878-105-3
Figure 2. Effect of medium pH on the growth of Hericium erinaceus and Piptoporus betulinus strains.
Figure 3. Effect of medium pH on the growth of Agrocybe aegerita and Grifola frondosa strains.
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Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN968-878-105-3
Figure 4. Effect of medium pH on the growth of Ganoderma lucidum strains.
Figure 5. Effect of medium pH on the growth of Hypsizygus marmoreus (strain 1610).
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Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN968-878-105-3
Figure 6. Biomass production of different species of medicinal mushrooms in submerged culture on D medium. 1 – Auricularia auricula 961, 2 – A. auricula 975, 3 – Grifolafrondosa 962, 4 – Hericium erinaceus 965, 5 – Agrocybe aegerita 960.
Figure 7.Biomass production of different species of medicinal mushrooms in submerged culture on I medium.
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Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN968-878-105-3
Figure 8. Biomass production of different species of medicinal mushrooms in submerged culture on J medium.
Figure 9. Biomass production of different species of medicinal mushrooms in submerged culture on D medium.
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Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN968-878-105-3
ACKNOWLEDGMENTS
The authors would like to acknowledge Ms. Tina Ellor for editing the English text .
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