Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN 968-878-105-3
STUDIES OF MEDICINAL MUSHROOMS IN SUBMERGED CULTURES
M. L. Lomberh1, E. F. Solomko1, A. S. Buchalo1, B. Kirchhoff12
M.G. Kholodny Institute of Botany, National Academy of Sciences of Ukraine, 2 Tereschenkivska
St., Kiev 01601, Ukraine. < lomberh@hotmail.com >
2
WESER-CHAMPIGNON, Neue Heerstr. 35, Hessisch-Oldendorf 31840, Germany
1
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.
367
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
8.00.2, 1 liter, agar-agar, 20.0 g (Roth, Germany), pH 4.70.1 (data not shown).
2) Czapek’s medium (CzA) (g/l): sucrose, 30.0; NaNO3, 2.0; KH2PO4, 1.0; MgSO47H2O, 0.5;
FeSO47H2O, 0.01; KCL, 0.5; agar-agar, 20.0 (Roth, Germany); pH, 5.80.2 (data not
shown).
3) Malt extract agar (MEA): 48 g/l (Oxoid), pH 5.40.2.
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 6.50.2
5) Potato dextrose agar (PDA): 39 g/l (Oxoid), pH 5.60.1.
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 6.30.2
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):
Nutrient
Table 1. Composition of liquid media.
Concentration of nutrients in medium (g/l)
Dextrose
Peptone
Yeast extract
(NH4)2SO4
MgSO4*7H20
CaCl2*2H20
K2HPO4
KH2PO4
Corn steep liquor
Trace element solution
Concentrate
A
B
C
D
20.0
2.5
1,0
0
0.3
0
0
0.3
2 ml
10 ml
50.0
5.0
2.0
0
0.5
0.3
0
1.0
5 ml
10 ml
50.0
2.0
2.0
3.0
0.5
0
0-7.5
1.0-7.5
0
10 ml
25.0
10.0
1.0
0
0.25
0.15
0
0.44
2.5 ml
10 ml
368
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 121C for 20 min. The different concentrations of the
potassium dehydro- and hydrogen phosphate for medium C were sterilised separately. After cooling
to 40C trace metal solution concentrate (10 ml) was added to each litre of media. The trace metal
solution consisted (g per litre): 0.4 MnCl24H2O, 0.2 ZnCl2; 0.8 FeCl36H2O, 0.1 CuSO45H2O. The
original media A, B, and D had a pH of 4.80.1 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.
RESULTS AND 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 et al. 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.
369
Table 2. The optimal time to obtain inoculum of some species of medicinal mushrooms on different
agar media.
Time of cultivation, days
Species
Agrocybe aegerita
Auricularia auricula
Ganoderma applanatum
G. lucidum
G. lucidum
Grifola frondosa
Hypsizygus marmoreus
H. marmoreus
Piptoporus betulinus
P. betulinus
Pleurotus calyptratus
P. dryinus
P. eryngii
P. ostreatus
Lentinula edodes
Flammulina velutipes
strains
1503
961
920
921
1621
923
1611
1612
1649
1653
189
205
10
1016
1017
274
295
1637
1638
133
57
704
711
712
717
47
50
MEA
10
14
8
9
7
21
14
14
6
9
8
12
10
10
9
10
7
7
7
7
13
12
15
12
14
10
12
WA
10
10
—
12
7
19
15
14
6
*
5
6
8
6
6
5
7
9
6
6
14
12
11
12
12
10
9
OA
—
—
7
6
6
—
14
12
19
10
7
9
10
6
6
5
6
6
6
6
*
*
*
*
*
8
15
PDA
12
10
14
20
6
14
14
13
10
9
7
11
9
6
7
7
6
7
6
7
11
10
10
9
9
9
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
370
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 26C 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.
Dry weight of mycelium, g/l
Strains
А
В
С
Agrocybe aegerita
A. aegerita
A. aegerita
Hericium erinaceus
H.erinaceus
H.erinaceus
Ganoderma applanatum
G.lucidum
G.lucidum
G.lucidum
Grifola frondosa
Lentinula edodes
L. edodes
197
168
960
991
965
963
920*
921*
922*
1621*
976*
713
374
6.4
4.5
7.2
2.7
3.2
3.0
3.4
2.7
1.0
4.4
-
17.0
12.5
14.0
5.8
8.9
9.2
2.5
5.7
5.6
8.0
2.7
4.2
4.0
4.2
7.5
6.4
6.0
6.8
4.4
6.0
5.2
8.2
6.1
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.
371
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).
7
A. auricula
975
A. auricula
961
Biomass, g/l
6
5
4
3
2
1
0
3
3 .3
3 .9
4 .4
5 .1
5 .5
5 .7
6 .6
6 .7
7 .4
pH
Figure 1. Effect of medium pH on the growth of Auricularia auricula strains.
372
12
H. erinaceus 991
H. erinaceus 963
10
P. betulinus 1653
Biomass, g/l
8
6
4
2
0
3.2
3.3
3.9
4.5
5
5.5
6.1
6.6
6.7
pH
Figure 2. Effect of medium pH on the growth of Hericium erinaceus and Piptoporus betulinus
strains.
pH
3.2
3.4
4.5
5.1
5.4
5.5
6.3
6.7
0
1
l/g , s samoiB
2
3
4
G. frondosa 976
5
G. frondosa 923
6
A. aegerita 197
7
Figure 3. Effect of medium pH on the growth of Agrocybe aegerita and Grifola frondosa strains.
373
De
11
G. lucidum 1621
10
G. lucidum 921
Biomass, g/l
9
8
7
6
5
4
3
3.2
3.7
4.1
4.9
5.5
6.1
6.3
6.7
pH
Figure 4. Effect of medium pH on the growth of Ganoderma lucidum strains.
Biomass, g/l
10
8
6
4
2
0
6.8
7.2
7.6
7.8
8
pH
Figure 5. Effect of medium pH on the growth of Hypsizygus marmoreus (strain 1610).
374
biomass, g/l
14
1
12
2
10
3
8
4
6
5
4
2
0
2
5
7
9
12
time of cultivation, days
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 – Grifola frondosa 962, 4 – Hericium
erinaceus 965, 5 – Agrocybe aegerita 960.
2.5
A. auricula 975
A. auricula 961
2
H. erinaceus 965
1.5
biomass, g/l
1
0.5
0
2
5
7
9
12
time of cultivation, days
Figure 7. Biomass production of different species of medicinal mushrooms in submerged culture
on I medium.
375
H. marmoreus
1611
H. erinaceus 965
7
biomass, g/l
6
5
L. edodes 55
4
L. edodes 704
3
2
1
0
2
3
5
7
9
12
14
time of cultivation, days
Figure 8. Biomass production of different species of medicinal mushrooms in submerged culture on
J medium.
G. lucidum
1621
G.lucidum 921
35
biomass, g/l
30
H. marmoreus
1611
25
20
15
10
5
0
days
2
5
7
9
12
14
Figure 9. Biomass production of different species of medicinal mushrooms in submerged culture on
D medium.
376
ACKNOWLEDGMENTS
The authors would like to acknowledge Ms. Tina Ellor for editing the English text .
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