Screening of medicinal mushroom strains with antimicrobial activity and polysaccharides production

Objectives: This study aimed to determine the antimicrobial activity of culture ﬂ uid and mycelial mass of selected Hericium strains from the IBK Mushroom Culture Collection (Kyiv, Ukraine) against bacteria and fungi, as well as to evaluate the potential of these mushrooms for biomass and extracellular polysaccharides production. Methods: The antimicrobial activity of ethyl acetate extracts of culture ﬂ uid and aqueous-ethyl extracts of mycelial mass for three Hericium species: Hericium cir-rhatum IBK 2393, Hericium coralloides IBK 2332, and Heri-cium erinaceus IBK 2530 against Gram-positive Bacillus subtilis , Staphylococcus aureus , and Gram-negative Escherichia coli , Klebsiella pneumoniae , Pseudomonas aur-eginosa bacteria as well as Aspergillus niger , Penicillium polonicum , and Mucor globosus fungi by the disk di ﬀ usion method was conducted. Analysis of the minimum inhibitory concentration was carried out according to the broth dilution method. Results: High antimicrobial activity of culture ﬂ uid extracts of Hericium mushroom species against S. aureus was established after 14 – 21 days of cultivation. No e ﬀ ects of Hericium extracts against A. niger , P. polonicum , and M. globosus were observed. On the 7th day of submerged cultivation, the studied strains produced the maximum yield of extracellular polysaccharides. The most promising extracellular polysaccharide (EPS) producers were strains of H. coralloides IBK 2332 (1.89 g/L) and H. erinaceus IBK 2530 (1.90 g/L). The maximal value of biomass was produced by H. cirrhatum IBK 2393 on the 21st day of submerged cultivation (17.45 g/L). Conclusions: Hericium mushrooms have signi ﬁ cant potential for the development of new drugs with health bene ﬁ ts properties.


Introduction
Edible mushrooms have not only great nutritional value and taste but also various medicinal properties [1,2].Most basidiomycetes contain biologically active substances with antibacterial effects, such as polysaccharides, carotenoids, phenolic compounds, terpenoids, etc., in their fruiting bodies, mycelium, and culture fluid [3,4].
One of the priority areas in biotechnology and experimental mycology is the search for new producers of biologically active substances from edible mushrooms of different ecological and trophic groups.One of the wellknown edible mushrooms with medicinal properties confirmed by clinical studies is Hericium erinaceus (Bull.: Fr.) Pers.This fungus has been used in folk medicine, mainly in South-East Asia countries (China, Japan).Purified bioactive metabolites from the fruiting bodies or mycelium of H. erinaceus have biological activities such as antitumor, neuro-and gastroprotective, immunomodulatory, neurotrophic, antioxidant, antibacterial, hypoglycemic, and hepatoprotective [5,6].Among biologically active substances isolated from Hericium fungi are hericenones, erinacins, extracellular and endopolysaccharides, phenols, sterols, and others [2,7].From the moment of isolation of oncostatic polysaccharides from the fruiting bodies of some basidiomycetes, an active study of these compounds is being begun, as well as the search for their producers [8][9][10].Polysaccharides obtained not only from fruiting bodies but also from submerged mycelium and culture fluid of Hericium fungi attracted much attention due to their strong pharmacological effect [11][12][13][14].
At present, among Hericium fungi, H. erinaceus is the most studied.At the same time, there is little information about the secondary metabolites produced by other species of Hericium mushrooms.Only data about erinacine isolation from H. flagellum and corallocins from Hericium coralloides which are potential therapeutic agents for neurodegenerative disorders were reported [7,15].
Antimicrobial properties of macromycetes, including Hericium fungi, have been studied in numerous works [16][17][18][19][20].It was found that aqueous, ethanol, methanol, ethyl acetate extracts of fruit bodies, and mycelium of H. erinaceus have antimicrobial activity against a number of bacteria [16,17,[19][20][21][22].The antibacterial activity of the culture liquid of H. erinaceus was also found [22].Besides, H. erinaceus extract showed strong activity against Staphylococcus aureus [16,20,23].It should be noted that most data provide information about the antibacterial activity of extracts from the H. erinaceus fruiting bodies, while there is little information about the antimicrobial activity of extracts from mycelium and liquid fluid of other species of Hericium fungi [22].Along with the antibacterial effect, the antifungal activity of extracts of mycelium of Hericium species against Candida albicans and Cryptococcus neoformans has also been reported [24].
In search of new sources of antimicrobial producers, the aim of our work was to study the antimicrobial activity of culture fluid and mycelial mass of three strains of three Hericium species from the ІВК mushroom culture collection (Kyiv, Ukraine), selected after preliminary screening, as well as to evaluate the ability of Hericium fungi for biomass and extracellular polysaccharides production under submerged cultivation.

Mushroom strains
Pure cultures of three species of Hericium fungi (Hericium cirrhatum 2393, H. coralloides 2332, H. erinaceus 2530) were obtained from the IBK Mushroom Culture Collection of the M.G.Kholodny Institute of Botany of the National Academy of Sciences of Ukraine (Table 1) [25].All strains were maintained on agar wort medium and stored at 4 °C.
The inoculum preparation was well described in our previous study [4].Mycelium cultivation was carried out in 500 mL Erlenmeyer flasks with 100 mL of GPY liquid nutrient medium on an orbital shaker (120 rpm) at 25 ± 0.1 °C for 28 days.

Test microorganisms
Five bacterial strains and three fungal strains were tested in this research.Three Gram-negative bacteria include Escherichia coli (B-906), Klebsiella pneumoniae (M-123), and Pseudomonas aureginosa (B-900), two Gram-positive bacteria include S. aureus (B-918), Bacillus subtilis (B-901).All bacterial strains were obtained from the collection of the Faculty of Biotechnology and Microbiology of the National University of Food Technologies (Kyiv, Ukraine).
Fungal strains include Aspergillus niger (VURV-F 822), Penicillium polonicum (VURV-F 823) from the Collection of Microorganisms of Crop Research Institute (Prague, Czech Republic), and Mucor globosus (N-O18) from the collection of microorganisms of the Ukrainian Institute of Botany.

Preparation of mushroom extracts
Strains of Hericium were cultivated for 7, 14, 21, and 28 days in submerged conditions on an orbital shaker.After that, mycelial biomass was separated from the liquid by filtration, washed with distilled water, and dried to constant weight at 60 °C.Obtained fungal biomass was extracted with 70 % ethanol in a ratio of 1:50 (w/v, biomass to ethanol) in an ultrasonic bath at 40 °C for 30 min and left for 24 h at 4 °C.After that, the extract was filtered and centrifuged for 20 min at 3,500 rpm to remove the biomass.The resulting extract was tested for antimicrobial activity [26].
To concentrate the antimicrobial substances in the culture fluid, the method of liquid-liquid extraction was used.Ethyl acetate was added at a ratio of 2:1.The resulting mixture was shaken for 10 min and left for 20 h at 4 °C.The ethyl acetate fraction was evaporated to dryness on a rotary evaporator [27].The procedure was repeated three times.The resulting residue was weighed, dissolved in 70 % ethanol, and adjusted to a volume equal to 1/10 of the original culture liquid.

Antimicrobial assay
The antibacterial activity of biomass and culture fluid extracts of the studied strains was assayed by the disk diffusion method [27].A small number of bacteria from a freshly grown colony was transferred by a microbiological loop into test tubes with sterile saline, shaken until a homogeneous suspension was obtained, and the density of the inoculum was adjusted to exactly 0.5 in accordance with the McFarland standard (5 × 10 6 cells/mL) [26].The resulting suspension of bacteria from the calculation of 0.2 mL was applied evenly to the surface of the Muller-Hinton medium.Extracts (10 μL) were applied to standard Bio Merieux firm discs (6 mm in diameter), dried at 40 °C for 30 min, and placed on the surface of an agar medium, pre-inoculated with a bacterial or fungal test culture.After that, the agar plates were incubated under appropriate conditions depending on the studied microorganism (24 h at 37 °C for bacteria and 72 h at 26 °C for fungi).Antibacterial/antifungal analysis was performed by measuring the growth inhibition zone around the filter discs [27].In all assays, antibiotic Gentamycin sulphate (40 mg/mL, Ukraine) was used as a positive control.The ethyl acetate for the experiment with culture fluid and 70 % ethanol for the experiment with mycelial biomass were used as negative controls.
Analysis of the minimum inhibitory concentration (MIC) was carried out according to the broth microdilution method [28].The test was performed in a 96-well polystyrene microtiter plate (Colstar, USA).50 μL of the Muller-Hinton medium containing the appropriate concentration of the mushroom extract was added to each well, except for the control ones.The extracts were diluted in a range from 2 up to 768 times.After that, a 50 μL suspension of microbial cells (5 × 10 6 cells/mL) was added to each well; the sterility control well contained 100 mL of the Muller-Hinton medium, and the growth control well had 50 mL of medium and 50 mL of the bacterial suspension.The test was carried out in triplicate; MIC was defined as the minimum extract concentration at which no growth was observed in any repeats.

Polysaccharide determination
The mycelial biomass (MB) was harvested by filtration through Whatman No.1 filter paper.It was dried in a hot air oven at 60 °C until constant weights were recorded.For the determination of EPS, the obtained culture fluid after mycelia removal was concentrated in a vacuum evaporator three times from the initial volume, precipitated with 96 % cooled ethanol in a ratio of 1:1 and placed in a refrigerator at 4 °C for 24 h.The precipitate was isolated from the supernatant by centrifugation at 8,000 g for 15 min.After separation, EPS was dried at 60 °С to constant weight.The yields of MB and EPS were expressed as the g dry weight/L of the culture liquid [29,30].

Statistical analysis
All experiments were carried out in triplicate.All data were statistically processed for analysis.The Student's t-test was applied to determine significance, and a p-value of <0.05 was considered statistically significant.The results were expressed as mean ± standard deviation using Excel 2010 (Microsoft Corporation, USA).
According to the results, none of the extracts showed any activity against test micromycete: A. niger, P. polonicum, and M. globosus under conducted cultivation conditions.
For all of the strains, we observed no antimicrobial activity of the homogenized mycelium and culture fluid after 7 days of submerged cultivation.It was also found that studied Hericium sp.strains did not show antimicrobial activity against test bacteria Pseudomonas aeruginosa and E. coli.An exception was only the culture fluid extracts from H. coralloides IBK 2332 against E. coli on the 21st day of incubation.
Insignificant activity against Gram-negative bacteria K. pneumoniae was observed in strains H. coralloides IBK 2332 and H. cirrhatum IBK 2393 on the 14th -21st days of incubation; the diameters of the growth retardation zone were less than 10 mm.The antimicrobial activity of fungal extracts against Gram-positive B. subtilis was also manifested on the 14th -21st day of cultivation.The highest antimicrobial activity against B. subtilis was found in the culture fluid of the H. erinaceus IBK 2530 on the 14th day of cultivation; the diameter of the growth retardation zone was 28.0 ± 0.1 mm.
Among Gram-positive bacteria, S. aureus was the most sensitive to fungal extracts (Table 2, Figure 1).Ethyl acetate extracts of the culture fluid of H. cirrhatum ІВК 2393 and H. erinaceus ІВК 2530 showed the highest antimicrobial activity on the 14th day of cultivation, the diameter of the growth retardation zone was around 30 mm.The best results with the highest antimicrobial activity in growth suppression of S. aureus demonstrated the culture fluid extracts of H. coralloides IBK 2332 on the 21st day of cultivation; the diameter of the growth retardation zone was 32.1 ± 0.1 mm.Overall, these results indicate that in all investigated strains on the 14th and 21st day of cultivation, the growth retardation zone for S. aureus exceeded the positive control values.In contrast to the culture fluid, aqueous-ethyl extracts of the mycelial mass showed twice less antimicrobial activity, except H. cirrhatum ІВК 2393 (Table 2).

The minimum inhibitory concentration of aqueous-ethyl extracts of Hericium fungi
For a better understanding of the prospects for the use of these extracts against bacterial pathogens, it is important to establish the minimum inhibitory concentrations of the extracts.The MIC is the lowest concentration of a sample that inhibits the visible growth of microorganisms.Since Hericium fungal extracts showed the maximal effect against S. aureus, therefore, MIC of this test culture was determined (Table 3).It was established that the extract of the H. cirrhatum biomass has the least inhibitory activity.So, the MIC for this extract was determined when it was diluted 6 times, which corresponds to the content of dry substances at the level of 1900 μg/mL.Extracts obtained from the biomass of H. Coralloides and H. erinaceus inhibited the growth of S. aureus at much higher dilutions, namely 96 times, the content of dry substances in these extracts was 130 μg/mL.The investigated extract of the culture liquid of H. erinaceus was more effective than the biomass extract of this mushroom; its MIC was 50 μg/mL.

Accumulation of biomass and extracellular polysaccharide production of Hericium fungi
Analyzing the data obtained, we note that all studied Hericium sp.strains synthesized extracellular polysaccharides in the glucose-peptone-yeast nutrient medium (Table 4).
In general, it is shown that for all strains, an increase in the accumulation of biomass was observed from 7 to 21 days of the cultivation period.Another important finding was that, at the same time, with an increase in the duration of cultivation, a decrease in the content of EPS was observed.The maximal value of biomass was produced by H. cirrhatum IBK 2393 on the 21st day of submerged cultivation -17.45 ± 0.71 g/L.The maximum concentration of biomass produced by H. coralloides IBK 2332-11.53 ± 1.09 g/L and 12.10 ± 0.64 g/L was obtained on the 14th and 21st days of growth, respectively.
The data obtained indicate that after 7th day of submerged cultivation, all the strains studied produced the maximum EPS yield.The most promising EPS producers were strains of H. coralloides IBK-2332 (1.89 ± 0.12 g/L) and H. erinaceus IBK-2530 (1.90 ± 0.07 g/L).Studies have shown that some of these metabolites have antibacterial and antifungal properties [21,24].It was found that higher antimicrobial activity of mushroom extracts was observed against Gram-positive bacteria, including drug-resistant strains, such as methicillin-resistant [23].Okamoto et al. established the antimicrobial potential of H. erinaceus and identified some bioactive molecules with antimicrobial activity against S. aureus, Salmonella enteritidis, Vibrio parahaemolyticus, and E. coli [37].Our results correspond to the literature.Analyzing the data obtained, we note that the studied strains  All obtained samples had weak antibacterial activity (MIC from 250 to 500 μg/mL/mL) [38].After purification and separation of the extracts by chromatography, an increase in the MIC to 12.5-50 μg/mL and 6.25-25 μg/mL was observed for fractions 1 and 2, respectively.Based on the obtained results, it can be assumed that during subsequent purifications of H. erinaceus IBK 2530 ethyl extract with MIC of 50 and 130 μg/mL to S. aureus, the antibacterial activity of the obtained samples increased.These data must be interpreted cautiously because the compositions of four fractions of H. coralloides ethyl acetate extract obtained in another research did not exhibit antibacterial activity against B. subtilis, S. aureus, E. coli, Botrytis cinerea, and Rhizoctonia solani [39].
Thus, comparing the obtained results with the literature data, it should be noted that H. cirrhatum IBK 2393, H. coralloides IBK 2332, and H. erinaceus IBK 2530 culture fluid extracts, as well as their biomass extracts, exhibit high antibacterial effect against Gram-positive bacteria such as S. aureus, as well as B. subtilis, which makes it possible to talk about the prospects of using these mushroom strains in the future as antibacterial agents.However, this requires further study and is another potential area of future research.
According to the literature data, the antibacterial effect of basidiomycetes is due to the presence of different biologically active compounds, including polysaccharides [1,4,7].Previous studies have reported that the yield of extracellular polysaccharides during the cultivation of H. erinaceus ranges from 0.33 to 2.77 g/L depending on the carbon and nitrogen sources, minerals, vitamins, and the pH of the nutrient medium.Under optimal cultivation conditions, the maximum biomass yield in H. erinaceus reached 14.24 ± 0.45 g/L.At the same time, the maximum EPS production was observed on the 8th day of fungal cultivation (2.75 ± 0.27 g/L) [13].In our research, the maximum biomass yield in H. erinaceus IBK 2530 was 8.04 ± 0.42 g/L, and the maximum EPS production we also observed after 7 days of fungal cultivation was 1.90 ± 0.07 g/ L. Compared with literature data on other mushrooms, the maximum EPS production in Lentinus edodes was 0.8 g/L, in Pleurotus ostreatus 1.3 g/L, and in P. eryngii 1.0 g/L [29].The results strongly depend on the effect of various sources of carbon and nitrogen on the productivity of biomass and extracellular polysaccharides, as was shown by Elisashvili et al. [29].The highest EPS productivity was obtained on media with glucose or sodium gluconate as the carbon source and peptone оr liquid corn extract as the nitrogen source [29].Our study did not aim to optimize the medium and increase biomass and EPS yield.Therefore, we used the composition of the media components whose effectiveness was proven by previous researchers: glucose 25.0 g/L, peptone 3.0 g/L, and yeast extract 2.0 g/L [29,30].It was a screening study of promising Hericium species and strains selected at the previous stage of our research.The most promising results were 17.45 ± 0.71 g/L and 12.10 ± 0.64 g/L biomass produced by H. cirrhatum IBK 2393 and H. coralloides IBK 2332, respectively, on the 21st day of submerged cultivation.According to our data, the EPS accumulation in culture liquid rather correlated with mushroom biomass yield.Interestingly, long-term submerged cultivation of these cultures to obtain EPS is not advisable.Among mushrooms tested, the maximum EPS we observed on the 7th of submerged cultivation.Similar results have been reported by Elisashvili after 7 days of cultivation of Agaricus sp. and Oudemansiella canarii [3].
In conclusion, Hericium extracts exhibit strong antibacterial activity against the Gram-positive bacteria S. aureus.Our results demonstrate that mushroom extracts had no effect against A. niger, P. polonicum, and M. globosus.On the 7th day of submerged cultivation, all the strains studied produced the maximum EPS yield (1.55-1.9g/L).However, the EPS productivity of each strain decreased with the length of cultivation time.At the same time, an inverse correlation was observed for the accumulation of mycelial biomass.The maximum amount of biomass was accumulated on 14-21 days of fungal cultivation.Thus, Hericium fungi have significant potential for the development of new drugs with health benefits properties.

Figure 1 :
Figure 1: Antibacterial activity of ethyl acetate extract of Hericium species culture fluid against Staphylococcus aureus (negative control, the 7th, 14th, 21st days of cultivation).
Research funding: This research was funded by the Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan (Grant No. AP09258296).Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.Competing interests: Authors state no conflict of interest.Informed consent: Informed consent was obtained from all individuals included in this study.Ethical approval: The local Institutional Review Board deemed the study exempt from review.

Table  :
The studied species and strains of Hericium.

Table  :
Antimicrobial activity of culture fluid ethyl acetate extracts and mycelial mass aqueous-ethyl extracts of Hericium strains against Staphylococcus aureus.CF, ethyl acetate extracts of the culture fluid; MB, aqueous-ethyl extract of mycelial biomass.a Gentamycin sulphate, antibiotic solution; b ethyl acetate for experiment with culture fluid,  % ethanol for experiment with mycelial biomass.The maximum value is indicated in bold.All results are expressed as mean ± SD from three experiments (n=).c The values within the columns between  and  days of cultivation are significantly different (p≤.).
[6,16,23,[34][35][36]ies, H. coralloides IBK 2332, H. cirrhatum IBK 2393, and H. erinaceus IBK 2530 extracts demonstrate strong antibacterial effects against bacterial test cultures.At the same time, analyzing the available literature, we did not find data indicating the antimicrobial activity of H. cirrhatum fungi.Previous studies demonstrate that Hericium fungi are producers of secondary metabolites with antimicrobial properties[2,[32][33][34]].An aqueous extract from the fruiting bodies of H. erinaceus is known to accelerate wound healing.Various researchers have confirmed the antimicrobial effect of extracts of fruiting bodies of H. erinaceus that inhibited the growth of Bacillus cereus, B. subtilis, Enterococcus faecalis, S. aureus, P. aeruginosa, E. coli and Plesiomonas shigelloides[6,16,23,[34][35][36].Data obtained by Han et al. and Song et al. show that ethyl acetate and methanol extracts from H. erinaceus fruiting bodies exhibit a high antibacterial effect against Micrococcus luteus and S. aureus, and moderate activity against Enterobacter cloacae and Streptococcus mutans.

Table  :
Minimum inhibitory concentration (MIC) of extracts from biomass and liquid culture of Hericium fungi against Staphylococcus aureus ( days of cultivation).
a Concentration was calculated in accordance with the content of dry matter in extracts.

Table  :
Accumulation of biomass and exopolysaccharides in Hericium fungi.The maximum value is indicated in bold.All results are expressed as mean ± SD from three experiments (n=).aThevalues within the rows in comparison with data obtained on the th day are significantly different (p≤.).synthesizebiologicallyactive substances that are effective against the resistance to methicillin-resistant test bacterium S. aureus, which is known by various antibiotic resistance spectra.Liu et al. studied various fractions of ethyl extracts of H. erinaceus mycelial biomass against Helicobacter pylori.