Myceial growth on solid and liquid nutrient media
P. ostreatus is the object of intensive research due to its unique attributes, which open up broad prospects for its application in biotechnology and various sectors of modern industry [6]. Finding the optimal growing medium for successful mushroom growth is one of the first steps to ensure that they can be used efficiently and cost-effectively in the future. The present comparative studies on the growth of P. ostreatus strains on five agar media revealed that the cultures generally grew fast and differently on all the solid media tested. Differences in mycelial growth of P. ostreatus strains may be related to their availability of appropriate nutrients, primarily sources of carbon, nitrogen, also vitamins, macro and microelements. This study allowed us to establish favorable and alternative solid agar media for the growth of each strain. The studied strains, according to the degree of greater adaptive ability to the same grow on the used solid nutrient media, can be presented in the following order: 2462 > 2460 > 2461 = 1685 > 551. In general, GPYA medium supported the growth of all strains used better than others. This result confirmed previous evidence of the ability of glucose to be rapidly catabolized by fungi for easy cellular energy production [20]. However, the highest growth rate of most strains was observed on PDA medium. The best growth rate was found for the strain P. ostreatus 2462 (15.0 ± 0.8 mm/day on PDA). It was previously found that this medium is also best suited for mycelial growth of other strains of P. ostreatus under different study [14, 21, 22, 23]. In contrast to our observations, the best mycelial growth of P. ostreatus was observed in MEA [24, 25].
One of the most pressing tasks of biotechnology is to find ways to increase the fungal yield when they are grown in artificial conditions. To solve this problem, culture media are saturated with various additives of natural or synthetic origin. Recently, synthetic low-molecular-weight heterocyclic compounds represented by pyridine, pyrimidine, pyrazole and oxazole derivatives have been under extensive research due to their high efficiency at low concentrations and lack of toxic effects on plant and animal cells, which ensures environmental safety [26, 27, 28, 29, 30, 31]. To the best of our knowledge, in this study we examined the effect of synthetic plant growth regulators Ivin, Methyur and Kamethur on fungal growth is the first time. The results obtained showed positive, negative and neutral effects of these chemical compounds on the yield of P. ostreatus mycelium.These data demonstrate that the effect of these chemical compounds was highly dependent on the strain, as well as the duration of cultivation and the concentration of the respective compound used. It should be noted that no difference in the influence of the concentration of the chemical compounds used was found only when P. ostreatus cultures were cultivated for two weeks. The studied synthetic compounds can be arranged in the following order (according to the degree of fungal growth stimulation): Kamethur > Ivin > Methyur. A certain chemical similarity between Methyur and Kamethur at all concentration used had the same growth-regulating effect on the 7th day of the experiment for strains 551, 1685 and 2462, on the 14th day of the experiment for strains 551, 2460, 2461, and on the 21st day of the experiment only for strain 1685. In addition, all three chemical compounds used at all concentrations had the same growth-regulating effect on the P. ostreatus strains 551, 2460, 2461 when cultivated for two weeks and on the P. ostreatus strain 1685 when cultivated for three weeks. Among all studied cultures, P. ostreatus strains 551 and 1685 were more receptive to positive assimilation of all investigated compounds used in nanomolar concentrations. However, the growth-stimulating activity of the synthetic plant growth regulators Ivin, Methyur and Kamethur was previously established for various crops [28, 29, 30, 32, 33, 34]. In general, concentrations of 10− 5, 10− 6 and 10-7M of these synthetic plant growth regulators showed the highest growth-regulating effect [28, 29, 30, 32, 33, 34].
Despite a certain increase in the stimulating effect under the influence of synthetic plant growth regulators, the second strategy used to increase the biomass yield turned out to be more effective. According to the results obtained, the use of nutrient media based on a combination of natural waste makes it possible to increase the yield of mycelium of P. ostreatus strains by 2.2–2.9 times compared to the control. It is well known that fungi need carbon, nitrogen, and inorganic compounds as a source of nutrition. The medium prepared on the basis of natural waste allows fungal cultures to receive the necessary nutrients in an accessible form, such as crude protein, crude fiber, nitrogen, vitamins, minerals, which can support their growth. The bioavailability of nutrients in the medium is determined by the unique physiological and biological characteristics (above all, ability to secrete specific enzymes) of the cultures and has a strain-specific dependence. It should be noted that strain 2462, which had the maximum growth on solid PDA medium, also showed the best results of biomass production when growing on most of the studied liquid media. In general, the studied wastes can be a good alternative basis for the production of mycelium as a seed (spawn) and biomass, and therefore as well as an effective additional component to substrates for the production of fruiting bodies. Since substrates with a high biomass return, which can pose environmental problems, can be a good alternative for the cultivation of P. ostreatus. In this regard, the potential use of such studied waste as an alternative medium for mycelial production deserves to be explored also in industry conditions and confirmed through further research in this field. Successful bioconversion of amaranth flour into mycelial biomass was recorded in our previously studies [35, 36, 37]. Meanwhile, according to our early observations, mycelium of higher fungi obtained using amaranth flour had antibacterial [36], antiviral [38], anticancer [39] and wound healing activities [40]. Our current study also demonstrated for the first time positive effect of amaranth flour waste in combination with other wastes studied on the increase in the mycelial mass production of used P. ostreatus strains. The possibility and effectiveness of using natural waste (as a monobase or as a component of a liquid culture medium) to increase the yield of P. ostreatus mycelium has been shown in some studies [41, 42].
Taking into account the data obtained in this work, a promising issue of our further research is the study of the combined effect of synthetic plant growth regulators Ivin, Methyur and Kamethur and natural waste to increase the biomass yield of P. ostreatus strains.
Biological activities
In addition to its high food and industrial value, P. ostreatus continues to play a significant role in medicine and pharmacy. Infectious diseases leaded to fungi and bacteria represent an important cause of morbidity and mortality among the general population. The use of crude extracts of Pleurotus species and phytochemicals, of known antimicrobial properties, can be of great significance in the therapeutic treatments. Although a certain P. ostreatus strain have been early studied with respect to their phytochemical constitutes and antimicrobial potential, their physiological interactions between microbes remain poorly understood. Strong antagonistic activity was recorded for all used P. ostreatus strains against potential harmful micromycetes Aspergillus niger, Candida albicans, Issatchenkia orientalis, Fusarium poae, and Microdochium nivalе. The absence of variability in the types of reactions, the level of their visualization, as well as morphological changes in the contacting colonies and the presence of the same level of antagonistic index indicate that fungal-fungal interactions were independent of the strain. The results of the study may not reflect true ecological relationships due to the possible influence of abiotic and biotic factors in the environment, but this study offers a preliminary but rapid way to study and predict how the tested strains of P. ostreatus will interact with pathogenic fungi. Understanding the response of microbial communities and possible changes in the habitat can provide valuable information on microbial competition of fungi. To the best of our knowledge the antagonistic activity of P. ostreatus against F. poae and M. nivale was studied in this study for the first time in dual culture. Our results are consistent with the data on the successful antagonistic activity and the ability of P. ostreatus mycelium to replace the mycelium of Pythium spp. [43], Bipolaris sorpkiniana, Fusarium culmorum, Gaeumannomyces graminis var. tritici, and Rhizoctonia cerealis [16], Clonostachys rosea and Trichoderma pseudokoningii [17], Issatchenkia orientalis and Candida albicans strains [44], Aspergillus niger and Penicillium polonicum [45] under similar co-culture conditions. Moreover, further study of the effects on living organisms will be important to deepen our understanding of these compounds and their potential therapeutic use.
The preliminary screening for antimicrobial activity in this study showed that only the EtOAc extract of two P. ostreatus strains, namely 2460 and 2462, showed good growth inhibition of Staphylococcus aureus ATCC 65388 and Escherichia coli ATCC 6633, respectively. The obtained results in agreement with the findings to those performed by Vamanu [46] regarding the ethanolic mycelium extracts from P. ostreatus strain PQMZ91109 also possesses antibacterial effect against E. coli CBAB 2, S. aureus ATCC 6588. Similarly, it was previously observed by Younis et al [47] that an aqueous extract of P. ostreatus mycelium inhibited the growth of S. aureus RCMB (B001001՚՚3՚՚), while it had no effect against E. coli RCMB (B004001՚՚4՚՚), but led to inhibition of Klebsiella pneumoniae RCMB growth (B008001՚՚2՚՚). In addition, our results are in line with the report of El-Rahman et al. [48], who observed inhibition of the growth of S. aureus ATCC 6588 as well as Escherichia coli CBAB2 when applying P. ostreatus extract prepared using a solvent mixture consisting of methanol: glycerol: water (1:1:1 v/v). Also, hot-water mycelial extract from Pleurotus sp. strain CCEBI-3024 led to the autolytic enzyme system of the microbial cell of S. aureus ATCC 25953 [49]. However, our results are contrary to those performed by Owaid et al. [50] regarding to absent of antibacterial action against E. coli ATCC 25922 and S. aureus HIP10267. The native mycelium of P. ostreatus strain 551 in our previous study [38] was also inactive against E. coli 06 and S. aureus 209.
The analysis of DPPH free radical scavenging activity is one of the most common methods for determining antioxidant capacity, can accept an electron or hydrogen atom to become a stable diamagnetic molecule. Our result shows that all extracts of P. ostreatus strains have DPPH radical scavenging activity, but the percentage of inhibition depends on the strain of P. ostreatus as well as on the solvents used. The highest radical scavenging effect of P. ostreatus 1685 (61%) was quite close to the results of previous studies with hot water extracts of P. ostreatus [51] and some results obtained with ethanol extracts of mycelium of P. ostreatus strain PQMZ91109 [46], but lower than the results obtained in other studies using hot water extract. However, all extracts of studied P. ostreatus strains possessed more antioxidant activity compare to observation performed by El-Rahman et al. [48] mentioned influence of mycelial extract prepared with solvents mixture (methanol: glycerol: water).
The antioxidant capacity is a way of reflecting the effect of reducing compounds in the fungal extract. The observed free radical scavenging activity can be attributed to the presence of phytochemicals, in particular, phenolic compounds. TPC in both mycelial extracts of P. ostreatus strain 1685 and EtOH extract of P. ostreatus strain 2461 was similar to reported amounts in other P. ostreatus extract prepared from mycelium [46, 51]. In addition, all the extracts of the studied P. ostreatus strains had a higher TPC compared to the results of El-Rahman et al. [48] when exposed to mycelial extract prepared with a mixture of solvents (methanol: glycerol: water). It is well known that each solvent has its own specific selectivity for the extraction of different compounds that contribute to the overall antioxidant activity. However, it is important to note that even if the EtOAc extract the highest amount of TPC from P. ostreatus strain 2461, it does not provide the best antiradical scavenging activity compared to the other fungal strains. However, it should be borne in mind that the diversity and effective extract yield of different compounds can vary greatly depending on various factors. Due to this tendency, the antioxidant activity of an extract cannot be attributed to the individual contribution of a particular compound. Because, other significant compounds that were not extracted in this study may have a greater contribution to the antioxidant activity.
Our results emphasize the crucial importance of studying specific strains of cultures, as well as raising awareness of the sustainable use of waste for mushroom cultivation. Some difference in the results of mycelial growth of P. ostreatus strains used on solid and liquid nutrient media, antagonistic, antibacterial and radical scavenging activities as well as in total phenolic content of this investigation and compare to literature data of other researchers may be due to the use of different P. ostreatus strains, different methods of media preparing, extraction solvents or different assayed microorganisms, and expressed results in other measurement units.