The Effect of Supplementing Mushroom Growing Substrates on the Bioactive Compounds, Antimicrobial Activity, and Antioxidant Activity of Pleurotus ostreatus (2024)

2.1. P. ostreatus Mushroom Cultivation Procedure

P. ostreatus mushrooms were cultivated on locally available substrate/waste materials namely sugar cane bagasse and sugar cane tops. Few modifications from the methods by [18] were incorporated in the process of mushroom cultivation. The four-step process was followed for the cultivation of P. ostreatus: (a) preculturing of P. ostreatus on PDA, (b) spawn preparation, (c) substrate preparation and inoculation, and (d) P. ostreatus fruiting. The test P. ostreatus mushroom was obtained from Cedara College of Agriculture at Pietermaritzburg in Kwa-Zulu Natal (South Africa), where it was previously identified and characterised.

The P. ostreatus mushroom strain was initially precultured on potato dextrose agar (PDA) and thereafter incubated under dark environment at ±25°C till mycelia fully covered the PDA plate. The precultured P. ostreatus strain was then stored at 4°C, hence maintained as mother culture for further processing. P. ostreatus mushroom spawn was prepared following a modified method outlined by [22] using birds seed grains that was soaked in overnight in distilled. Briefly 4 g of the soaked birds seed grains were mixed with 1g gypsum (CaSO. 4·2H₂O) and 300 g calcium carbonate (CaCO3) and autoclaved. The grains were then inoculated aseptically with the previously grown mushroom cultures and thereafter incubated under dark environment at ±25°C, until mycelia fully colonized the bird seed grains. The prepared mushroom spawn was also stored at 4°C for further processing. The locally available substrates (sugarcane bagasse and sugar cane tops) were sparingly sprinkled with H₂O till 65% moisture was achieved. Thereafter, the substrates were supplemented with wheat bran at levels of 0%, 2%, 18%, and 20% wheat bran (WB), respectively. These levels of supplements were thoroughly mixed with the substrates and then pasteurised at 60–65°C for six hours and allowed to cool at room temperature [23]. The pasteurised substrates were then inoculated with the previously prepared spawn, which was later incubated under dark till mycelia fully colonised the substrates. Once the substrates were fully colonized by mushroom mycelia, they were removed from dark environment into the fruiting room that was made of 30% shade cloth. The P. ostreatus mushrooms fruited under ambient temperatures at constant fogging to achieve 60% moisture which is ideal for fruiting of oyster mushrooms.

2.2. Carbon to Nitrogen Ratio (C/N) of Supplemented Substrates and Mushroom Yield/Productivity

The supplemented substrates (sugar cane tops and sugar cane bagasse) were analysed for total carbon and nitrogen composition following a modified method by [24]. The C and N within substrates were analysed using CHN analyser (Leco, Moenchengladbach, Germany) following the combustion method, whereby 3 mg of dried substrates were analysed in triplicate. Thereafter, the C/N ratio was calculated from the mean of the results obtained and used to confirm the degree of condensation of organic compounds. The yield of the produced mushrooms was calculated according to method extrapolated from [18]; hence, the following equation was used:

2.3. Preparation of Mushroom Extracts

A slightly modified method by Chowdhury et al. [25] was used for the extraction and preparation of mushroom extract. The freshly harvested mushrooms were sundried under transparent tunnel with 30% shade cloth. Then the dried mushrooms were milled into powder using a Scientec hammer mill, resulting into a 2 mm mesh powder. Thereafter, 100 g of powdered mushroom was dissolved into 250 ml methanol and incubated in shaker set at 200 rpm for 24 h at 25°C. The mushroom extract was filtered using Whatman No. 1 filter paper and evaporated to semidryness using fume mode. The semidry P. ostreatus mushroom extract was then stored at 4°C for further analysis.

2.4. Screening of Bioactive Compounds Using Gas Chromatography-Mass Spectrometry (GCMS)

Well established method by Daffodil et al. [26] was used to screen for the bioactive compounds within the mushrooms. For GCMS analysis, 3 mg of extract was mixed with 10% methanol and 90% dichloromethane (DCM) and shaken to dissolve into a hom*ogenous mixture. The GC-MS analysis of the extract was conducted using the Shimadzu GC-MS solutions system, whereby the gas chromatography interfaced with the mass spectrometer (GC-MS) which had Elitel and a fused silica capillary column (30 mm × 0.25 mm 1D X1 μMdf, consist of 100% Dimethyl poly siloxane). Electron ionization system with ionizing energy of 70 eV was used for the GCMS detection. The carrier gas named Helium (99.999%) was used at the constant flowrate of 1 ml/min, with the injection volume of 8.00 μl, and split ratio of 10 : 1.250°C was the injector temperature and 28°C being the ion source temperature. The oven was programmed at 110°C (2 min isothermal) and was increasing at 10°C/min to 200°C and 5°C/min to 280°C, then ended with 9 min isothermal which was at 280°C. The 70 eV was used for the mass spectra with scanning interval of 0.5 seconds, having fragments from 45 to 450 Da. The GC had the running time of 36 min in total at which the relative amount in percentage of each component was calculated using comparison of the average peak area with the total areas. The GC-MS mass spectrum was interpreted using database of National Institute Standard Technology (NIST), which has more than 62000 patterns in their library.

2.5. Antimicrobial Activity Assay

The minimum inhibitory concentration (MIC) of mushroom extracts was determined for Escherichia Coli, Staphylococcus aureus, Candida albicans, and Cryptococcus neoformans using the microplate dilution assay [27]. About 100 μl of nutrient broth was added to all the 96 well microtitter plates. About 100 μl of mushroom extract (5 mg/ml) dissolved in 1% DMSO was thereafter serially diluted throughout the rows in the 96 well microtitter plates. About 100 μl bacterial culture set at 0.5 McFarland standard was then added to all the wells of the 96 well microtitter plates which were incubated at 37°C for 24 hours. The p-Iodo-nitrotetrazolium violet (INT) solution (40 μl of 0.2 mg/ml) was thereafter added into 96 well microtitter plates and incubated at 37°C for 30 min. The red colour indicated the growth of the microorganism, fermenting INT into formazan. MIC was recorded as the lowest concentration of mushroom extract that completely inhibited the microbial growth of organisms.

2.6. Reducing Power Assay

The P. ostreatus ability to reduce free radicals was evaluated using modified method reported by Ayeni et al. [28]. About 2.5 mL of the methanolic mushroom extracts at various concentrations (10–800 μg/ml) were mixed with 0.2 M phosphate buffer (pH 6.6) together with 1% potassium ferricyanide. The mixture was incubated at 50°C for 20 min, and thereafter, 10% trichloroacetic was added, and the solution was centrifuged for 10 min at 1000 rpm. Immediately after centrifugation, 2.5 ml of the upper layer of the solution was taken out and mixed with equal ratio of distilled water and FeCl₃ (0.5 mL, 0.1%). Absorbance was measured at 700 nm (96 well plate reader); hence, higher absorbance indicated better reducing power of mushroom extracts.

2.7. Statistical Analysis of Results

All experiments were repeated independently, in triplicate. Data generated were calculated using SPSS original 6.0 and Graph Pad Prism. The results are reported as mean ± S.E.M. The statistical differences were determined using one-way analysis of variance (ANOVA), followed by Tukey-Kramer multiple comparison test. The values were considered statistically significant where p ≤ 0.05.

3.1. The C/N Ratio of Growing Substrates Together with P. ostreatus Yield

The Pleurotus mushrooms have the greatest advantage of being cultivated in a simple way within tropical climates. Such mushrooms can be grown on both lignin and cellulose rich substrates which are readily obtainable [29]. Even though the Pleurotus mushrooms have such advantages, it should be emphasised that the growth and yield performance of P. otreatus are dependent on the carbon to nitrogen ratio (C/N) of the growing substrates [30]. Hence, previous studies have reported that substrates should be supplemented with nitrogen and carbon source to obtain optimum C/N for the growth of mushroom [31]. Therefore, mushroom growing substrates should be formulated in such a way that they contains balanced C/N ratio [30]. In our study, two different substrates (sugar cane tops and sugar cane bagasse) were supplemented with varying levels of wheat bran (0%, 2%, 14%, 18%, and 20%) in order to obtain an optimum C/N ratio which would improve the growth and yield performance of P. otreatus mushrooms. From the results on Figure 1(a), it is noted that the unsupplemented substrates (0%) and lower levels of WB supplemented substrates (2%) had significantly higher C/N, when compared to higher levels of supplementations (14%, 18% and 20%). This was especially observed for bagasse supplemented with 0% and 2% WB, which had significantly (p < 0.05) higher C/N ratio (96 : 1 and 94 : 1), when compared to bagasse supplemented with 14%, 18%, and 20% WB. Similar trend was also observed for the sugar cane supplemented substrates, with the exception being the fact that sugar cane substrates had lower C/N ratio when compared to bagasse substrates. The obtained C/N ratio for higher levels of supplementation for bagasse substrates was in line with an ideal C/N value reported by Duprat [32] who stated that an ideal C/N value for P. ostreatus grown agro-industrial waste should range from 25 to 50 : 1. The sugar cane substrates had an ideal range for C/N ratio regardless of the effect of wheat bran supplementation. Our study confirms that as C/N ratio decreases, the yield could potentially increase; hence, it was noted that higher levels of supplementation that had lower C/N ration and hence produced better yield (Figure 1(b)), when compared to the lower levels of supplementation which had higher C/N ratio. Therefore, increasing the levels of wheat bran supplement in mushroom growing substrates might have probably resulted in balanced or optimum C/N, hence influencing the yield performance of Pleurotus spp. mushroom. Our findings were in line with the results reported by [33] who stated that substrates with lower C/N ratios result in higher mushroom yield. The C/N ratios that were obtained, were influenced by the level of wheat bran supplemented into the substrates; hence, it was observed that for both substrates, an increase in wheat bran level resulted in the increase in mushroom yield (Figure 1(b)). Such findings corroborates with the findings of [34–36], who reported that an increase supplementation level within the base substrates result in increased mushroom yield, which is of great interest to mushroom farmers.

3.2. The Antioxidant Property of P. ostreatus Mushroom

Mushrooms have been reported to have protective role just like many plants, they potentially protect organisms against oxidative stress; hence, they produce phytocompounds with antioxidant activity, which prevent formation of free radicals, which forms diseases due to the reactive oxygen species (ROS) and reactive nitrogen species (RNS) [37]. Hence, previous authors have stated that the consumption of Oyster mushrooms may protect the human body since Oyster mushrooms have radical scavenging activity [38]. The present study revealed that methanolic extract of P. ostreatus mushroom grown on both unsupplemented and wheat bran supplemented substrates (sugar cane tops and sugar cane bagasse) have antioxidant property. Figures 2(a) and 2(b) indicate that P. ostreatus mushrooms have reducing power, which was concentration-dependent; hence, the reducing power was observed to be increasing as the concentration increased throughout. Such trend of concentration dependency of reducing power of P. ostreatus mushroom is in line with the results obtained by [39], who also demonstrated that the reducing power of methanol extract from five ear mushrooms was dose-dependent. Thus, the observed reducing power (ability) of P. ostreatus mushroom might be due to the fact that mushrooms have the hydrogen donating ability which breaks the free radical chain [40]. When comparing the reducing power of different supplement levels, it was observed from Figures 2(a) and 2(b) that unsupplemented (0%) substrates had higher reducing power followed by lower supplementation (2% WB) compared to the rest of supplemented substrates. The highest reducing power was observed at the concentration of 800 μg/ml, with a reducing power of 42% observed on unsupplemented (0%) sugar cane bagasse. The unsupplemented (0%) sugar cane tops were also observed to have higher reducing power of 61% with a concentration of 800 μg/ml, which was similar to the reducing power of the control namely ascorbic acid (A. A), which also had a reducing power of 61% at 800 μg/ml. Such variations in reducing power of differently supplemented substrates was probably due to the differences in the amount of reductones such as phenolics and flavonoids, which have antioxidant ability through breaking free radical chain by donating a hydrogen atom [41].

3.3. Antimicrobial Activity of P. ostreatus Mushroom Extracts

Besides the antioxidant property (reducing power) of mushrooms, the current study also confirmed that the P. ostreatus mushroom also have the antibacterial and antifungal activity as observed on Tables ​Tables11 and ​and2.2. The results in Tables ​Tables11 and ​and22 reveal that P. ostreatus grown on wheat bran supplemented substrates have the antimicrobial potential. The methanolic extract of P. ostreatus mushroom grown on differently supplemented substrates inhibited Staphylococcus aureus, Escherichia coli, Candida albicans, and Cryptococcus neoformans with MIC values ranging from 2.5 mg/ml to 0.08 mg/ml. The results in Table 1 indicate that mushroom extracts grown on sugarcane tops had moderate antibacterial activity for S. aureus (MIC range from 0.31–0.16 mg/ml) in all levels of WB supplementation, and 20% WB showed moderate antibacterial activity towards C. albicans (0.31 mg/ml). However, mushroom extracts grown from sugarcane bagasse had good antibacterial activity for supplements such as 14% (0.02 mg/ml) and 20% WB (0.08 mg/ml) for E. coli bacteria. Furthermore, moderate antibacterial activity was also noted for sugar cane bagasse on bacteria such as S. aureus (0.31–0.63 mg/ml), C. albicans (0.31–0.63 mg/ml), and C. neoformans (0.16, 0.31, and 0.63 mg/ml). These results indicate that P. ostreatus extract had better activity; hence, the auth [27] have reported that the lower the MIC, the better the activity, and other researchers have stipulated and classified the antimicrobial activity of plant extract as good (MIC < 0.1 mg/mL), moderate (0.1 ≤ MIC ≤ 0.625 mg/mL), and weak (MIC > 0.625 mg/mL) [42]. Thus, such findings support the reports of previous studies which stipulated that Pleurotus ostreatus mushrooms have antibacterial and antifungal properties [43].

In general, it was noted that supplementation of substrates with wheat bran had some impact on the antimicrobial activity (antibacterial and antifungal) of P. ostreatus as observed that the mushrooms grown on unsupplemented substrates (0%) had MIC values which were different to other supplemented substrates; however, other mushrooms grown on supplemented substrates had MIC values which were similar to the MIC values of unsupplemented substrates. For example, it is observed in Table 2 that Staphylococcus areus was equally inhibited by P. ostreatus extract grown on unsupplemented (0%) and supplemented (2% and 18% WB) bagasse substrates since the MIC value of 0.63 mg/ml was noted; however, the only exception was only observed on 14% WB (MIC of 0.16 mg/ml) and 20% WB (MIC of 0.31 mg/ml). Such similarities and differences in terms of inhibitory activities of P. ostreatus extract were probably due to their content in total phenols and flavonoids as Barros et al. [44] had previously stipulated that the antimicrobial activity of different mushrooms was directly correlated with their content of total phenols and flavonoids.

3.4. GCMS Analysis of Compounds within P. ostreatus

The GC-MS results in Tables ​Tables33 and ​and44 also testify that the mushrooms grown on various supplemented substrates have varying and some have similar compounds which might have played a role in both antimicrobial activity and antioxidant activity, which was observed in our study. Therefore, it could be stipulated that the high antibacterial potency observed for mushrooms grown in bagasse supplemented with 14% WB (MIC of 0.02 mg/ml) and 18% WB (MIC of 0.08 mg/ml) could be due to the presence of compounds such as Pentadecane, Hexadecane, Heptadecane, and Phenol, 2,2′-methylenebis [6-(1,1-dimethylethyl), which are known to have antibacterial activity. Furthermore, the GC-MS analysis have detected several compounds from Pleurotus ostreatus mushroom grown on different substrates (sugar cane tops and sugar cane bagasse) with varying levels of wheat bran supplements. Some of these compounds have the known biological activities; however, others do not have known biological activities. The results in Tables ​Tables33 and ​and44 indicate compounds of known activities; hence, compounds which did not have known activities were not included within the Tables.

However, the GCMS screening on Tables ​Tables33 and ​and44 indicates that Pleurotus ostreatus grown on different substrates with various levels of wheat bran supplementation produced many compounds of which some are similar. The mushrooms grown on sugar cane (Table 3) supplemented with 0%, 18%, and 20% WB had similar compounds such as vitamin E compounds namely beta Tocopherol, gamma Tocopherol, and Alpha-Tocopherol, which are known to have antioxidant property [45]. Furthermore, mushrooms grown on sugarcane bagasse (Table 4) supplemented with 0%, 2%, and 20% WB also had vitamin E compounds. Besides the vitamin E compounds, there were plenty of other bioactive compounds detected by GCMS analyzer, including phenols, terpernoids, fatty acids, and fatty acids derivates as profile chromatogram on Figures ​Figures33 and ​and44 have highlighted some of these compounds found on methanolic extract of P. ostreatus mushroom. Compounds such as heptadecane, hexadecane, pentadecane, dibutyl phthalate, cholest-22-ene-21-ol, 3,5-dehydro-6-methoxy- and ß-sitosterol which were detected on some of the Pleurotus ostreatus mushrooms are well known to have antimicrobial activity [46–51]. Such findings prove that some of the activities observed such antioxidant and antimicrobial were mainly due to the presence of the above-mentioned compounds. However, in vivo studies need to be explored to validate such activities.

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