The Influence of Substrate Composition on Nutritional Content and Biological Activity of Some Pleurotus Mushrooms Extracts

Abstract

Oyster mushrooms (Pleurotus spp.) are a nutrient-rich functional food, packed with protein, fiber, and bioactive compounds, offering a broad range of therapeutic qualities. This paper reports the findings in terms of crude protein, crude fiber, total polyphenols, total flavones, and some phenolic compounds along with the antimicrobial and antioxidant activities of some Pleurotus mushrooms extracts: P. eryngii, P. ostreatus, and P. columbinus. The integration of brewery-spent grains (BSG) into the nutrient media and culture substrate induced a major statistical increase (p < 0.05) for crude protein, total polyphenols, total flavones, and chlorogenic and caffeic acids as well as antioxidant activity. The lowest inhibition concentration IC₅₀ was recorded for P. ostreatus, followed by P. eryngii and P. columbinus. Among the strains, only P. ostreatus and P. columbinus exerted antimicrobial activity against two pathogens: Staphylococcus aureus and Streptococcus pyogenes. These results add and provide evidence of oyster mushrooms’ nutritional properties and possible positive effects on human health.

1. Introduction

Edible and medicinal macromycetes (EMMs) represent a valuable alimentary resource that is consumed for their culinary appeal alongside their nutritive and medicinal properties [1]. Intensive mushroom cultivation represents a widely recognized biotechnological process that generates nutrient-dense foods by myco-valorization of the lignocellulosic biomasses and embodies a profitable industry [2,3,4]. High-quality proteins can be synthesized by EMMS more biologically efficiently in comparison to those generated by livestock. In addition, they provide considerable amounts of fiber, mineral elements, and vitamins, are calorie-limited, and contain a large percentage of polyunsaturated fatty acids compared to the overall amount of fatty acids [5,6].

Numerous species of macromycetes have been studied over the years and their biologically active compounds have been effectively used to alleviate or avoid a wide range of medical conditions. The broad health-promoting and therapeutic applications of EMMs are attributed to the content of the metabolic products that have been extracted from mycelia and mushrooms. Among the numerous biologically active molecules synthesized by EMMS, the most frequently reported in the literature are polysaccharides, phenolic compounds, vitamins, terpenoids, proteins, and amino acids, many of which have valuable biotechnological potential that can be applied in the food, pharmaceutical, or medical industries [7,8,9]. These biocompounds possess antioxidant, antidiabetic, antineoplastic, immunostimulatory, and an array of other properties that favor human health [10,11,12]. Pleurotus mushrooms have been reported for their antioxidant, hypocholesterolemic, hypoglycemic, and hypolipidemic properties alongside their antitumor activity against cancer cell proliferation [13,14]. Among the biocompounds found in EMMS, phenolic compounds attract great interest considering their antioxidant, anti-inflammatory, or antitumor qualities [15].

Bacterial resistance to first-choice antibiotics has risen substantially considering the vast array of antimicrobial substances. EMMs possess significant antimicrobial properties due to their bioactive compounds, which have been demonstrated to prevent the growth of different bacterial and fungal pathogens, making them a promising substitute for developing antimicrobial products for food safety and healthcare. Over the past few years, renewable supplies have been investigated and Pleurotus mushrooms might represent a potential source for novel antimicrobials [16,17]. Endogenous factors including reactive oxygen species (hydroxyl radicals, superoxide anion radicals, hydrogen peroxide, nitric oxide radicals, etc.) and exogenous factors such as smoking, ionizing radiation, pollution, and pesticides can cause oxidative stress, which might affect proteins, enzymes, nucleic acids, and other molecules, impairing normal functions and leading to various diseases. Pleurotus spp. mushrooms are known to exert an antioxidant activity that is more pronounced in comparison with other therapeutic characteristics [18,19].

Lignocellulosic residues have tremendous potential for producing various goods used in a multitude of applications such as the food, beverage, and feed industries, textiles, obtaining antioxidants, enzymes, and low-cost chemicals, etc. [20,21]. P. ostreatus is grown on a vast array of lignocellulosic materials, and its global cultivation has been approximated to be over 4.1 million tons in the past few years [22], as the management of these organic resources can be implemented by cultivating this group of superior fungi in an ecologically and financially feasible approach [23].

Consequently, this research was carried out to assess the effect that a BSG-enriched substrate might have on the chemical quality of mushrooms by evaluating the nutritional content alongside the antioxidant and antimicrobial capacities of three Pleurotus spp. mushrooms extracts. The research was carried out between 2023 and 2024.

2. Materials and Methods

2.1. Biologic Material

The biological material belongs to the fungal germoplasm collection of the RDIVFG Vidra, Romania, employing the mycelia of three Pleurotus species: P. eryngii strain PeM-41, P. ostreatus strain PoM-77, and P. columbinus strain PcM-98.

2.2. Mushroom Cultivation

The technological flow of in vitro propagation and fruiting of mycelia was identical to that presented by Rusu et al. [24], from which the best results provided the framework for investigating the effect on the chemical content of mushrooms generated through various agro-industrial by-products that were integrated in the composition of both nutrient media and culture substrates in relation to the method of obtaining the spawn. In this paper, P. ostreatus and P. columbinus employed liquid inoculum on granular support in order to generate the grain spawn, while P. eryngii strain relied on solid inoculum on granular support to produce the spawn, since these spawn variations were the most productive, leading to better harvests. For fruiting, the control sample (V0) consisted of 100% wheat straws versus the V1 sample, which consisted of 75% wheat straws + 25% BSG. Both substrate variants were mixed with 2% CaCO₃, 6% CaSO₄ and supplemented with wheat bran, sunflower middlings, and maize bran, 3% each. Calcium-based amendments and nutritional supplements were calculated to the wet substrate.

2.3. Sample Preparation

After harvesting, the mushrooms had their stipes separated from the pileus and then were oven-dried at 37 °C for 36 h. After this step, the dried biomasses were grounded to fine powders, distributed into properly sealed Falcon tubes and labeled (

Table 1. Sample labeling.

Sample	Species/Strain	Substrate Variant	Morphological Part
P1	Pleurotus eryngii PeM-41	V0	Stipes
P2	Pleurotus eryngii PeM-41	V1	Stipes
P3	Pleurotus eryngii PeM-41	V0	Pileus
P4	Pleurotus eryngii PeM-41	V1	Pileus
P5	Pleurotus ostreatus PoM-77	V0	Stipes
P6	Pleurotus ostreatus PoM-77	V1	Stipes
P7	Pleurotus ostreatus PoM-77	V0	Pileus
P8	Pleurotus ostreatus PoM-77	V1	Pileus
P9	Pleurotus columbinus PcM-98	V0	Stipes
P10	Pleurotus columbinus PcM-98	V1	Stipes
P11	Pleurotus columbinus PcM-98	V0	Pileus
P12	Pleurotus columbinus PcM-98	V1	Pileus

).

2.4. Crude Protein and Crude Fiber Content Determination

Total nitrogen (N) and crude protein content (CPC) (N_6.25) were evaluated by the Macro Kjeldahl Assay [25]. The content of crude fiber (CFC) of the samples was determined through the FiberBags filtration system (from Gerhardt Analytical Systems, Königswinter, Germany) in accordance with the method of Oprea et al. [26].

2.5. Total Phenolic Content Analysis

The content of total phenolic compounds (TPC) was evaluated using a modified Folin–Ciocâlteu method, following the protocols by Arlet et al. [27] and Mulțescu et al. [28]. After adding 0.5 mL of Folin–Ciocâlteu reagent to the diluted samples, they were left to incubate in the dark for 8′. Following the addition of a 7.5% sodium carbonate solution, the samples were incubated for two additional hours in the dark. The measurement of absorbance was taken at 765 nm. Gallic acid solutions with concentrations ranging from 0.005 to 0.5 mg/mL were used to generate a standard curve. The TPC was reported as milligrams of gallic acid equivalents (mg GAE) per gram of the sample’s dry mass.

2.6. Total Flavonoid Content Evaluation

The content of total flavonoid compounds (TFC) was determined by employing a modified aluminum chloride complexation assay [29,30]. After being diluted with 10% sodium acetate, the samples were centrifuged, filtered, and combined with 2 milliliters of a 2.5% aluminum chloride solution. After 45 min of dark incubation, the mixture’s absorbance at 420 nm was measured. A standard curve was generated using rutin solutions with concentrations ranging from 0.01 to 0.16 mg/mL. TFC was expressed as milligrams of rutin equivalents (mg RE) per gram of dry mass of the examined sample.

2.7. Phenolic Compounds by High-Performance Liquid Chromatography Analysis

The assessment of individual phenolic compounds was performed according to the protocol described by Pascariu et al. [31]. Reversed-phase separation was achieved using a 5 µm SunFire Column (3.9 × 150 mm) with a binary elution gradient of 0.5% orthophosphoric acid in ultrapure water (A) and acetonitrile (B) at 40 °C. Chromatograms were recorded at 300 nm wavelength. Before separation, the samples were diluted, filtered through 0.45 µm membrane syringe filters, and 2 µL was injected. Phenolic compounds were identified by comparing their retention times to those obtained by injecting standard solutions (4.27′ for gallic acid, 9.70′ for chlorogenic acid, 12.19′ for caffeic acid, 13.01′ for syringic acid, 17.43′ for 4-coumaric acid, 19.03′ for rutin, and 24.31′ for quercetin). Peak area was calculated for the analyte quantification sample using the calibration curves of the corresponding standards (r2 was 0.9949 for gallic acid, 0.9989 for chlorogenic acid, 0.9992 for caffeic acid, 0.9979 for syringic acid, 0.9989 for p-coumaric acid, 0.9994 for rutin, and 0.9909 for quercetin).

2.8. Antimicrobial Activity

The working protocols were after Balouiri et al. [32]. Briefly, the extracts were prepared by weighing 1 g of each sample to which 10 mL EtOH (50%) was added, subsequently being ultrasonicated for 30′ and centrifuged at 6000 rpm for 10′. The extracts were filtered using 0.22 μm Millipore filters. The well diffusion method was used in order to evaluate the extracts’ antimicrobial activity. The pathogenic reference strains were inoculated into soybean tryptic agar (TSA, melted and cooled at 45 °C) and distributed in Petri dishes. After solidification, 13 (12 samples + 1 control) wells (6–7 mm in diameter, 3–4 cm apart) were made in the media containing the pathogen strain using a sterile device. In each well, 100 µL of extract was added, and the Petri dishes were incubated at 37 °C for 24–48 h.

The antimicrobial activity was tested against a number of bacteria and yeasts, namely: Bacillus cereus ATCC 11778, Enterococcus faecalis ATCC 29212, Enterococcus faecium ATCC 6057, Enterococcus hirae ATCC 10541, Listeria innocua ATCC 33090, Listeria ivanovii ATCC 19119, Listeria monocytogenes ATCC 7644, Staphylococcus aureus ATCC 33592, Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis ATCC 51625, Staphylococcus epidermidis ATCC 12228, Streptococcus pyogenes ATCC 19615, Rhodococcus equi ATCC 8939, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 27853, Salmonella enterica subsp. typhimurium ATCC 14028, Salmonella enterica subsp. enteritidis ATCC 13076, Serratia marcescens ATCC 14756, Candida albicans ATCC 10231, Candida glabrata ATCC 2001, Candida parapsilopsis ATCC 20019, and Candida tropicalis ATCC 44508.

The microdilution technique was used to determine the minimum inhibitory concentration (MIC). Reference strains were grown in soybean casein broth at 30–35 °C for 24 h. The viable load was determined by the modified colony forming unit technique (CFU/mL) and considering that 10 μL was inoculated, the calculation formula was:

$$\mathrm{N}=m\times c\ast 100$$

where

• N = CFU-colony forming units or number of viable cells per mL;
• m = arithmetic means of colonies measured using three plates that were inoculated with the same dilution;
• c = dilution inverse factor from which inoculation was performed;
• 100 = result ratio to 1 mL.

A series of Eppendorf tubes each containing 50 µL culture medium (Mueller–Hinton broth) was prepared. The first Eppendorf tube contained only 50 µL extract with a concentration of 0.2 µg/mL. In the following tubes, the extract was diluted by a dilution factor of 2 directly into the Mueller–Hinton liquid culture medium. In the penultimate tube, no extract was added as a positive control. In the last tube, the standardized inoculum of microorganisms was not added as the negative control. All prepared tubes were inoculated with 5 µL standardized inoculum (except the last tube). The Eppendorf tubes were incubated at the optimum growth temperature for 24 h. The minimum bactericidal concentration (MBC) represents the minimum amount of antibiotic capable of destroying ≥ 99.99% of the bacteria. From each dilution, a volume of 10 µL was deposited as a drop (“spot”) on TSA medium, distributed in Petri plates, and incubated at 37 °C for 24–48 h.

2.9. Antioxidant Activity

The total antioxidant activity was determined by employing a protocol adapted after Irimescu et al. [33], in which DPPH (2,2-diphenyl-1-picrylhydrazyl), a stable free radical, is reduced by antioxidants, a reaction that is observed with the change in color from deep violet to yellow or colorless [34]. The assay involved using different concentrations of samples combined with 0.1 M DPPH solution (prepared in 80% methanol) and incubated in the dark for 30 min. The absorbance of the sample was measured at 515 nm following the incubation period, with the DPPH solution serving as a control. The antioxidant activity was expressed as a radical scavenging activity (% RSA) employing the calculation:

$$\%\mathrm{RSA}=(1-{\displaystyle \frac{A sample}{A control}})\ast 100$$

RSA was used for calculating the IC₅₀ value for each sample, which represents the concentration of the sample required to scavenge 50% of DPPH free radicals.

2.10. Statistical Data Analysis

Every test was run in triplicate, and three samples were used. Values for extraction yield, bioactive compounds, and antioxidant activity were reported as mean ± SD. Using GraphPad Prism 8.0.1 software (San Diego, CA, USA), a one-way analysis of variance (ANOVA) was used to determine the statistical significance of the data. Post hoc Tukey’s multiple comparison tests with α = 0.05 were then conducted.

3. Results

3.1. Crude Protein and Crude Fiber Content

The substrate variants had a substantial impact (p < 0.05) on the cpc (Figure 1), with the computed p-value nearly equal to 0.01. For P. eryngii and P. ostreatus, the BSG-enriched substrate led to differences of at least +40% for both morphological parts, while P. columbinus led to variations of about +30%. The CPC was highest for P. ostreatus with an average of 34.47 ± 9.02% d.w., followed by P. columbinus with 25.54 ± 6.59% d.w. and P. eryngii with 19.48 ± 7.31% d.w. For all samples, the CPC of pileus was higher than that of stipe.

Substrate variations did not statistically (p > 0.05) affect the CFC (Figure 1). The CFC recorded the highest values for P. columbinus with an average of 14.95 ± 2.11% d.w., followed by P. ostreatus with 12.59 ± 1.82% d.w. and P. eryngii with 11.14 ± 1.37% d.w. In contrast to the other two strains, which displayed a higher CFC in the stipes, P. eryngii had a higher CFC at the pileus level.

3.2. Phenolic and Polyphenol Compounds Content

TPC (Figure 2) was statistically significant (p < 0.05), improved by the substrate variations. The BSG-supplemented substrate led to differences of +18% and +22% for the stipes and pileus of P. eryngii, differences of +36% and +45% for P. ostreatus and differences of −11% and +17% for P. columbinus. The TPC recorded the highest values for P. ostreatus with an average of 42.32 ± 11.82 mg GAE/g d.w., followed by P. columbinus with 30.17 ± 5.94 mg GAE/g d.w. and, finally, P. eryngii with 24.55 ± 7.99 mg GAE/g d.w. For all three strains, pileus had a higher TPC than stipes.

TFC (Figure 2) was statistically significant (p < 0.05) impacted by the substrate composition. The BSG-supplemented substrate led to differences of +33% and +22% for the stipes, respectively, pileus of P. eryngii, variations of +28% and +31% for P. ostreatus, and differences of −31% and −12% for P. columbinus. The TFC was highest for P. ostreatus with an average of 25.54 ± 3.76 mg RE/g d.w., followed by P. columbinus with 22.92 ± 3.65 mg RE/g d.w. and P. eryngii with 10.48 ± 7.02 mg RE/g d.w. P. eryngii displayed the highest TFC in the pileus, for P. ostreatus it was almost identical between the two morphological parts, while for P. columbinus, it varied according to the substrate variant, higher for stipes on V0 and higher for pileus on V1.

The gallic acid content (

Table 2. Phenolic compounds by HPLC analysis.

Compound	Gallic Acid	Chlorogenic Acid	Caffeic Acid	Syringic Acid	p-Coumaric Acid	Rutin	Quercetin
Sample	μg/g d.w.	μg/g d.w.	μg/g d.w.	μg/g d.w.	μg/g d.w.	μg/g d.w.	μg/g d.w.
P1	113.35 ± 1.88	0.866 ± 0.07	0.10 ± 0.04	0.50 ± 0.04	0.16 ± 0.01	1.50 ± 0.01	<LoD
P2	84.57 ± 0.04	0.56 ± 0.06	0.89 ± 0.05	0.49 ± 0.03	0.16 ± 0.02	1.60 ± 0.0	<LoD
P3	119.62 ± 0.86	0.66 ± 0.12	1.36 ± 0.05	0.10 ± 0.01	0.97 ± 0.07	1.25 ± 0.22	<LoD
P4	121.22 ± 3.11	0.60 ± 0.01	1.05 ± 0.05	1.47 ± 0.07	2.33 ± 0.09	1.12 ± 0.05	<LoD
P5	140.27 ± 1.55	4.84 ± 0.45	1.83 ± 0.07	1.74 ± 0.32	1.03 ± 0.03	1.45 ± 0.06	0.15 ± 0.04
P6	121.60 ± 8.46	4.88 ± 0.13	1.71 ± 0.04	1.97 ± 0.02	1.95 ± 0.03	0.53 ± 0.08	0.16 ± 0.01
P7	155.32 ± 4.21	11.06 ± 0.28	3.38 ± 0.05	1.55 ± 0.24	1.73 ± 0.22	0.70 ± 0.02	0.47 ± 0.07
P8	183.33 ± 2.18	9.42 ± 0.02	2.45 ± 0.06	1.52 ± 0.10	6.95 ± 0.01	0.68 ± 0.23	0.57 ± 0.02
P9	126.50 ± 2.62	2.58 ± 0.08	1.52 ± 0.27	1.97 ± 0.01	0.62 ± 0.08	0.25 ± 0.07	0.47 ± 0.064
P10	89.41 ± 0.96	2.06 ± 0.01	0.10 ± 0.01	0.51 ± 0.01	0.81 ± 0.04	0.03 ± 0.03	2.65 ± 0.11
P11	146.90 ± 1.90	3.09 ± 0.06	2.28 ± 0.04	0.19 ± 0.19	2.13 ± 0.06	0.98 ± 0.08	1.37 ± 0.19
P12	104.25 ± 5.38	2.22 ± 0.08	1.67 ± 0.05	0.16 ± 0.16	1.58 ± 0.01	0.46 ± 0.01	2.12 ± 0.01

LoD = Limit of Detection.

) was not statistically influenced (p > 0.05) by the substrate variants. Of the five phenolic acids quantified, gallic acid had by far the highest values, tens or, in some cases, several hundred times higher in comparison. The gallic acid content displayed the largest values for P. ostreatus with an average of 150.13 ± 26.08 μg/g d.w., pursued by P. columbinus with 116.77 ± 25.22 μg/g d.w. and P. eryngii with 109.69 ± 17.09 μg/g d.w. For all the samples, the gallic acid content was higher in the pileus compared to the stipes.

The chlorogenic acid content (Table 2) was statistically significantly influenced (p < 0.05) by the substrate variants, thus V1 produced the lowest values. The BSG-enriched substrate variant led to differences of −35%, respectively, −10% for the stipes and pileus of P. eryngii, variations of +1%, respectively, −15% for P. ostreatus, while P. columbinus displayed differences of −20%, respectively, −28%. The chlorogenic acid content had the highest values generated by P. ostreatus with an average of 7.55 ± 3.18 μg/g d.w., followed by P. columbinus with 2.49 ± 0.46 μg/g d.w. and P. eryngii with 0.67 ± 0.14 μg/g d.w. The chlorogenic acid content was higher in the pileus compared to the stipe for P. ostreatus and P. columbinus strains.

The caffeic acid content (Table 2) was statistically significantly influenced (p < 0.05) by the substrate variants; thus, the control samples led to better results compared to V1 for all strains. The BSG-enriched substrate led to negative differences of −11% for stipes and −22% for pileus for P. eryngii, variations of −7% and −27% for P. ostreatus, and differences of −34% and −27% for P. columbinus. The caffeic acid content showed the highest values for P. ostreatus with an average of 2.34 ± 0.77 μg/g d.w., pursued by P. columbinus with 1.62 ± 0.53 μg/g d.w. and P. eryngii with 1.07 ± 0.20 μg/g d.w. Caffeic acid content was higher at the pileus level compared to stipes for all three species.

Syringic acid content (Table 2) was not statistically significantly influenced (p > 0.05) by substrate variants. The syringic acid content had the highest values produced by P. ostreatus with an average of 1.69 ± 0.21 μg/g d.w., followed by P. eryngii with 0.87 ± 0.47 μg/g d.w. and P. columbinus with 0.71 ± 0.86 μg/g d.w. The syringic acid content was higher in the pileus for P. eryngii and in the stipe for P. ostreatus and P. columbinus.

The p-coumaric acid content (Table 2) was not statistically significantly influenced (p > 0.05) by the substrate variants. The p-coumaric acid content showed the highest values produced by P. ostreatus with an average of 2.91 ± 2.72 μg/g d.w., followed by P. columbinus with 1.28 ± 0.70 μg/g d.w. and, finally, P. eryngii with 0.90 ± 1.02 μg/g d.w. The p-coumaric acid content was higher at the pileus level for all samples.

Rutin content (Table 2) was not statistically significantly influenced (p > 0.05) by substrate variants. The rutin content recorded the highest values for P. eryngii with an average of 1.37 ± 0.22 μg/g d.w., followed by P. ostreatus 0.84 ± 0.41 μg/g d.w. and, finally, P. columbinus with 0.43 ± 0.41 μg/g d.w. The rutin content was higher in the stipe compared to the pileus for most samples.

Quercetin content (Table 2) was not statistically significantly influenced (p > 0.05) by the substrate variants and could not be quantified for P. eryngii, being below the detection limit. The quercetin content recorded the highest values for P. columbinus with an average of 1.65 ± 0.95 μg/g d.w. followed by P. ostreatus with 0.34 ± 0.21 μg/g d.w. Quercetin content was higher at the pileus level for most samples.

3.3. Antimicrobial Activity

Of the three strains observed, only P. ostreatus and P. columbinus exerted antimicrobial activity against two pathogens, namely: Staphylococcus aureus ATCC 6538 and Streptococcus pyogenes ATCC 19615. The zones of inhibition were circular, very evident, and of different sizes, directly proportional to the antimicrobial activity exerted.

Table 3. Inhibition zones growth (mm) and their significance.

Sample	P1	P2	P3	P4	P5	P6	P7	P8	P9	P10	P11	P12
Pathogen
S. aureus ATCC 6538 S. pyogenes ATCC 19615	- -	- -	- -	- -	4 + 13 +++	7 ++ 13 +++	6 ++ 10 +++	8 ++ 15 +++	5 ++ 14 +++	3 + 8 ++	11 +++ 12 +++	7 ++ 6 ++

- 0 mm; + 1–5 mm; ++ 5–10 mm; +++ >10 mm.

displays the growth and significance of the inhibition zones while

Table 4. Minimal Bactericidal Concentration (MBC, mg/mL).

Sample	P1	P2	P3	P4	P5	P6	P7	P8	P9	P10	P11	P12
Pathogen
S. aureus 6538	-	-	-	-	6.25	6.25	1.25	6.25	6.25	6.25	6.25	1.25
S. pyogenes 19615	-	-	-	-	1.25	1.25	6.25	1.25	1.25	-	1.25	6.25

displays the MBC.

The antimicrobial activity was not statistically influenced (p > 0.05) by substrate variants. Against S. aureus, it was more strongly exerted by the extracts prepared from pileus for both Pleurotus strains. As regards S. pyogenes, antimicrobial activity displayed higher values on most of the extracts from stipes.

3.4. Antioxidant Activity

The antioxidant capacity was statistically significantly impacted (p < 0.05) by the substrate variants; hence, the V0 control variant led to IC₅₀ (Figure 3) higher values compared to the V1 variant, with higher values representing a lower antioxidant activity. BSG-enhanced substrate resulted in differences of −30%, respectively, −44% for the stipes and pileus of P. eryngii, −10% and −42% for P. ostreatus, respectively, −8% and −37% for P. columbinus. The lowest inhibition concentration IC₅₀ was recorded for P. ostreatus with an average of 9.82 ± 2.39 mg/mL, followed by P. eryngii with 11.48 ± 4.83 mg/mL and, finally, P. columbinus with 14.26 ± 5.06. mg/mL. Extracts from pileus led to lower IC₅₀ index values compared to those prepared from stipes.

4. Discussion

The values determined for CPC were fairly similar to those found and reported by other researchers, such as Cueva et al. [35] for P. ostreatus cultivated on several lignocellulosic substrates or Irshad et al. [36] for P. ostreatus and P. columbinus. For P. eryngii cultivated on wheat straws, Krüzsely et al. [37] reported a 30.25 ± 0.26% d.w. CPC, while Ogundare et al. [38] reported a CPC of 23.07 ± 0.40% d.w. (P. ostreatus), 25.74 ± 0.34% d.w. (P. columbinus) and 24.66 ± 0.33% d.w. (P. eryngii).

For P. eryngii generated on wheat straws, Krüzsely et al. [37] reported a CFC of 12.7 ± 0.71% d.w., while for P. columbinus cultivated on grass hay and wheat straws, Tshinyangu [39] reported a CFC ranging between 17.9 and 19.7 ± 1.12% d.w. Ogundare et al. [38] reported a CFC of 2.00 ± 0.11% d.w. (P. ostreatus), 1.09 ± 0.14% d.w. (P. columbinus), and 2.22 ± 0.17% d.w. (P. eryngii).

Lower amounts of TPC were reported by Rezaeian et al. [40], with quantities of 1.9 ± 0.37 mg GAE/g d.w. for P. ostreatus and 2.5 ± 0.25 mg GAE/g d.w. for P. eryngii. For P. columbinus, Elhusseiny et al. [41] found and reported a TPC of 22.50 ± 1.53 mg GAE/g d.w.

Lower amounts of TFC were reported by El-Razek et al. [42] for P. ostreatus depending on the substrate variant used, values of 2.53 ± 0.07 mg RE/g d.w., respectively, 2.83 ± 0.02 mg RE/g d.w., while Gąsecka et al. [13] reported a TFC of 2.11 ± 0.19 mg RE/g d.w. for P. ostreatus generated on wheat straws and 1.26 ± 0.17 mg RE/g d.w. for P. eryngii generated on a blend of flax shives and beech sawdust (3:1).

The level of different phenolic compounds reported for Pleurotus sp. varies a lot according to the species, substrate, and cultivation condition. Larger amounts of gallic acid found in P. ostreatus mushrooms have been reported by other authors, with values up to 290.34 µg/g d.w. (Palacios et al. [43] or 333.37 µg/g d.w. Jabłońska-Ryś et al. [44]. Kim et al. [45] reported a chlorogenic acid content of 19 μg/g d.w. for P. ostreatus while for P. columbinus, Angelini et al. [46] reported amounts of 1.62 ± 0.13 µg/g d.w. cultivated on wheat straws in a 4:2:1 w/w/w ratio with oak sawdust and coffee grinds and 1.10 ± 0.01 µg/g d.w. on wheat straws in 3:2:1 w/w/w ratio with beech sawdust and soybeans. Radzki et al. [47] reported a caffeic acid content of 0.12 ± 0.01 µg/g d.w. for P. ostreatus while Matkovits et al. [48] reported values ranging from <0.01 to 1.54 ± 0.54 µg/g d.w. for a total of 14 P. ostreatus cultivars. Radzki et al. [47] reported a syringic acid content of 0.09 ± 0.01 µg/g d.w. for P. eryngii and 0.10 ± 0.00 µg/g d.w. for P. ostreatus while Matkovits et al. [48] reported amounts of syringic acid smaller than 0.05 µg/g d.w. for all 14 P. ostreatus cultivars examined. Gąsecka et al. [13] reported higher amounts of p-coumaric acid, with values of 7.17 ± 0.23 µg/g d.w. and 9.12 ± 0.12 µg/g d.w. for P. ostreatus and 6.49 ± 0.22 µg/g d.w. and 8.00 ± 0.18 µg/g d.w. for P. eryngii, while Reis et al. [49] reported much lower amounts of p-coumaric acid, with values recorded as 0.81 ± 0.03 µg/g d.w. for P. ostreatus and 1.04 ± 0.04 µg/g d.w. for P. eryngii. For all the four P. ostreatus strains examined, Vamanu [50] did not record any values for rutin while Jayakumar et al. [51] recorded a content of 31.20 mg/100 g. For both P. ostreatus and P. eryngii, Akyüz et al. [52] reported a 0.25 µg/g d.w. quercetin content.

Phenolic compounds, including flavones and phenolic acids, exhibit antimicrobial activity by disrupting microbial membranes, inhibiting enzymes, generating oxidative stress, or preventing biofilm formation. Their effectiveness varies based on structure and microorganism type, making them valuable natural antimicrobial agents [53]. Many articles published by different researchers reported the antimicrobial activity of Pleurotus mushrooms against several pathogens, such as Khalil et al. [54], Akyüz et al. [55], Younis et al. [56], and Singh et al. [57].

The IC₅₀ index values determined in this paper are in the range of those reported by other studies. A level of 8.88 mg/mL was reported by Arbaayah et al. [58] for P. ostreatus while for P. columbinus, Elhusseiny et al. [41] recorded an IC₅₀ value of 35.13 ± 3.27 mg/mL alongside Angelini et al. [46], which, depending on the culture substrate used, obtained much lower values as 2.25 and 4.98 mg/mL.

5. Conclusions

The study reveals that oyster mushroom strains differed in their therapeutic potential, with P. ostreatus having the strongest antioxidant activity and only P. ostreatus and P. columbinus displaying antimicrobial effects. Furthermore, antimicrobial testing was limited to a couple of species, requiring broader research on other pathogens. While BSG-supplemented substrate enhanced the mushrooms’ nutritional value, its large-scale feasibility and sustainability remain yet to be assessed. The study does not examine long-term safety, particularly regarding potential risks from consuming BSG-grown mushrooms. Since the findings are based on specific strains and substrates, further investigation is necessary to establish whether these benefits are more widely applicable.