Gamma-Polyglutamic Acid Reduces Heavy Metal Uptake and Stabilize Microbial Biosafety in Edible Mushroom Cultures

Abstract

This study evaluated the influence of gamma-polyglutamic acid (γ-PGA) on the heavy metal sorption efficiency of three edible mushroom species—Pleurotus ostreatus, Agaricus bisporus, and Boletus edulis—exposed to cadmium (Cd), lead (Pb), and their mixtures (in a 1:1 ratio). The experiment presented an innovative approach—combining the natural sorption properties of mushrooms with the addition of γ-PGA, which has strong chelating properties. Sorption assays were performed using escalating γ-PGA concentrations (1, 2, and 5 mL to 250 mL of liquid medium), with data analyzed via generalized linear models employing a gamma distribution and a log link function. Results revealed that sorption efficiency was highest at the lowest γ-PGA concentration and decreased significantly with increasing γ-PGA levels across all species and metal treatments. Notably, P. ostreatus and A. bisporus demonstrated superior and more stable sorption capacities relative to B. edulis, which exhibited greater variability. These findings suggest that while edible mushrooms are effective biosorbents for heavy metals, higher γ-PGA concentrations may inhibit metal uptake by chelating metals in solution and reducing their bioavailability. Additionally, γ-PGA at 1 mM markedly enhanced antimicrobial activity against Salmonella enteritidis and Escherichia coli (with a 20% increase in growth inhibition zone compared to the control group) in mushroom cultures and when applied independently. This dual functionality underscores the potential of γ-PGA–mushroom systems for bioremediation and food safety applications, highlighting the need for the careful optimization of γ-PGA concentration to maximize biosorption efficacy and antimicrobial benefits.

1. Introduction

Heavy metal contamination poses a significant threat to environmental and human health due to the persistence, toxicity, and bioaccumulation of metals in ecosystems [1], particularly with food chains, with cadmium (Cd) and lead (Pb) being significant toxicants that impact the environment and the health of living organisms, even at low concentrations [2]. Conventional remediation techniques, including chemical precipitation and ion exchange, are often cost-prohibitive and may generate secondary pollution [3]. In this context, biosorption, the passive sequestration of contaminants by biological materials (through various physicochemical mechanisms, including adsorption, ion exchange, complexation, or electrostatic bonds), has emerged as a promising, eco-friendly alternative for the removal of heavy metals from contaminated environments [4].

Edible mushrooms are increasingly recognized for their remarkable biosorption capacities, attributed to the abundance of functional groups (e.g., carboxyl, amino, and phosphate) in their cell wall polysaccharides and proteins, which can bind metal ions effectively [5]. Species such as Pleurotus ostreatus, Agaricus bisporus, and Boletus edulis have been widely studied for their metal uptake abilities, making them valuable candidates both for bioremediation [6] and as indicators of environmental pollution [7]. The demonstrated ability of edible mushrooms, namely Pleurotus ostreatus, Agaricus bisporus, and Boletus edulis to sorb heavy metals such as Pb and Cd, highlights their potential utility as sustainable and cost-effective biosorbents in bioremediation technologies. Biosorption by fungi is increasingly recognized as a promising alternative to conventional heavy metal removal methods due to its eco-friendly nature, specificity, and capacity to operate under mild conditions [8,9].

In parallel, gamma-polyglutamic acid (γ-PGA), a naturally occurring [10,11], biodegradable polymer produced by certain Bacillus species [12,13], has attracted attention for its strong metal-chelating properties [14,15]. γ-PGA is capable of forming stable complexes with various metal ions, potentially affecting the bioavailability and mobility of metals in the environment [13]. While γ-PGA has been explored for its potential to immobilize toxic metals and reduce their bioavailability, its impact on the biosorption efficiency of edible fungi remains unclear. Understanding the interaction between γ-PGA and mushroom biosorption [16] is crucial for optimizing bioremediation strategies and ensuring food safety [17], especially given the growing use of mushrooms in both environmental and dietary applications [18,19,20,21]. This study aimed to evaluate the effect of different concentrations of γ-PGA on the sorption efficiency of Cd and Pb by three edible mushroom species, providing insights into the implications for bioremediation and the safety of mushroom-derived foods.

Moreover, the enhanced antimicrobial efficacy against Salmonella enteritidis and Escherichia coli observed at low γ-PGA concentrations reinforces the feasibility of employing γ-PGA–mushroom systems not only for detoxification but also for improving microbial safety in mushroom cultivation and related food products [22,23]. Controlling pathogenic bacterial contamination is critical for public health and for maintaining product quality in the edible mushroom industry, where humid cultivation conditions can favor bacterial proliferation. The ability of γ-PGA to inhibit key pathogens aligns with prior reports confirming its antimicrobial properties and supports its application as a biocontrol agent in agricultural and food safety contexts [10,24].

Bacterial biosafety is a significant concern in the cultivation of edible mushrooms due to the susceptibility of mushroom-growing environments to bacterial contamination [20]. Mushrooms are cultivated in humid, nutrient-rich conditions, which can promote the proliferation of both beneficial and harmful bacteria. During routine food safety investigations in the UK, Salmonella enterica serovar Kedougou was found in commercially grown mushrooms, as well as in mushroom compost and casing materials [21]. No human illness was recorded. However, the incident highlighted how non-sterile cultivation inputs, such as sugar beet lime (used as a substrate additive), could introduce Salmonella enteritidis into the mushroom production chain. Studies on pre-cut, ready-to-eat sliced mushrooms from the Italian market revealed high levels of Escherichia coli, with counts exceeding 5 log CFU/g—well above regulatory limits for such products [22]. These findings signal poor hygienic conditions during processing associated with the direct or indirect fecal contamination of substrates and indicate that E. coli contamination can originate both pre- and postharvest due to contact with contaminated soil, water [23], or handling equipment [24,25]. Studies across several countries confirm the susceptibility of mushrooms to acquiring E. coli at various stages from cultivation through market handling [26]. The microbial infestation by pathogenic microorganisms also influence yield, product quality, and consumer safety, making strict bacterial management essential in commercial mushroom cultivation [27,28,29].

This study investigated the dual functionality of gamma-polyglutamic acid (γ-PGA) in the biosorption of heavy metals and microbial biosafety enhancement within edible mushroom cultures. The research focused on three widely cultivated mushroom species—Pleurotus ostreatus, Agaricus bisporus, and Boletus edulis—selected for their distinct morphological and physicochemical characteristics relevant to metal uptake. The primary objective was to assess the impact of varying concentrations of γ-PGA on the sorption efficiency of cadmium (Cd), lead (Pb), and their binary mixtures by these species. It was hypothesized that all three mushrooms would demonstrate substantial sorption capabilities, albeit with species- and metal-specific differences. A further hypothesis posited that elevated concentrations of γ-PGA would decrease metal uptake due to its potent metal-chelating properties, which reduced the bioavailability of free metal ions in the growth medium. Notwithstanding this inhibitory effect, the extent of the reduction was expected to vary in relation to mushroom species and metal treatment.

In addition to metal sorption, the investigation extended to evaluating the protective role of γ-PGA on fungal biomass growth under heavy metal stress. It was anticipated that γ-PGA supplementation may mitigate metal toxicity, thereby sustaining or enhancing biomass production. To elucidate potential human health implications, metal bioavailability from mushroom biomass would be examined under simulated gastrointestinal digestion, determining whether γ-PGA modulated metal release. The study also explored the antimicrobial efficacy of γ-PGA when administered alone and in combination with mushroom cultures against two significant foodborne pathogens: Salmonella enteritidis ATCC 13076 and Escherichia coli ATCC 25922. It was expected that γ-PGA enhanced antimicrobial activity in a manner dependent on the interaction between bacterial species, fungal host, and polymer concentration. By integrating these objectives, this research aimed to provide comprehensive insights into the multifunctional role of γ-PGA in improving the safety and sustainability of edible mushroom production. The findings will inform strategies for optimizing γ-PGA application to simultaneously maximize heavy metal biosorption and microbial control, thus contributing to the broader fields of environmental biotechnology and food safety.

2. Materials and Methods

The scheme of the experiment

In order to investigate the effect of poly-gamma glutamic acid (γ-PGA) addition on the efficiency of permanent binding of toxic metals (Cd and Pb) to reduce their availability to the mycelium and consequently to the human body, the experimental project was divided into four main stages. The research scheme is presented in Figure 1:

Stage 1: Preparation

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Step 1: Cultivation of in vitro mushroom mycelia with or without metals.

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Step 2: Biosynthesis and purification of γ-PGA.

Stage 2: Treatment

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Cultivation with metals and increasing γ-PGA concentrations (1, 2, 5 mL/250 mL medium).

Stage 3: Analysis

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Sorption and desorption efficiency of metals in biomass.

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Bioavailability assessment via artificial digestive juices (gastric and intestinal phases).

Stage 4: Antimicrobial testing

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Kirby–Bauer disk diffusion assay with Salmonella enteritidis and E. coli strains on mushroom cultures ± γ-PGA.

2.1. Mushrooms Biomass

In vitro cultures of Agaricus bisporus, Boletus edulis, and Pleurotus ostreatus (Preparation—Stage 1) were used.

Step 1

In the presented experiment, studies were conducted on three species of mushrooms, differing in their natural habitats: Agaricus bisporus, Boletus edulis, and Pleurotus ostreatus. In order to establish experimental in vitro cultures, 0.2 g of inoculum was passaged from the initial strains obtained from shaking in vitro cultures with confirmed genotype. In vitro cultures were conducted in 500 mL Erlenmeyer conical flasks filled with 250 mL of liquid medium prepared according to the recipe by Oddoux. In vitro cultures were grown under physicochemical conditions (temperature 22 °C ± 2 °C, lighting 200 lux with a light-dark cycle (12–16 h light/8–12 h dark)) optimized in previously undertaken research [30].

The experimental material was the biomass of A. bisporus, B. edulis, and P. ostreatus obtained on a liquid medium according to Oddoux fortified with toxic metals Cd and Pb (in the form of their inorganic salts: CdCl₂ and PbCl₂) in an amount of 4 mg/L (the given value corresponds to the pure metal content) of liquid medium and with the addition of poly-gamma glutamic acid. The cultures were carried out for each of the toxic metals separately and as their mixture (each case in three independent repetitions). Additionally, the in vitro cultures were enriched with the addition of γ-PGA at concentrations of 1 mL, 2 mL, and 5 mL to 250 mL liquid medium (the culture variants are presented in Figure 1).

Each culture was performed in five independent replicates simultaneously. After 21 days (optimal mycelium growth time), the mycelium was separated from the liquid medium by filtration (using a Pyrex Büchner funnel with a perforated plate from Merck, Darmstad, Germany). Half of the obtained biomass was subjected to a desorption test. The remaining biomass was freeze-dried at −40 °C (Freezone freeze-dryer no. 4.5 from Labconco, Kansas City, MO, USA). The resulting lyophilized biomass was mineralized (each sample in three independent replicates) and then analyzed for metal content using atomic absorption spectrometry (AAS, Waltham, MA, USA). The results are presented as mean values ± standard deviations.

2.2. Poly-Gamma Glutamic Acid (γ-PGA)

Synthesis of γ-PGA (Preparation—Stage 1):

Step 2

The optimized medium composition for γ-polyglutamic acid (γ-PGA) production by Bacillus subtilis in shake flask cultures had been characterized in recent studies [12]. A typical formulation includes the following components: glucose at 4% (w/v), yeast extract at 1% (w/v), sodium glutamate at 1% (w/v), magnesium sulfate heptahydrate (MgSO₄·7H₂O) at 0.025% (w/v), potassium hydrogen phosphate (K₂HPO₄) at 0.2% (w/v), and ammonium chloride (NH₄Cl) at 0.3% (w/v), with the initial pH adjusted to 6.0.

Cultures were incubated at 42 °C with shaking at 220 rpm in 250 mL Erlenmeyer flasks containing 30 mL working volume, inoculated with 2% (v/v) seed culture, and fermented for 72 h. Under these conditions, γ-PGA production reached approximately 125 g/L.

Following fermentation, the isolation and purification of γ-PGA were performed to ensure a high-quality polymer suitable for subsequent applications. The culture broth was first centrifuged to remove bacterial cells and debris. The γ-PGA was then precipitated by adding three volumes of cold ethanol or isopropanol, followed by incubation at 4 °C for 12–24 h. The precipitate was recovered by centrifugation, washed with alcohol to remove impurities, and subsequently dried under vacuum. Additional purification steps, such as dialysis against distilled water and lyophilization, were conducted to eliminate residual low molecular weight compounds and salts, thereby improving the polymer’s purity and functional properties [31]. These isolation and purification procedures are critical in obtaining γ-PGA with consistent molecular weight, purity, and bioactivity, which directly impact its metal chelation efficacy and antimicrobial performance in mushroom cultures.

These conditions provide both sufficient carbon and nitrogen sources as well as essential minerals, enabling efficient Bacillus subtilis growth and γ-PGA biosynthesis [32,33]. Glucose served as the primary carbon source while sodium glutamate supplied glutamate monomers for polymer synthesis. Yeast extract contributed vitamins and growth factors and mineral salts maintained cellular metabolism and buffer capacity. The slightly acidic to neutral pH (around 6.0) was chosen as optimal for enzymatic activities associated with γ-PGA production.

In the experimental setup, γ-PGA was added to the liquid culture medium by volume at three levels: 1 mL, 2 mL, and 5 mL per 250 mL of medium. These volume-based concentrations corresponded approximately to molar concentrations near 1 mM, based on the γ-PGA stock solution concentration obtained after fermentation and purification. In antimicrobial activity assays, γ-PGA concentrations are directly expressed in terms of molarity (1 mM) to reflect the precise biochemical exposure conditions in those tests, where defined γ-PGA solutions were applied.

2.3. Reagents

Liquid culture media:

CdCl₂ and PbCl₂ salts were purchased from Sigma-Aldrich (Darmstadt, Germany).

Mineralization of freeze-dried biomass:

We used 65% HNO₃(V) acid and 30% H₂O₂ Suprapure^® (Merck, Darmstadt, Germany) and quadruple-distilled water (conductivity <1 μS/cm, HLP 5 distiller, Hydrolab, Straszyn, Poland).

AAS method measurement:

Standard solutions of metals: Cd(II), Pb(II) at a concentration of 1 g/L (Sigma-Aldrich, Darmstadt, Germany).

Artificial digestive juices:

Artificial digestive fluids—saliva, gastric juice, and intestinal juice—used in the simulated gastrointestinal model were formulated based on protocols described in the literature [34,35,36]. The preparation involved the use of the following analytical-grade reagents: KH₂PO₄, Na₂HPO₄, KHCO₃, MgCl₂, C₆H₈O₇, CaCl₂, NaCl, pepsin, HCl, pancreas extract, bile salts, and NaHCO₃ (all sourced from Sigma-Aldrich, Darmstadt, Germany). The accurately weighed compounds were placed into 1 L flat-bottom flasks and diluted with quadruple-distilled water to the required volume.

2.4. Analysis Effectiveness of Metal Adsorption and Desorption of Toxic Metals in the Biomass Obtained in Stage 1 from In Vitro Cultures with Added Poly-Gamma Glutamic Acid (γ-PGA) (Stage 2)

In vitro cultures

To evaluate the efficiency of heavy metal adsorption by selected fungal species, the chemical composition of both the growth substrate after cultivation and the resulting fungal biomass was analyzed. Biomass of Agaricus bisporus, Boletus edulis, and representatives of the Pleurotus genus was homogenized using a porcelain mortar. From the homogenized material, three independent subsamples of 0.200 g each were accurately weighed using an analytical balance. The samples were transferred into Teflon vessels and subjected to wet digestion using a mixture of 2 mL of 30% hydrogen peroxide (H₂O₂) and 6 mL of 65% nitric acid (HNO₃) under closed-system conditions at a pressure of 30 bar (digestion system: Anton Paar, Baden, Switzerland). To remove excess reagents, the digests were heated at 100 °C for 120 min. After digestion, the resulting solutions were quantitatively transferred to 10 mL volumetric flasks using quadruple-distilled water. Additionally, the degree of metal desorption from the biomass was determined. For this purpose, half of the obtained biomass was extracted in quadruple-distilled water for 60 min (temperature 23 ± 2 °C). The biomass was then separated from the rest of the solution by filtering it on a glass Buchner funnel and the obtained filtrate was analyzed.

2.5. Assessment of Metal Extraction by Simulated Digestive Juices from Mushroom Biomass Obtained Under Cultivation Conditions with and Without γ-PGA (Stage 3)

To determine the bioavailability of heavy metals, such as cadmium (Cd) and lead (Pb), originating from biomass obtained from in vitro cultures, an extraction procedure using simulated digestive fluids was conducted. The study included the analysis of both biomass from three mushroom species as well as biomass from their respective cultivated forms, to which 2 mL of γ-polyglutamic acid (γ-PGA) was added as a supplement to the growth medium. Each determination was carried out in triplicate. Biomass samples weighing 0.5 g were placed in conical flasks. Subsequently, 5 mL of artificial saliva was added, and the samples were subjected to digestion in a model gastrointestinal system (Gastroel-2014) using 20 mL of simulated gastric juice for 60 min, following the protocol described by Opoka et al. (2016) [36]. After completion of the gastric phase, the mixture was filtered through 0.22 μm pore-size membrane filters (Millex, Millipore^®, Burlington, MA, USA). The resulting filtrate was supplemented with 20 mL of artificial intestinal juice and incubated for an additional 150 min. In the final step, the mixture was filtered again, and the concentrations of heavy metals were determined in the obtained filtrates [34,35,36].

2.6. Testing Material Properties (Biomass and γ-PGA)

Surface characterization: Specific surface area, total pore volume, and average pore diameter were measured by nitrogen adsorption techniques, employing the Brunauer–Emmett–Teller (BET) and Langmuir models (ASAP 2010, Micromeritics, Norcross, GA, USA). The microstructural features and surface morphology of both mycelial samples and γ-PGA powder were examined using scanning electron microscopy (SEM, FEI Nova NanoSEM 200, Hillsboro, OR, USA). Imaging was performed at a magnification of 20,000× for γ-PGA powders and 10,000× for in vitro cultured mycelium, with an accelerating voltage of 18 kV.

2.7. Antimicrobial Susceptibility Testing

The antimicrobial activity of mushroom biomass, both alone and in combination with γ-PGA, was assessed using the Kirby–Bauer disk diffusion method on Mueller–Hinton Agar (bioMérieux, Warsaw, Poland). For quality control and antimicrobial susceptibility testing, the reference strains Escherichia coli ATCC 25922 and Salmonella enteritidis ATCC 13076 were utilized. Reference strains were suspended in a 0.85% saline solution (bioMérieux, Warsaw, Poland) before being plated onto the culture medium. Subsequently, diffusion discs containing samples of pure biomass from in vitro cultures of A. bisporus, B. edulis, and P. ostreatus, as well as γ-PGA and their combinations, were placed on the inoculated plates.

The plates were incubated at 37 °C for 24 h. Following incubation, the diameters of the inhibition zones were measured. Results were compared with to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria (Breakpoint table ver.15.0, valid from 2025-01-01).

2.8. Statistical Analysis

The effects of gamma-polyglutamic acid (γ-PGA) concentration, mushroom species, and heavy metal treatments (cadmium, lead, and their mixtures) on sorption efficiency and antimicrobial activity were analyzed using generalized linear modeling (GLM). Given that response variables such as sorption efficiency percentages and inhibition zone diameters are continuous, strictly positive, and often positively skewed, a gamma distribution with a log link function was employed. This approach effectively managed heteroscedasticity and non-normality in the data, providing interpretable parameter estimates through maximum likelihood estimation [37,38].

The experimental design included multiple independent replicates (biological replicates or batches of in vitro cultures) for each treatment combination. To account for inherent variability among replicates and avoid pseudo-replication, a generalized linear mixed modeling (GLMM) framework incorporating both fixed effects (γ-PGA concentration, mushroom species, metal treatment, and their interactions) and random effects (replicate identity) was considered appropriate. Inclusion of random intercepts for replicate identity enabled the model to generalize findings beyond specific experimental runs, providing more robust and accurate effect estimates while properly controlling for within-group correlations.

Prior to final model selection, exploratory analyses assessed variance attributable to replicate-level effects. If replicate variability was found to be minimal or negligible, and the experimental replicates were balanced and controlled, a fixed-effects-only GLM model was implemented to maximize model parsimony and interpretability. This decision was justified based on statistical diagnostics indicating limited random variation and the absence of convergence issues or biased inference in fixed-effects models.

Model adequacy and assumptions were rigorously evaluated. Residual diagnostics, including inspection of quantile–quantile plots and plots of residuals versus fitted values, confirmed the appropriateness of the gamma distribution with log link. Multicollinearity among explanatory variables and interaction terms was assessed using variance inflation factors (VIFs) and condition indices. Where moderate multicollinearity was detected, careful interpretation focused on overall interaction patterns rather than isolated parameter effects to ensure robust conclusions.

Significance of fixed effects and interactions was tested using Wald chi-square (χ²) statistics with a significance threshold of α = 0.05 [39,40]. To mitigate inflated type I error rates due to multiple pairwise comparisons, post hoc analyses employed Tukey’s multiple comparison tests with Bonferroni correction, enhancing the stringency and reliability of statistical inference.

All statistical analyses were conducted using Statistica TIBCO version 12.0 (TIBCO Software Inc., Palo Alto, CA, USA). Visualization of results included plotting predicted means with 95% confidence intervals to facilitate clear interpretation of treatment effects and interaction dynamics.

3. Results and Discussion

3.1. Characterization Materials (Biomass and γ-PGA)

Using the BET method (the method assumes that molecules can form multiple adsorption layers), the specific surface areas of the examined biomass from in vitro cultures were determined as follows: A. bisporus (Ab) 11.4 m²/g, B. edulis (Be) 1.3 m²/g, P. ostreatus (Po) 13.2 m²/g, and 15.4 m²/g γ-PGA powder. The surface areas obtained from the Langmuir equation (the method assumes only one adsorption layer) were accordingly 12.3 m²/g, 1.6 m²/g, and 13.1 m²/g for Ab, Be, and Po and 15.8 m²/g for γ-PGA powder. The obtained BET surface area values gave a lower surface area than the Langmuir surface area. These discrepancies may have resulted from applying the Langmuir model outside its scope—the Langmuir model assumes single-layer adsorption on a uniform surface. When the Langmuir isotherm is fitted to data that also include multilayer adsorption (i.e., outside its scope of applicability), the model incorrectly “overinterprets” the data—and overestimates the amount of adsorbate.

Total pore volume (V_micro), micropore area (S_micro), and average pore diameter (A_pore) of nanopowders are shown in

Table 1. Parameters characteristic of biomaterials.

Type of Biomaterials/Parameters	Ab	Be	Po	γ-PGA
Vmicro, cm3/g	0.0020	0.0001	0.0028	0.031
Smicro, m2/g	3.5	0.4	4.6	8.1
Apore, nm	8	6	13	15

Abbreviations: Po—P. ostreatus, Ab—A. bisporus, Be—B. edulis, V_micro—pore volume, A_pore—average pore diameter.

.

3.2. Structure Examination of Mycelium Biomass

The morphology analysis of P. ostreatus, A. bisporus, and B. edulis mycelia was examined using SEM microscopy. The resulting images obtained at an accelerating voltage of 5 kV and a magnification of 10,000 are presented in Figure 2.

In Figure 2A, showing the SEM images of P. ostreatus, a compact structure of a single fibril can be observed, which may indicate chitin and glucan elements of the mushroom cell wall. The EDX analysis of P. ostreatus (Figure 2D) of the chemical composition indicated carbon and oxygen as the main components (70.1 ± 0.09 wt.% and 28.2 ± 0.1 wt.%, respectively) with an admixture of potassium, phosphorus, and magnesium (0.9 ± 0.04 wt.%, 0.4 ± 0.02 wt.%, and 0.1 ± 0.02 wt.%, respectively). The SEM image of A. bisporus (Figure 2B) possesses a finer fibrous structure but contains a significant number of void spaces. The EDX analysis of the chemical composition of A. bisporus (Figure 2E) confirmed the main components as carbon (64.8 ± 0.12 wt.%) and oxygen (27.8 ± 0.13 wt.%), but nitrogen was also present (6.0 ± 0.41 wt.%). Potassium and chlorine (0.4 ± 0.04 wt.% and 0.5 ± 0.03 wt.%, respectively) were visible in the present in the admixture, while in comparison to Po, no magnesium content was observed. The structure of B. edulis (Figure 2C) was finely fibrous and compact; no voids were visible. Agglomerated spherical structures could be observed on the fibers. The EDX analysis of the chemical composition of B. edulis (Figure 2F) indicated the presence of carbon and oxygen (69.3 ± 0.14 wt.% and 28.7 ± 0.14 wt.%, respectively), with admixture of chlorine, phosphorus, and potassium (0.4 ± 0.04 wt.%, 0.5 ± 0.03 wt.%, and 0.4 ± 0.05 wt.%, respectively).

3.3. Sorption and Desorption by Mycelia In Vitro Cultures

Figure 3 presents the efficiency of sorption (left panels) and desorption (right panels) for three mushrooms—P. ostreatus (Po), A. bisporus (Ab), and B. edulis (Be)—in both single-metal and mixed-metal conditions from generalized linear mixed models. In single-metal conditions (top left), all mushrooms showed high sorption efficiency (>80%), but B. edulis (Be) had a notably lower efficiency compared to the other two species, especially for one of the bars (likely representing a specific metal or treatment). In mixed-metal conditions (bottom left), sorption efficiency generally decreased for all mushrooms, with B. edulis (Be) showing the most pronounced drop (efficiency drops close to 60% for one bar). Statistical analysis using the Wald chi-square test revealed significant differences in sorption efficiency among mushroom species and metal treatments, with Wald statistics of W = 111 and W = 9.5, respectively (p < 0.001). These values indicate strong support for the effects of these factors on metal sorption. Post hoc pairwise comparisons using Tukey’s test with Bonferroni correction identified that both Pleurotus ostreatus and Agaricus bisporus exhibited significantly higher sorption efficiencies compared to Boletus edulis, particularly under single-metal exposure conditions. Moreover, sorption efficiencies differed significantly between cadmium and lead treatments. These detailed comparisons clarified the specific group differences underlying the overall statistical effects observed.

For single metals (top right), desorption efficiency was relatively high and similar among fungi, with no significant differences (W = 0.35; ns). For mixed metals (bottom right), desorption efficiency varied more, with P. ostreatus (Po) and B. edulis (Be) showing higher efficiencies than A. bisporus (Ab). Significant differences were observed here (W = 7.9, p < 0.001).

Figure 4 demonstrates the protective effect of γ-polyglutamic acid (γ-PGA) supplementation on fungal growth under heavy metal stress conditions. The red squares represent treatments with γ-PGA addition while the blue circles represent control treatments without γ-PGA supplementation. All three fungal species showed reduced biomass under heavy metal stress (PbCl₂ and CdCl₂) compared to control conditions. γ-PGA supplementation (red line) consistently provided protection, maintaining higher biomass levels across all treatment conditions and mushroom species. The protective effect of γ-PGA was most pronounced under stress conditions, with smaller differences observed in control treatments. P. ostreatus showed the highest biomass production overall, reaching approximately 4.8 g/250 mL in control with γ-PGA treatment. It showed the most dramatic response to heavy metal stress, with biomass dropping to around 2.4 g/250 mL under PbCl₂ stress. γ-PGA treatment maintained biomass at 2.4–2.6 g/250 mL under both metal stress conditions while untreated fungi dropped to 1.4–1.7 g/250 mL. A. bisporus demonstrated a more gradual decline in biomass with increasing metal stress. It showed consistent protective effects of γ-PGA across all treatments, with treated fungi maintaining 3.6, 2.6, and 2.3 g/250 mL for control, PbCl₂, and CdCl₂, respectively. Untreated mushrooms showed lower biomass values of 2.9, 2.2, and 1.5 g/250 mL for the same conditions. B. edulis exhibited the lowest overall biomass production among the three species. It showed similar patterns to other species, with γ-PGA providing consistent protection. The difference between treated and untreated conditions became more pronounced under CdCl₂ stress.

The GLM with log link function demonstrates that γ-PGA concentration is a significant negative predictor of sorption efficiency, with each increment leading to a multiplicative decrease in metal uptake (

Table 2. The summary of generalized linear mixed models with gamma distribution and log link function for sorption efficiency of heavy metals and its mixtures for three species of mushrooms and four concentration of γ-polyglutamic acid (γ-PGA).

	df	Walds Stat.	p
Lead
Intercept	1	29,449.37	0.000
Mushroom species	2	19.34	0.000
PGA conc	3	1925.94	0.000
Mushroom species*PGA conc	6	46.2	0.000
Cadmium
Intercept	1	23,071.05	0.000
Mushroom species	3	1359.29	0.000
PGA conc	2	17.55	0.000
Mushroom species*PGA conc	6	24.27	0.000
Lead mix
Intercept	1	27,834.16	0.000
Mushroom species	3	1703.95	0.000
PGA conc	2	13.42	0.001
Mushroom species*PGA conc	6	26.77	0.000
Cadmium mix
Intercept	1	9910.183	0.000
Mushroom species	3	816.978	0.000
PGA conc	2	34.887	0.000
Mushroom species*PGA conc	6	11.912	0.044

Abbreviations: df—degrees of freedom, Walds Stat.—Wald coefficient of statistics, * indicates interaction between factors.

). Across all species and treatments, higher concentrations of γ-PGA (darker bars) generally corresponded to lower sorption efficiency (Figure 5). Sorption efficiency decreased as γ-PGA concentration increased for all metals and all mushroom species. The highest sorption occurred in the absence of γ-PGA while the lowest was observed at 5 mL γ-PGA. This trend was consistent across Pb, Cd, and their mixtures, indicating that γ-PGA addition effectively reduces the uptake of heavy metals. P. ostreatus (Po) and A. bisporus (Ab) generally showed a higher baseline sorption of heavy metals compared to B. edulis (Be). Despite species differences, increased γ-PGA consistently reduced sorption in all three mushrooms. Adding γ-PGA during cultivation or processing can be an effective strategy to minimize the accumulation of toxic metals in mushrooms. As can be seen, γ-PGA is a polyanionic chelating agent for metals (Cd and Pb), containing numerous carboxyl groups (–COO⁻) that can release protons, especially at pH values above 4. The negatively charged fragments form binding sites for heavy metal cations, enabling either direct metal–OOC interaction (coordination with carboxylates) or electrostatic attraction, creating a local field at the carboxylates that bind heavy metals. At a higher pH, around 6 (which was the pH observed in the present studies), γ-PGA occurs in a random coil, with a larger number of available carboxyl groups, which increases the ability to bind metals.

Sorption efficiency was consistently higher in gastric juice (g.j) than in intestinal juice (i.j) across all species and metal treatments (Figure 6). A marked decrease in efficiency was observed when transitioning from gastric to intestinal conditions for all mushrooms and metals. The addition of 2 mL γ-PGA (red lines) did not consistently improve or reduce efficiency compared to reference conditions (blue lines); the effect varied by species and metal (Table 2).

Species and metal—specific observations

For both single metals (Pb, Cd) and metal mixtures (Pb_mix, Cd_mix), sorption efficiency of Pleurotus ostreatus in gastric juice was high (often above 60%) and dropped sharply in intestinal juice (below 40%). The effect of γ-PGA addition was minimal, with red and blue lines closely overlapping in most cases. B. edulis showed a greater variability of sorption (larger confidence interval), especially in gastric juice. Sorption efficiency also declined from gastric to intestinal conditions. The impact of γ-PGA addition was inconsistent for this species. In some cases (e.g., Pb in gastric juice), γ-PGA slightly increased efficiency, while in others (e.g., Cd_mix), it had little or no effect. A. bisporus exhibited similar patterns to the other species, with higher efficiency in gastric juice and a pronounced decline in intestinal juice. The difference between γ-PGA-treated and reference samples was generally small, though in some cases (e.g., Pb in gastric juice), γ-PGA addition resulted in marginally higher efficiency.

The digestive phase was the dominant factor influencing sorption efficiency, with all mushrooms showing much higher metal binding in gastric conditions than in intestinal conditions. γ-PGA addition (2 mL) did not have a uniform effect: its influence on sorption efficiency was minor and varied depending on mushroom species and metal treatment. Variability was highest for B. edulis, particularly under gastric conditions. These results suggest that while fungal species can effectively bind metals in acidic gastric conditions, this ability is substantially reduced in the more neutral intestinal environment, and the benefit of γ-PGA supplementation is limited under the tested conditions.

Sorption abilities of different mushroom species

Our results demonstrate that all three tested mushroom species—P. ostreatus, A. bisporus, and B. edulis—possess significant abilities to sorb heavy metals, with notable interspecies differences. P. ostreatus and A. bisporus generally exhibited higher sorption efficiencies compared to B. edulis, especially under single-metal exposure [41,42,43,44]. This was consistent with previous findings, where P. ostreatus and A. bisporus had been reported as efficient biosorbents for metals such as Cd, Pb, and Cu due to their high contents of functional groups (e.g., carboxyl, amino, and phosphate) in their cell walls [43,44]. B. edulis, while still capable of metal uptake, showed greater variability and lower efficiency, particularly in mixed-metal scenarios [45].

Influence of gamma-polyglutamic acid (γ-PGA) on metal sorption

The addition of γ-polyglutamic acid (γ-PGA) had a complex and generally inhibitory effect on metal sorption efficiency across all mushrooms and treatments. Increasing the γ-PGA concentration from 1 mL to 5 mL led to a consistent reduction in sorption efficiency for all tested species. This may be attributed to γ-PGA’s strong metal-chelating properties, which can immobilize metals in solution and prevent their interaction with the mushroom cell wall binding sites [46,47]. While γ-PGA is known for its ability to bind and immobilize heavy metals [10,46,47,48], in the context of biosorption by mushrooms, its presence likely reduces the availability of free metal ions for mushroom uptake, thus lowering overall sorption efficiency.

γ-PGA and metal sorption under simulated digestive conditions

Sorption efficiency was consistently higher in simulated gastric juice (acidic pH) than in intestinal juice (neutral pH), regardless of the fungal species or metal type. This was consistent with the known pH dependence of biosorption [49,50,51], where lower pH enhances the protonation of functional groups on fungal cell walls, promoting metal binding [3]. The addition of 2 mL of γ-PGA reduced the sorption efficiency of toxic metals in the mycelium (its effect was species-dependent for mycelium morphological properties, see Figure 4). This suggests that the addition of γ-PGA may immobilize metals by forming complexes with metals with a larger spatial conformation—micelles, whose desorption from the mycelium is hindered. Similar results had been obtained in studies on vascular plants, where exogenous chelators, such as γ-PGA, can reduce the bioavailability of metals but can also inhibit their uptake and translocation by plants [10].

Comparison to vascular plants

In vascular plants, γ-PGA and other organic acids are known to play dual roles: they can chelate and immobilize metals in the rhizosphere, reducing toxicity, but may also limit the plant’s ability to take up essential and non-essential metals [13]. Our findings in mushrooms mirror this phenomenon; γ-PGA addition reduces metal uptake (sorption efficiency), likely by sequestering metals in a form that is less accessible to biological binding sites. This highlights a general principle applicable across biological kingdoms: while chelators like γ-PGA can mitigate metal toxicity, they may also inadvertently lower the efficiency of bioremediation agents, whether mushroom- or plant-based.

Microbiological activity

The generalized linear model (GLM) analysis revealed significant antimicrobial effects of γ-polyglutamic acid (γ-PGA) on Salmonella enteritidis ATCC 13076 and Escherichia coli in cultures of the edible mushrooms P. ostreatus (Po), A. bisporus (Ab), and B. edulis (Be) (

Table 3. Generalized linear three-way interaction modeling of the inhibition zone diameter of examined pathogen strains in relation to culture of edible mushroom and presence of γ-PGA.

Effect	df	Wald’s Stat.	p
Intercept	1	46,139.6	0
Pathogen	1	0.8	0.371141
Fungi	3	2453.7	0
γ-PGA	1	4.27	0.038761
Pathogen*Fungi	3	168.83	0
Pathogen*PGA	1	20.93	0.000005
Fungi*PGA	2	629.36	0
Pathogen*Fungi*PGA	2	40.29	0

Abbreviations: df—degrees of freedom, Wald’s Stat.—Wald coefficient of statistics, * indicates interaction between factors.

). The main effect of fungal species on inhibition zone diameter was highly significant (Wald χ² = 2453.7, p < 0.001), indicating that the type of mushroom culture strongly influenced antibacterial activity. In contrast, the reference strains identity alone did not significantly affect the inhibition zone size (Wald χ² = 0.8, p = 0.37). The application of γ-PGA was associated with a significant increase in antimicrobial activity (Wald χ² = 4.27, p = 0.039), confirming its overall inhibitory effect on bacterial growth. Significant interaction effects were observed between the reference strains and fungal species (Wald χ² = 168.83, p < 0.001), bacteria and γ-PGA treatment (Wald χ² = 20.93, p < 0.00001), fungal species and γ-PGA (Wald χ² = 629.36, p < 0.001), and the three-way interaction among the reference strains fungi, and γ-PGA (Wald χ² = 40.29, p < 0.001). These interactions suggest that the antimicrobial efficacy of γ-PGA depends on a complex interplay between the bacterial species and the specific mushroom host (Table 3).

Figure 7 illustrates the mean diameters of bacterial growth inhibition zones (±95% confidence intervals) for Salmonella enteritidis ATCC 13076 (left panel) and Escherichia coli (right panel) following treatment with γ-PGA at 1 mM concentration (red symbols) compared with untreated controls (0 mM γ-PGA, blue symbols). The data are presented for three edible mushroom cultures—Pleurotus ostreatus (Po), Boletus edulis (Be), and Agaricus bisporus (Ab)—as well as for γ-PGA treatment alone (PGA). For Salmonella enteritidis ATCC 13076, the application of 1 mM γ-PGA significantly increased the inhibition zone diameter in all mushroom cultures compared to controls. Specifically, P. ostreatus and A. bisporus exhibited inhibition zones of approximately 25 mm under γ-PGA treatment compared to roughly 20 mm or less in controls. Boletus edulis showed an increase from a minimal inhibition zone (~6 mm) in the absence of γ-PGA to approximately 14 mm with 1 mM γ-PGA, indicating a notable enhancement of antimicrobial activity.

Similarly, for Escherichia coli, γ-PGA treatment enhanced bacterial inhibition across all tested cultures and in γ-PGA alone. In P. ostreatus cultures, inhibition zones increased from roughly 16 mm in controls to near 23 mm with γ-PGA. Boletus edulis showed a marked increase from minimal inhibition (~6 mm) to approximately 15–16 mm upon γ-PGA treatment. A. bisporus and γ-PGA alone also exhibited significant increases in the inhibition zone size, reaching approximately 20–22 mm compared to lower values without γ-PGA.

The results clearly demonstrate that γ-polyglutamic acid (γ-PGA) at a concentration of 1 mM significantly enhances antimicrobial activity against both Salmonella enteritidis and Escherichia coli within cultures of the edible mushrooms Pleurotus ostreatus (Po), Agaricus bisporus (Ab), and Boletus edulis (Be), as well as when applied alone (PGA). The diameter of bacterial growth inhibition zones serves as a robust quantitative metric of this antibacterial efficacy, with data revealing that the magnitude of inhibition varies considerably across mushroom species and bacterial pathogens. For Salmonella enteritidis, the addition of 1 mM γ-PGA induced a substantial increase in inhibition zone diameters for all fungal treatments relative to non-γ-PGA controls. Notably, P. ostreatus and A. bisporus exhibited the largest absolute inhibition zones, averaging approximately 25 mm when combined with γ-PGA, compared to roughly 20 mm or less without γ-PGA supplementation. These results suggest that these species may provide a bioactive microenvironment that potentiates γ-PGA’s antibacterial properties. Conversely, B. edulis, which displayed a relatively low basal inhibition zone (~6 mm) without γ-PGA, exhibited a pronounced relative increase to approximately 14 mm upon γ-PGA treatment. This marked enhancement supports the hypothesis that γ-PGA compensates for weaker intrinsic antimicrobial activity, indicating a potential synergistic effect between γ-PGA and mushroom-specific factors. Similarly, inhibition zones for E. coli expanded significantly with γ-PGA treatment. The P. ostreatus culture showed an increase from approximately 16 mm to 23 mm in the presence of γ-PGA whereas B. edulis exhibited a near tripling of inhibition zone diameter from ~6 mm to 15–16 mm. A. bisporus and treatment with γ-PGA alone also demonstrated considerable improvements, achieving inhibition zones in the range of 20–22 mm compared to lower baseline values. Such consistent enhancement aligns with previous studies demonstrating that γ-PGA disrupts Gram-negative bacterial cell membranes and induces oxidative stress through reactive oxygen species, thereby inhibiting bacterial growth [52].

The observed variation in antibacterial efficacy among the mushroom species is likely attributable to the differential production of bioactive metabolites and distinct physicochemical microenvironments within each fungal culture. Previous research documented that Pleurotus and Agaricus species contain antimicrobial compounds such as polysaccharides, phenolic compounds, and proteins, which may act synergistically alongside γ-PGA to amplify bacterial inhibition [53,54]. In contrast, the lower inherent antibacterial activity observed for Boletus edulis underscores its limited natural antimicrobial defenses; however, the substantial improvement with γ-PGA highlights the polymer’s broad-spectrum antimicrobial potential as an additive in mushroom cultivation. From a practical perspective, these findings underscore the potential utility of γ-PGA supplementation in mushroom production and postharvest management to mitigate contamination by significant foodborne pathogens such as Salmonella enteritidis and E. coli. Effective bacterial control is critical to enhancing product quality, ensuring consumer safety, and extending shelf life [55]. Furthermore, the differential responses observed across mushroom species highlight the necessity of tailoring biosafety strategies to specific fungal hosts and target bacterial contaminants, optimizing efficacy and applicability in commercial settings. Research has shown that the addition of γ-PGA (poly-γ-glutamate) can enhance antimicrobial properties in edible mushroom cultivation, primarily due to its unique physicochemical and biological properties. The addition of γ-PGA can disrupt the integrity of microbial cells—disrupting the structures of pathogen cells and damaging bacterial cell membranes. This may also be important in mushroom cultivation, where bacterial or competing pathogen control is necessary. When cultivating edible mushrooms (which are highly susceptible to drying and microbial growth), using γ-PGA as a coating can protect their surface from pathogens, oxidation, and dehydration—further limiting the growth of undesirable microorganisms.

The application of γ-polyglutamic acid (γ-PGA) as an antimicrobial agent in edible mushroom cultures presents promising benefits for microbial safety and biocontrol. However, the long-term use of any antimicrobial compound necessitates careful consideration of the potential development of microbial resistance and sustainability of its efficacy. The current literature suggests that γ-PGA exerts its antimicrobial effects primarily through mechanisms that do not directly target bacterial DNA replication or protein synthesis but instead involve the disruption of cell membranes and biofilm inhibition [56,57,58]. Such mechanisms are generally associated with a lower risk of inducing classical resistance compared to conventional antibiotics. Still, the potential for bacteria to adapt or develop tolerance via changes in membrane permeability, efflux pump activation, or altered biofilm architecture cannot be entirely excluded, especially under chronic exposure scenarios. To date, no clear evidence exists of bacterial resistance emerging specifically against γ-PGA in environmental or clinical settings, likely due to its mode of action and natural biodegradability. Additionally, γ-PGA is a naturally occurring, biodegradable polymer produced by non-pathogenic Bacillus species, which supports its environmental sustainability as a biocontrol agent [10]. Its integration into fungal cultivation media is thus anticipated to have minimal adverse ecological impacts compared to synthetic antimicrobials. Furthermore, the combined use of γ-PGA with edible mushrooms may provide a multifaceted barrier against pathogens by leveraging both the antimicrobial properties of γ-PGA and the intrinsic antimicrobial metabolites produced by fungi, potentially reducing selective pressure for resistance. This synergy favors a more sustainable approach to microbial management in agricultural and food systems [59]. Nonetheless, long-term field studies and monitoring are essential to assess the stability of γ-PGA’s antimicrobial efficacy over time [46], possible shifts in microbial community composition, and any subtle resistance development. Regular rotation or combination with other biocontrol agents may also be prudent to mitigate resistance risk and prolong effectiveness.

Practical implications for food safety regulations

The results of this study provide compelling evidence that gamma-polyglutamic acid (γ-PGA) significantly modulates both heavy metal biosorption and microbial safety in edible mushroom cultures [60]. Across all three species—Pleurotus ostreatus, Agaricus bisporus, and Boletus edulis—γ-PGA exhibited a concentration-dependent inhibitory effect on cadmium and lead uptake, corroborating previous findings on its metal-chelating capacity that reduces bioavailable metal ions in solution through complexation [44]. The enhanced sorption stability in P. ostreatus and A. bisporus, relative to B. edulis, underscores the role of species-specific morphological and biochemical traits influencing biosorption efficacy, consistent with earlier reports showing differential metal accumulation linked to cell wall composition and porosity [49]. The pH-dependent sorption patterns observed under simulated gastrointestinal conditions align with established biosorption mechanisms, where acidic gastric juice favors metal binding via the protonation of fungal cell wall functional groups while neutral intestinal juice reduces binding efficiency [53].

Concomitantly, the antimicrobial activity of γ-PGA at 1 mM concentrations against Salmonella enteritidis and Escherichia coli—two principal foodborne pathogens—demonstrates its potential as a natural biocontrol agent for improving food safety in mushroom production. The synergistic enhancement of antimicrobial efficacy when γ-PGA is combined with mushroom cultures suggests the production of bioactive metabolites by fungi that act complementarily to γ-PGA’s membrane-disruptive and oxidative-stress-inducing mechanisms. Such multifaceted antimicrobial activity is especially valuable given the documented vulnerability of mushroom cultivation environments to bacterial contamination from substrate inputs and postharvest handling.

From a regulatory perspective, these findings have immediate relevance for food safety control frameworks concerning edible mushrooms. Heavy metal contamination in food products is subject to strict maximum allowable limits mandated by agencies such as the European Food Safety Authority (EFSA) [61] and the United States Food and Drug Administration (FDA) [60]. The demonstrated ability of γ-PGA to reduce metal uptake offers a promising postharvest or cultivation-stage intervention to ensure compliance with these regulations, thereby mitigating health risks associated with chronic heavy metal exposure [2]. Furthermore, the antimicrobial properties of γ-PGA could contribute to reduced reliance on synthetic preservatives or chemical sanitizers, aligning with consumer demand for minimally processed and ‘clean label’ foods [59,62,63].

The implementation of γ-PGA supplementation strategies in commercial mushroom cultivation could therefore support integrated hazard analysis and critical control point (HACCP) programs by targeting microbial contamination at the production stage [64]. Regulatory guidelines focused on microbial safety could consider endorsing biopolymers such as γ-PGA as part of approved food additive lists or Good Agricultural Practices (GAPs) to enhance shelf life and reduce foodborne illness outbreaks from mushrooms. Additionally, the natural biodegradability and minimal environmental impact of γ-PGA favor its acceptance in sustainable agricultural models encouraged by food safety authorities worldwide [65,66].

Future research should explore optimal dosing regimens, potential effects on organoleptic properties, and long-term efficacy under commercial-scale conditions [12]. Moreover, regulatory bodies may require comprehensive risk assessments covering toxicological profiles, allergenicity, and environmental persistence to establish safety dossiers and facilitate market approval [67]. Nonetheless, this paper paves the way for practical, environmentally friendly interventions that simultaneously address toxic metal contamination and microbial hazards in edible mushrooms, thereby reinforcing public health protection and food quality assurance [63,68].

4. Conclusions

Heavy Metal Sorption by Edible Mushrooms and γ-PGA Influence

All three mushroom species—Pleurotus ostreatus, Agaricus bisporus, and Boletus edulis—demonstrated high sorption efficiency for cadmium (Cd), lead (Pb), and their mixtures, with efficiencies generally exceeding 84%. Sorption efficiency was highest at the lowest γ-polyglutamic acid (γ-PGA) concentration (1 mL) and decreased significantly as the γ-PGA concentration increased to 2 mL and 5 mL across all species and metal treatments. This inhibitory effect of increasing γ-PGA concentration on metal uptake was consistently observed and statistically confirmed via generalized linear mixed modeling. Among the species, P. ostreatus and A. bisporus maintained higher and more stable sorption efficiencies compared to B. edulis, which showed greater variability and slightly lower overall efficiency. Metal mixtures did not significantly alter these trends. These findings underscore the necessity of optimizing γ-PGA concentrations to balance biosorption efficacy in practical bioremediation or food safety applications involving edible mushrooms.

Antimicrobial Activity of γ-PGA in Combination with Edible Mushrooms

γ-Polyglutamic acid (γ-PGA) at a 1 mM concentration significantly enhanced antimicrobial activity against Salmonella enteritidis ATCC 13076 and Escherichia coli ATCC 25922 in cultures of the edible mushrooms Pleurotus ostreatus, Agaricus bisporus, and Boletus edulis, as well as when applied alone. Inhibition zone diameters increased from approximately 20 mm to 25 mm for Salmonella enteritidis ATCC 13076 in P. ostreatus and A. bisporus and from about 6 mm to 14 mm in B. edulis. Similarly, for Escherichia coli ATCC 25922, zones expanded from roughly 16 mm to 23 mm in P. ostreatus, about 6 mm to 15–16 mm in B. edulis, and from 18 mm up to 21 mm in A. bisporus. These quantitative increases demonstrate the effective antimicrobial enhancement afforded by γ-PGA, underscoring its potential as a natural additive to improve microbial safety and product shelf life in mushroom cultivation and postharvest management. Future research should explore the underlying molecular mechanisms driving the synergistic interactions and assess in vivo effectiveness in cultivation and storage settings to optimize γ-PGA utilization for biosafety enhancement in edible mushroom production.