Optimizing Ergothioneine Biosynthesis and Antioxidant Activity in Agaricus spp. Through Amino Acid Supplementation and Yeast–Peptone Mixtures

Abstract

With increasing demand for antioxidant-rich foods, research has focused on cost-effective methods to produce natural antioxidants. Mushrooms, especially Agaricus species, are rich in bioactive compounds like ergothioneine, a potent antioxidant. Ergothioneine has been shown to offer significant health benefits, such as protecting against oxidative stress, cardiovascular diseases, and premature aging. This study explores the effects of amino acid supplementation (methionine, cysteine, and histidine) and yeast–peptone mixtures on ergothioneine production, antioxidant activity, total phenolic content, and growth rate in various Agaricus species; this was conducted through two distinct experiments within a completely randomized design. In the first experiment, 13 treatment combinations were tested, involving varying concentrations of individual amino acids (methionine, cysteine, and histidine) at 0.5, 1, and 2 mM, as well as their combined concentrations (0.5 + 0.5 + 0.5, 1 + 1 + 1, and 2 + 2 + 2 mM), compared to a control (no amino acids). The second experiment tested yeast extract and peptone mixtures at seven concentrations: control (no supplementation), yeast (2 and 4 g/L), peptone (2 and 4 g/L), and combinations of yeast and peptone (2 + 2 and 4 + 4 g/L). Results revealed that supplementation with amino acids at 1 + 1 + 1 mM significantly enhanced ergothioneine content and antioxidant activity, though it resulted in decreased growth rates. In contrast, lower concentrations of amino acids (0.5 + 0.5 + 0.5 mM) increased ergothioneine production, with minimal impact on growth. Yeast and peptone supplementation at 2 + 2 g/L yielded the highest ergothioneine content, antioxidant activity, and growth rates across all Agaricus species tested. The most effective combination for maximizing ergothioneine production, antioxidant activity, and growth was found to be 0.5 mM of methionine, cysteine, and histidine, combined with 2 g/L of yeast extract and 2 g/L of peptone. Agaricus bitorquis (Quél.) Sacc. emerged as a promising candidate for ergothioneine production due to its genetic potential and metabolic efficiency. However, the strong responsiveness of Agaricus bisporus (white) to optimized culture conditions offers a viable alternative to A. bitorquis, which may require more complex and costly cultivation strategies.

1. Introduction

In recent years, increasing concerns regarding human health have led to a significant surge in demand for foods and supplements rich in antioxidants. This growing interest has spurred research into cost-effective methods for the synthetic or natural production of antioxidants for human consumption [1]. Among these efforts, considerable attention has been directed toward plant-based antioxidants, particularly phytochemicals, which play a pivotal role in the development of functional foods [2,3]. Mushrooms, as a distinct group of edible fungi, are recognized for their rich phytochemical content, including phenolic compounds that exhibit notable antioxidant activity [4]. Despite their considerable nutritional potential, mushrooms have historically received less attention than other vegetables regarding their dietary significance. However, their distinctive bioactive composition has increasingly attracted scientific interest, particularly in the realm of functional foods and disease prevention. Among the various mushroom species, Agaricus bisporus (button mushroom) is the most widely consumed worldwide, valued not only for its accessibility and culinary adaptability but also for its rich array of bioactive compounds. These include ergothioneine (2-mercaptohistidine trimethylbetaine, Ergo), a sulfur-containing antioxidant with exceptional cytoprotective properties, as well as polysaccharides, amino acids, phenolic compounds, dietary fiber, vitamins, and essential minerals [5]. This study primarily focuses on Ergo, a potent antioxidant found in mushrooms, exploring its potential for human health and functional food development.

Ergo is a naturally occurring amino acid analogue that is primarily biosynthesized by mycobacteria and fungi, including certain mushroom species [6]. This water-soluble compound exhibits antioxidant properties through multiple mechanisms, with its most prominent function being the scavenging of free radicals [5,7]. As a potent antioxidant, Ergo interacts with reactive oxygen species (ROS) and free radicals, thereby preventing cellular damage and protecting biological structures from oxidative harm. These properties have positioned Ergo as a potential protective agent against chronic conditions, including cardiovascular diseases, type 2 diabetes, and premature aging [8]. The primary mechanism by which Ergo exerts its antioxidant effects is by reducing oxidative stress and preventing damage to crucial macromolecules, such as DNA and proteins, thereby mitigating the risk of cellular degradation [9]. Notably, Ergo is highly concentrated within mitochondria, where it plays a critical role in protecting these vital organelles from oxidative damage [10]. This protection helps prevent genetic mutations and protein degradation typically induced by oxidative stress, underscoring Ergo’s significance in cellular defense and longevity. Given its substantial health benefits, Ergo has been associated with a range of therapeutic effects, including cataract prevention, enhanced metabolic energy, reduced inflammation, and protection against cardiovascular diseases [5,11]. However, despite its dietary importance, A. bisporus contains lower concentrations of Ergo compared to certain other mushroom species, highlighting the need for optimization strategies to enhance its biosynthesis. Since humans lack the enzymatic pathways necessary to synthesize Ergo endogenously, dietary intake is the sole determinant of its physiological availability, making mushrooms—particularly A. bisporus—a vital dietary source. Among various food sources, mushrooms are the richest contributors, containing between 0.1 and 1 mg/g of Ergo in dried material [12,13]. This underscores their crucial role as a natural dietary reservoir of this potent antioxidant.

To elucidate the biosynthetic pathway of Ergo, researchers have employed various isotopic and competition experiments. Strong evidence supports the idea that Ergo is derived from the amino acids methionine (Met), histidine (His), and cysteine (Cys), which serve as intermediates in its biosynthesis [13,14]. Several studies have explored the manipulation of culture media components to enhance Ergo accumulation in mushroom fruiting bodies and mycelia [15,16,17]. Among different cultivation techniques, submerged fermentation has gained attention as a promising method due to its shorter production time and higher yield compared to traditional fruiting body cultivation. This technique enables controlled environmental conditions, leading to more efficient secondary metabolite production. In the present study, we developed a submerged fermentation approach to enhance Ergo production in mushroom mycelia. However, despite the growing body of research on submerged fermentation, there remains a notable gap in the systematic optimization of culture media components to maximize Ergo biosynthesis. Specifically, limited studies have comprehensively examined the precise modulation of the carbon-to-nitrogen (C/N) ratio, trace element supplementation, and pH regulation—factors that are known to significantly influence secondary metabolite production in fungi. Additionally, the interactive effects of these parameters on Ergo yield remain largely unexplored; thus, the study of these is crucial for achieving an optimal metabolic state in fungal cultures. Given that Ergo is widely recognized for its potent antioxidant, neuroprotective, and immunomodulatory properties, optimizing its production not only enhances its pharmaceutical potential but also improves large-scale commercial viability [6]. Addressing these knowledge gaps will contribute to the advancement of fungal biotechnology and ensure the sustainable and efficient production of Ergo through submerged fermentation.

The composition of culture media is a critical factor in fungal cultivation, directly influencing metabolic activity, the biosynthesis of bioactive compounds, and overall growth dynamics [18]. Among the essential constituents, yeast extract serves as a highly nutritious supplement, providing key biomolecules such as amino acids, peptides, B vitamins, and nucleotides [19]. As a complex organic source, yeast extract plays a pivotal role in regulating metabolic pathways, facilitating the synthesis of secondary metabolites, and ultimately enhancing fungal growth and physiological performance [20]. Another vital constituent of culture media is peptone, a hydrolyzed protein derivative obtained through the enzymatic or chemical hydrolysis of various protein sources, including casein, gelatin, meat, and soy [21]. Peptones are rich in peptides, free amino acids, vitamins, and trace elements, serving as a primary nitrogen source that supports microbial growth, enzymatic biosynthesis, and metabolic efficiency [22]. The combined presence of yeast extract and peptone establishes a highly effective nutrient matrix, facilitating the cultivation of fungi and other microorganisms across a broad spectrum of applications, including microbiological research, large-scale fermentation, pharmaceutical production, and biotechnological advancements [20,21].

Among various mushroom species, Agaricus spp. have been found to contain relatively low concentrations of Ergo [23]. As a result, there is a strong need for effective methods to enhance Ergo production in Agaricus spp., especially since button mushrooms (A. bisporus) are the most widely cultivated and consumed mushrooms worldwide. This study aims to optimize culture media for Ergo production and antioxidant activity in four Agaricus strains by supplementing them with specific amino acid precursors. Additionally, in a second experiment, we examine the impact of yeast extract and peptone, as organic nitrogen sources, on Ergo production and antioxidant activity in the same four Agaricus strains.

2. Materials and Methods

2.1. Microorganisms

The mycelial strains of various mushroom species were generously provided by Balot Co. (Tehran, Iran). The mushroom strains used in this study included Agaricus bitorquis (Quél.) Sacc., Agaricus blazei Murrill, Agaricus bisporus (white) (J.E.Lange) Imbach, and Agaricus bisporus (brown) (J.E.Lange) Imbach. Each strain was maintained on potato dextrose agar (PDA) plates (purchased from Merck, Darmstadt, Germany, purity ≥ 99%) and subcultured every two months. The plates were incubated at 25 °C for 12 days and subsequently stored at 4 °C.

2.2. Inoculation and Fermentation Conditions

All liquid cultivation experiments were conducted in 250vmL Erlenmeyer flasks containing 150 mL of medium and were incubated at 25 °C for 25 days with a rotational speed of 140 rpm on a rotary shaker. The FGM medium was used for culture optimization in this study. This medium consisted of 25 g/L of glucose (Sigma-Aldrich, Saint Louis, MO, USA, purity ≥ 99%), 2 g/L of yeast extract (Merck, Germany), 1 g/L of glutamic acid (Sigma-Aldrich, purity ≥ 98%), 0.5 mg/L of biotin (Sigma-Aldrich, purity ≥ 97%), 0.1 g/L of thiamine (Merck, Germany), 2 g/L of KH₂PO₄ (Sigma-Aldrich, purity ≥ 99%), 0.5 g/L of MgSO₄ (Merck, purity ≥ 98%), 5 mL of 0.1 M FeCl₃ (Sigma-Aldrich), and 5 mL of 0.1 M MnSO₄ (Sigma-Aldrich). The initial pH of the medium was adjusted to 5.5 before autoclaving at 121 °C for 15 min. Each mycelial strain was initially cultured on sterilized PDA medium in Petri dishes for 12 days. The PDA medium was then cut into approximately 5 mm × 5 mm squares using a sterilized blade and transferred to the seed culture medium.

Supplementation with Amino Acid

To evaluate the effect of amino acid precursor supplementation on Ergo production in four different Agaricus strains, a completely randomized design (CRD) with three replications was employed. Thirteen treatments with various amino acid combinations were tested. The selected amino acid concentrations were based on previous studies [24,25,26] and included the following: control (no amino acid), Met at 0.5, 1, and 2 mM; His at 0.5, 1, and 2 mM; Cys at 0.5, 1, and 2 mM; as well as combined treatments of Met, His, and Cys at concentrations of 0.5 + 0.5 + 0.5, 1 + 1 + 1, and 2 + 2 + 2 mM. The fermentation period for this experiment was set at 25 days based on preliminary studies and the existing literature [20,21,27], which suggest that amino acid assimilation and the subsequent biosynthesis of secondary metabolites, particularly Ergo, require an extended cultivation period.

2.3. Optimization of Different Organic Nitrogen Sources

This liquid cultivation experiment was conducted in 250 mL Erlenmeyer flasks containing 150 mL of medium, incubated at 25 °C for 15 days with a shaking speed of 140 rpm on a rotary shaker. To determine the optimal nitrogen source for four Agaricus strains, the most suitable mycelium and the best treatment from the previous experiment (0.5 mM amino acid combination) were cultured using the basic medium (FGM). A CRD with three replications was used. Seven different treatments incorporating yeast extract and peptone as organic nitrogen sources were applied, with concentration levels selected based on previous studies [19,20,22]. The treatments included a control group (no nitrogen supplementation), yeast extract at 2 and 4 g/L, peptone at 2 and 4 g/L, and combined treatments of yeast extract and peptone at 2 + 2 g/L and 4 + 4 g/L, respectively. The fermentation period was set at 15 days based on preliminary observations, which indicated that nitrogen assimilation for primary metabolism in submerged fungal cultures reached a plateau within this timeframe. This decision is further supported by prior studies and literature findings [16,23].

2.4. Harvesting of Mycelia and Determination of Mycelia Growth

At the end of each fermentation process, the cultured mycelia were separated from the medium by centrifugation at 5000 rpm for 20 min. The mycelia were then thoroughly washed with 500 mL of distilled water. The culture medium was separated from the mycelia using Whatman No. 1 filter paper. The washed mycelia were dried in an oven at 60 °C for 48 h to determine the dry weight (DW). The growth rate was calculated using the following formula:

$$\mathrm{Growth} \mathrm{rate} \left(\mathrm{g} \mathrm{DW}/\mathrm{L}\right)={\displaystyle \frac{\mathrm{dry} \mathrm{weight} \mathrm{of} \mathrm{mycelia} \left(\mathrm{g}\right)}{\mathrm{volume} \mathrm{of} \mathrm{culture} \left(\mathrm{L}\right)}}$$

(1)

2.5. Extraction Procedures

The samples were freeze-dried using a Christ Alpha 1–2 LDplus (Martin Christ, Osterode, Germany) under a vacuum pressure of 0.01 mbar at −50 °C and then finely ground into a uniform powder. For Soxhlet extraction, 10 g of mycelial powder was placed in a cellulose thimble and extracted with 200 mL of methanol (Merck, Germany) at 60 °C for 4 h. The extract was then filtered through Whatman No. 1 filter paper and concentrated using a rotary evaporator under reduced pressure at 40 °C [28]. The resulting methanolic extract was stored at −20 °C until further analysis and subsequently used for the determination of total phenolic content and antioxidant activity.

2.6. Antioxidant-Related Parameters

2.6.1. DPPH Radical Scavenging Activity

The DPPH free radical scavenging assay was conducted following the Method of Bozin et al. [29], with some modifications. Briefly, various concentrations of each extract were added to 1 mL of 90 µM DPPH solution, and the volume was adjusted to 3 mL with 95% v/v Methanol. The mixture was shaken immediately after the DPPH solution was added and then allowed to stand for 1 h at room temperature in the dark. The absorbance was measured at 517 nm against a blank (consisting of the same solution without the added extract). Three replicates were performed for each sample. Synthetic antioxidant BHT (butylated hydroxytoluene) was used as a positive control. The radical scavenging capacity (RSC) was calculated using the following equation:

$$\mathrm{R}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l} \mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g} \mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}={\displaystyle \frac{A_{\mathit{blank}}-A_{\mathit{sample}}}{A_{\mathit{blank}}}}\times 100$$

(2)

At the conclusion of the antioxidant activity measurement, the extract’s effectiveness was expressed in terms of IC50. IC50 represents the concentration of the extract that inhibits 50% of free radicals. This value was determined using linear regression analysis based on the antioxidant activity data at various extract concentrations [30].

2.6.2. Ferric Reducing Antioxidant Power (FRAP) Assay

The ability to reduce ferric ions was assessed using a modified version of the Benzie and Strain method [31]. To prepare the FRAP reagent, 100 mL of acetate buffer (300 mM, pH 3.6) was mixed with 10 mL of 2,4,6-tripyridyl-S-triazine (TPTZ) (10 mM in 40 mM HCl) and 10 mL of FeCl₃·6H₂O (20 mM) in a ratio of 10:1:1 and was incubated at 37 °C. The absorbance of the test tubes containing 2 mL of reagent and 200 µL of sample extract was measured at 593 nm after 5 min, using the FRAP solution as the blank. Trolox was used for calibration, with the absorbance range expected to be 0.2. The antioxidant capacity, based on the ability of the sample to reduce ferric ions, was calculated from the linear calibration curve and expressed as µmol Trolox equivalents per gram of plant dry weight (µmol T/g DW).

2.6.3. Total Phenolic Content

The total phenolic content of the extracts was determined using the Folin–Ciocalteu Method [32], with slight modifications. A 20 µL aliquot of each extract was mixed with 100 µL of Folin–Ciocalteu reagent and 1.6 mL of distilled water. After 3 min, 300 µL of 7% sodium carbonate solution was added to the mixture. The final solution was shaken for 2 h, after which the absorbance was measured at 765 nm. The results were calculated using a calibration curve generated with gallic acid (ranging from 0 to 1000 mg/L) and were expressed as milligrams of gallic acid equivalents per gram of dry weight (mg GAE/g DW).

2.7. Ergo Content

The method employed for quantifying Ergo in mushrooms was adapted from Dubost et al. [33] with some modifications. Freeze-dried mushroom powder (1 g) was mixed with 20 mL of the extraction solution, which consisted of 10 mmol/L of 1,4-Dithiothreitol, 100 mmol/L of betaine, and 100 mmol/L of 2-mercapto-1-Methylimidazole in 70% ethanol, and was vortexed for 90 s. To this mixture, 4 mL of a 10 g/L of sodium dodecyl sulfate solution was added, and the mixture was centrifuged at 3000× g for 10 min at 25 °C. The resulting supernatant was concentrated by rotary evaporation at 40 °C to a final volume of 5 mL and then filtered through a 0.45 µm CA non-sterile filter.

The analysis was conducted using high-performance liquid chromatography (HPLC), with separation performed on two Econosphere C18 columns (Alltech Associates, Deerfield, IL, USA), each measuring 250 mm × 4.6 mm with a 5 µm particle size, connected in tandem. The isocratic mobile phase consisted of 50 mM of sodium phosphate in water, with 3% acetonitrile and 0.1% triethylamine, adjusted to a pH of 7.3. The flow rate was set to 1 mL/min. A UV-VIS detector operating at 254 nm was used for detection. The injection volume was 10 µL, and the column temperature was maintained at ambient conditions. Ergo quantification was achieved by monitoring the absorbance at 254 nm and comparing the peak area of the sample to those of authentic standard concentrations. Ergo was identified by comparing the retention time of the peak with that of authentic standard. Quantification of the Ergo was based on multilevel external calibration curves. According to the results, the regression equation, R², LOD, LOQ, and concentration range were y = 107.02x − 110.88, 0.9898, 15 ng/mL, 53 ng/mL, and 0.32–108 µg/mL. All data were expressed as milligrams of Ergo per gram of dry weight (mg/g DW). For accuracy, triplicate analyses were performed for each sample.

2.8. Statistical Analyses

All analyses were conducted in triplicate to ensure reproducibility. Statistical analyses were performed using SAS 9.1 software. Data were subjected to analysis of variance (ANOVA), and mean comparisons were conducted using the least significant difference (LSD) test at p < 0.05.

3. Results

3.1. Effects of Different Amino Acids on Ergo Contents and Growth Rate

As shown in

Table 1. Effects of supplementing methionine, histidine, and cysteine on ergothioneine content and growth rate in various Agaricus species.

Amino Acid (mM)	Agaricus bitorquis		Agaricus blazei		Agaricus bisporus (White)		Agaricus bisporus (Brown)
Met * + Cys ** + His ***	Ergothioneine (mg/g DW)	Growth Rate (g DW/L)	Ergothioneine (mg/g DW)	Growth Rate (g DW/L)	Ergothioneine (mg/g DW)	Growth Rate (g DW/L)	Ergothioneine (mg/g DW)	Growth Rate (g DW/L)
0 + 0 + 0	1.03 ± 0.1 e ****	3.1 ± 0.43 a	0.89 ± 0.13 j	2.86 ± 0.48 a	0.46 ± 0.14 i	2.7 ± 0.37 a	0.57 ± 0.15 j	2.48 ± 0.35 a
0.5 + 0 + 0	1.24 ± 0.12 cd	2.87 ± 0.52 b	1.12 ± 0.18 g	2.64 ± 0.43 c	0.67 ± 0.16 ef	2.55 ± 0.5 cd	0.85 ± 0.19 fg	2.22 ± 0.43 de
1 + 0 + 0	1.45 ± 0.25 bcd	2.75 ± 0.4 cd	1.27 ± 0.21 e	2.55 ± 0.44 d	0.79 ± 0.15 cde	2.47 ± 0.25 ef	0.96 ± 0.16 d	2.22 ± 0. 5 de
2 + 0 + 0	1.65 ± 0.11 ab	2.66 ± 0.4 de	1.45 ± 0.23 c	2.54 ± 0.38 d	0.87 ± 0.23 c	2.42 ± 0.34 fg	1.05 ± 0.25 c	2.1 ± 0.37 ef
0 + 0.5 + 0	1.17 ± 0.12 d	2.93 ± 0.53 b	0.92 ± 0.15 i	2.74 ± 0.42 b	0.57 ± 0.16 h	2.67 ± 0.33 b	0.70 ± 0.14 i	2.36 ± 0.44 b
0 + 1 + 0	1.25 ± 0.12 cd	2.87 ± 0.41 b	1.11 ± 0.21 g	2.66 ± 0.56 c	0.67 ± 0.18 fg	2.58 ± 0.52 c	0.81 ± 0.18 gh	2.29 ± 0.43 bc
0 + 2 + 0	1.32 ± 0.11 cd	2.73 ± 0.3 de	1.16 ± 0.24 f	2.56 ± 0.45 d	0.72 ± 0.24 ef	2.46 ± 0.39 ef	0.85 ± 0.19 fg	2.19 ± 0.41 ef
0 + 0 + 0.5	1.19 ± 0.13 d	2.85 ± 0.4 bc	1.06 ± 0.16 h	2.66 ± 0.38 c	0.63 ± 0.12 gh	2.57 ± 0.4 cd	0.77 ± 0.17 h	2.29 ± 0.55 bc
0 + 0 + 1	1.33 ± 0.13 cd	2.7 ± 0.53 de	1.17 ± 0.14 f	2.53 ± 0.39 d	0.75 ± 0.16 ef	2.49 ± 0.35 fg	0.89 ± 0.22 ef	2.18 ± 0.38 ef
0 + 0 + 2	1.45 ± 0.2 bcd	2.64 ± 0.32 e	1.26 ± 0.23 e	2.47 ± 0.35 e	0.78 ± 0.13 e	2.39 ± 0.33 g	0.94 ± 0.23 de	2.14 ± 0.36 f
0.5 + 0.5 + 0.5	1.84 ± 0.14 a	2.89 ± 0.33 b	1.58 ± 0.26 b	2.64 ± 0.43 c	0.98 ± 0.25 b	2.68 ± 0.57 b	1.17 ± 0.28 b	2.41 ± 0.46 b
1 + 1 + 1	1.81 ± 0.14 a	2.38 ± 0.41 f	1.82 ± 0.27 a	2.15 ± 0.39 f	1.21 ± 0.27 a	2.05 ± 0.39 h	1.33 ± 0.23 a	1.11 ± 0.13 g
2 + 2 + 2	1.51 ± 0.15 abc	1.93 ± 0.28 g	1.38 ± 0.29 d	1.83 ± 0.27 g	0.86 ± 0.15 cd	1.74 ± 0.23 i	1.04 ± 0.25 c	1.06 ± 0.27 h

* Met: methionine; ** Cys: cysteine; *** His: histidine; **** means with similar letters in each column, based on an LSD test at a 0.05% probability level, are not significantly different.

, the results demonstrate that the highest Ergo levels in A. bitorquis were achieved with the Met + Cys + His treatment at concentrations of 0.5 + 0.5 + 0.5 mM and 1 + 1 + 1 mM, resulting in a 78.64% and 75.72% increase compared to the control group, respectively. Additionally, supplementation with Met + Cys + His at concentrations of 2 + 0 + 0 mM and 2 + 2 + 2 mM also led to high Ergo levels. In A. blazei, A. bisporus (white), and A. bisporus (brown), the highest Ergo levels were observed with the Met + Cys + His treatment at 1 + 1 + 1 mM, where the Ergo concentrations increased by 1.70-fold, 2.63-fold, and 2.33-fold compared to the control group. The lowest Ergo levels were found in the control treatment across all four Agaricus species. Furthermore, Table 1 shows that the maximum growth rate for all four Agaricus species was observed in the control group, while the minimum growth rate occurred with the Met + Cys + His supplementation at a concentration of 2 + 2 + 2 mM.

3.2. Effects of Yeast Extract and Peptone on Ergo Contents and Growth Rate

As shown in

Table 2. Effects of supplementing yeast and peptone on ergothioneine content and growth rate in various Agaricus species.

Yeast + Peptone (g/L)	Agaricus bitorquis		Agaricus blazei		Agaricus bisporus (White)		Agaricus bisporus (Brown)
	Ergothioneine (mg/g DW)	Growth Rate (g DW/L)	Ergothioneine (mg/g DW)	Growth Rate (g DW/L)	Ergothioneine (mg/g DW)	Growth Rate (g DW/L)	Ergothioneine (mg/g DW)	Growth Rate (g DW/L)
0 + 0	1.02 ± 0.11 d *	1.25 ± 0.15 d	0.92 ± 0.13 f	1.17 ± 0.25 d	0.49 ± 0.14 d	0.88 ± 0.13 d	0.58 ± 0.15 f	0.95 ± 0.21 d
2 + 0	2.15 ± 0.18 b	2.43 ± 0.27 b	1.84 ± 0.33 b	2.27 ± 0.32 b	1.56 ± 0.31 b	1.18 ± 0.18 b	1.53 ± 0.34 b	1.84 ± 0.34 b
4 + 0	1.91 ± 0.24 c	2.02 ± 0.35 bc	1.61 ± 0.24 d	1.91 ± 0.31 c	1.38 ± 0.23 c	1.43 ± 0.19 bc	1.34 ± 0.22 d	1.54 ± 0.33 bc
0 + 2	2.1 ± 0.25 b	1.92 ± 0.33 c	1.73 ± 0.33 c	1.79 ± 0.23 c	1.52 ± 0.27 b	1.35 ± 0.21 c	1.44 ± 0.21 c	1.45 ± 0.27 c
0 + 4	1.81 ± 0.21 c	2 ± 0.39 c	1.51 ± 0.24 e	1.88 ± 0.25 c	1.31 ± 0.22 c	1.41 ± 0.26 c	1.25 ± 0.29 e	1.52 ± 0.19 c
2 + 2	2.17 ± 0.38 a	3.05 ± 0.62 a	2.04 ± 0.29 a	2.85 ± 0.53 a	1.96 ± 0.26 a	2.14 ± 0.29 a	1.69 ± 0.28 a	2.31 ± 0.39 a
4 + 4	1.14 ± 0.26 d	1.26 ± 0.28 d	1.1 ± 0.18 e	1.18 ± 0.32 d	0.74 ± 0.21 d	0.89 ± 0.18 d	0.83 ± 0.13 e	0.93 ± 0.13 d

* Means with similar letters in each column, based on an LSD test at a 0.05% probability level, are not significantly different.

, the yeast + peptone treatment at a concentration of 2 + 2 g/L resulted in the highest Ergo content across all Agaricus species tested. This treatment led to a 2.12-fold, 2.21-fold, 4.00-fold, and 2.91-fold increase in Ergo content in A. bitorquis, A. blazei, A. bisporus (white), and A. bisporus (brown), respectively, compared to the control group. In contrast, the control group consistently exhibited the lowest Ergo content in A. blazei and A. bisporus (brown). For A. bitorquis and A. bisporus (white), both the control group and the yeast + peptone treatment at a concentration of 4 + 4 g/L resulted in the lowest Ergo content. A similar trend was observed in growth rate, with the yeast + peptone treatment at 2 + 2 g/L yielding the highest growth rates in all Agaricus species. Specifically, this treatment led to 2.44-fold increases in A. bitorquis, 2.43-fold increases in A. blazei, 2.43-fold increases in A. bisporus (white), and 2.43-fold increases in A. bisporus (brown) compared to the control group. In contrast, the lowest growth rates were consistently recorded in both the untreated control group and the yeast + peptone treatment at 4 + 4 g/L.

3.3. Effects of Different Amino Acids on Antioxidant-Related Parameters

The results demonstrated that all Agaricus species extracts exhibited varying degrees of free radical scavenging activity. The highest antioxidant activity, as measured by the DPPH assay, was observed in the treatment supplemented with a 1 mM combination of Met, Cys, and His across all Agaricus strains. This treatment showed the lowest IC50, indicating the strongest antioxidant properties. In contrast, the control group, which lacked amino acid supplementation, consistently exhibited the lowest antioxidant activity. Notably, in A. bitorquis, the treatments with 0.5 + 0 + 0 and 0 + 0.5 + 0 Met + Cys + His showed low antioxidant activity, with no significant difference compared to the control (

Table 3. Effects of supplementing methionine, histidine, and cysteine on antioxidant activity and total phenolic content in various Agaricus species.

Amino Acid (mM)	Agaricus bitorquis			Agaricus blazei			Agaricus bisporus (White)			Agaricus bisporus (Brown)
Met * + Cys ** + His ***	DPPH (mg/mL)	FRAP (µmol T/g DW)	Total Phenolic (mg GAE/g DW)	DPPH (mg/mL)	FRAP (µmol T/g DW)	Total Phenolic (mg GAE/g DW)	DPPH (mg/mL)	FRAP (µmol T/g DW)	Total Phenolic (mg GAE/g DW)	DPPH (mg/mL)	FRAP (µmol T/g DW)	Total Phenolic (mg GAE/g DW)
0 + 0 + 0	4.21 ± 0.5 a ****	320.3 ± 22.5 h	5.73 ± 1.12 f	4.73 ± 0.65 a	308.8 ± 22.6 i	5.42 ± 1.02 f	5.11 ± 0.94 a	257.2 ± 22.9 i	4.83 ± 0.93 c	4.85 ± 0.86 a	281.5 ± 24.9 i	5.00 ± 1.04 g
0.5 + 0 + 0	4.13 ± 0.45 ab	340.7 ± 28.5 g	5.95 ± 0.95 de	4.47 ± 0.67 b	324.6 ± 31.5 g	5.56 ± 0.82 de	4.86 ± 0.87 b	270.9 ± 26.6 gh	4.97 ± 1.04 bc	4.65 ± 0.99 b	297.4 ± 28.8 g	5.05 ± 1.33 fg
1 + 0 + 0	3.93 ± 0.39 c	367.9 ± 31.3 d	6.03 ± 1.24 cd	4.35 ± 0.72 c	346.0 ± 28.7 d	5.65 ± 0.91 bc	4.69 ± 0.82 de	291.1 ± 28.2 de	5.05 ± 1.11 abc	4.47 ± 0.93 de	319.1 ± 32.1 d	5.22 ± 1.11 bc
2 + 0 + 0	3.90 ± 0.54 c	368.4 ± 11.5 d	6.16 ± 1.05 b	4.26 ± 0.63 d	345.8 ± 29.4 d	5.68 ± 0.96 b	4.65 ± 0.94 e	294.8 ± 32.6 d	5.05 ± 0.84 abc	4.43 ± 1.14 e	320.2 ± 27.4 d	5.23 ± 1.22 bc
0 + 0.5 + 0	4.13 ± 0.65 ab	337.3 ± 16.7 g	6.06 ± 0.88 bc	4.47 ± 0.65 b	317.4 ± 33.5 h	5.62 ± 1.22 c	4.85 ± 1.01 b	266.3 ± 31.7 h	4.95 ± 0.87 c	4.55 ± 1.22 cd	292.2 ± 24.4 h	5.12 ± 1.29 def
0 + 1 + 0	4.07 ± 0.76 b	345.1 ± 33.4 fg	5.95 ± 1.11 de	4.44 ± 0.95 b	333.1 ± 37.6 f	5.57 ± 1.18 d	4.77 ± 0.71 c	276.8 ± 28.6 fg	6.63 ± 1.08 c	4.57 ± 0.74 bc	303.7 ± 23.3 f	5.12 ± 1.24 de
0 + 2 + 0	4.06 ± 0.73 b	337.9 ± 23.7 g	5.93 ± 0.85 de	4.49 ± 0.91 b	322.8 ± 26.9 g	5.52 ± 1.01 e	4.89 ± 0.79 b	269.2 ± 25.4 gh	4.92 ± 1.24 c	4.64 ± 0.77 b	296.1 ± 25.6 g	5.06 ± 1.35 efg
0 + 0 + 0.5	3.93 ± 0.55 c	349.4 ± 42.5 ef	6.07 ± 1.22 bc	4.37 ± 0.82 c	331.1 ± 25.5 f	5.62 ± 1.13 c	4.74 ± 0.77 cd	277.1 ± 20.4 fg	5.00 ± 1.33 bc	4.50 ± 0.82 cde	303.9 ± 29.9 f	5.15 ± 1.41 cd
0 + 0 + 1	3.95 ± 0.52 c	347.1 ± 38.7 fg	6.08 ± 1.16 bc	4.35 ± 0.75 c	334.6 ± 37.8 f	5.63 ± 1.06 c	4.69 ± 0.96 de	280.5 ± 22.6 f	5.03 ± 1.14 abc	4.49 ± 0.84 de	305.2 ± 21.3 f	5.20 ± 1.44 bc
0 + 0 + 2	3.91 ± 0.59 c	357.5 ± 25.9 e	5.95 ± 0.98 de	4.28 ± 0.62 d	341.1 ± 39.5 e	5.52 ± 0.88 d	4.65 ± 0.98 e	285.1 ± 22.6 ef	4.92 ± 1.33 c	4.43 ± 1.14 e	312.1 ± 32.3 e	5.21 ± 1.13 bc
0.5 + 0.5 + 0.5	3.71 ± 0.48 d	424.1 ± 23.4 b	6.37 ± 0.93 a	4.09 ± 0.96 e	407.2 ± 51.4 b	5.92 ± 1.02 a	4.42 ± 0.66 f	338.1 ± 20.9 b	5.26 ± 0.84 ab	4.21 ± 1.11 f	371.8 ± 33.1 b	5.48 ± 1.19 a
1 + 1 + 1	3.43 ± 0.44 e	448.2 ± 33.7 a	6.17 ± 1.21 b	3.75 ± 0.52 f	426.7 ± 42.4 a	5.66 ± 1.08 b	4.10 ± 0.69 g	361.3 ± 28.9 a	4.87 ± 0.88 c	3.90 ± 0.77 g	392.8 ± 26.6 a	5.28 ± 1.41 b
2 + 2 + 2	3.70 ± 0.45 d	394.1 ± 26. 5 c	5.85 ± 0.84 e	4.05 ± 0.55 e	375.1 ± 38.7 c	5.45 ± 1.01 f	4.44 ± 0.90 f	319.1 ± 32.7 c	4.85 ± 0.99 c	4.20 ± 0.84 f	346.5 ± 25.9 c	5.24 ± 1.03 b

* Met: methionine; ** Cys: cysteine; *** His: histidine; **** means with similar letters in each column, based on an LSD test at a 0.05% probability level, are not significantly different.

).

The results from the FRAP assay corroborated the findings from the DPPH assay, both revealing the reducing power of all tested strains. The highest antioxidant activity, as assessed by the FRAP assay, was again observed in the treatment supplemented with the 1 mM combination of Met, Cys, and His. Specifically, this treatment led to a 1.4-fold increase in antioxidant activity in A. bitorquis, 1.38-fold in A. blazei, 1.40-fold in A. bisporus (white), and 1.39-fold in A. bisporus (brown) compared to the control group. Conversely, the control group consistently exhibited the lowest antioxidant activity (Table 3).

The application of Met + Cys + His at a concentration of 0.5 + 0.5 + 0.5 mM resulted in the highest levels of phenolic content across all Agaricus strains. This treatment caused a significant increase in total phenolic content, with A. bitorquis showing an 11.16% increase, A. blazei a 9.22% increase, A. bisporus (white) an 8.90% increase, and A. bisporus (brown) a 9.60% increase compared to the control group. However, in A. bisporus (white), treatments with 1 + 0 + 0, 2 + 0 + 0, 0 + 0 + 1, and 0.5 + 0.5 + 0.5 mM Met + Cys + His also resulted in high phenolic content, with no significant difference from the 0.5 + 0.5 + 0.5 mM Met + Cys + His treatment (Table 3).

3.4. Effects of Yeast Extract and Peptone on Antioxidant-Related Parameters

The results demonstrated that the highest antioxidant activity, as measured by the DPPH assay, was observed in A. bitorquis, A. blazei, and A. bisporus (white) when yeast extract and peptone were applied at concentrations of 2 + 2 g/L, with IC50 values of 3.07, 3.72, and 4.51, respectively. In A. bisporus (brown), the greatest antioxidant activity in the DPPH assay occurred when yeast extract and peptone were applied at either 2 + 2 or 4 + 4 g/L, resulting in IC50 values of 4.27 and 4.24 mg/mL, respectively. For the FRAP assay, the highest antioxidant activity in both A. bitorquis and A. blazei was achieved with the 0 + 2 and 2 + 2 g/L yeast + peptone treatments. In A. bisporus (white) and A. bisporus (brown), the maximum antioxidant activity was observed with yeast + peptone concentrations of 0 + 2 and 0 + 4 g/L, with no significant difference compared to the 4 + 0 and 2 + 2 g/L treatments.

Regarding total phenol content, the application of 2 + 2 g/L yeast + peptone led to the highest increase in A. bitorquis and A. bisporus (white), with increases of 21.09% and 9.96%, respectively, compared to the control. The highest total phenol content in A. blazei was achieved with 4 + 4 and 2 + 2 g/L yeast + peptone treatments, reaching 6.17 and 6.22 mg GAE/g DW. In A. bisporus (brown), the highest total phenol content was observed with 0 + 2 and 2 + 2 g/L yeast + peptone treatments, though no significant difference was found when compared to the 4 + 0 and 4 + 4 g/L yeast + peptone treatments (

Table 4. Effects of supplementing yeast and peptone on antioxidant activity and total phenolic content in various Agaricus species.

Yeast + Peptone (g/L)	Agaricus bitorquis			Agaricus blazei			Agaricus bisporus (White)			Agaricus bisporus (Brown)
	DPPH (mg/mL)	FRAP (µmol T/g DW)	Total Phenolic (mg GAE/g DW)	DPPH (mg/mL)	FRAP (µmol T/g DW)	Total Phenolic (mg GAE/g DW)	DPPH (mg/mL)	FRAP (µmol T/g DW)	Total Phenolic (mg GAE/g DW)	DPPH (mg/mL)	FRAP (µmol T/g DW)	Total Phenolic (mg GAE/g DW)
0 + 0	4.06 ± 1.01 a *	328.8 ± 21.1 d	5.28 ± 1.07 e	4.63 ± 1.01 a	303.5 ± 31.1 c	5.45 ± 1.55 d	5.11 ± 1.09 a	251.1 ± 26 c	5.33 ± 1.11 c	4.85 ± 1.05 a	280.6 ± 32.9 c	5.02 ± 1.05 c
2 + 0	3.55 ± 0.84 c	359.7 ± 27.3 b	6.12 ± 1.05 d	4.27 ± 1.22 b	334.6 ± 33.5 b	5.76 ± 1.23 c	4.86 ± 1.05 b	273.3 ± 27 b	4.97 ± 1.16 d	4.79 ± 1.01 a	287.4 ± 33.4 c	5.22 ± 1.14 b
4 + 0	3.77 ± 1.01 b	347.4 ± 24.9 c	6.13 ± 1.41 d	4.15 ± 1.27 bc	335.2 ± 41.4 b	5.88 ± 1.07 bc	4.69 ± 1.44 c	288.4 ± 35 ab	5.25 ± 1.09 c	4.47 ± 1.22 b	322.1 ± 22.7 ab	5.31 ± 1.22 ab
0 + 2	3.45 ± 1.09 c	371.5 ± 26.8 a	6.43 ± 1.55 b	3.96 ± 1.08 c	348.1 ± 28.9 a	5.93 ± 1.09 b	4.65 ± 1.23 c	297.4 ± 33 a	5.22 ± 1.18 c	4.43 ± 1.11 b	333.2 ± 32.9 a	5.43 ± 1.32 a
0 + 4	3.68 ± 1.14 b	358.3 ± 31.4 b	6.1 ± 1.32 d	3.97 ± 1.09 c	327.3 ± 44.8 b	5.98 ± 1.02 b	4.85 ± 1.20 b	296.3 ± 31 a	5.35 ± 1.22 c	4.45 ± 0.89 b	333.7 ± 25.9 a	5.22 ± 1.38 b
2 + 2	3.07 ± 0.74 d	375.1 ± 41.7 a	6.71 ± 1.62 a	3.72 ± 0.81 d	353.4 ± 31.9 a	6.17 ± 1.28 a	4.51 ± 1.3 d	286.8 ± 37 ab	5.92 ± 1.31 a	4.27 ± 0.89 c	326.6 ± 32.6 ab	5.42 ± 1.09 a
4 + 4	3.76 ± 1.22 b	359.9 ± 40.1 b	6.23 ± 1.66 c	3.99 ± 0.86 c	332.4 ± 33.2 b	6.22 ± 1.27 a	4.89 ± 1.09 b	279.2 ± 44 b	5.63 ± 1.19 b	4.24 ± 1.02 c	312.2 ± 8.8 b	5.33 ± 1.15 ab

* Means with similar letters in each column, based on an LSD test at a 0.05% probability level, are not significantly different.

).

4. Discussion

Ergo is a naturally occurring amino acid analog produced by mycobacteria and various fungi, including specific mushroom species [34]. Recent advancements have improved our understanding of Ergo, particularly through the identification of a highly specific transporter in humans and higher organisms, as well as enhanced insights into its distribution due to improved analytical methods [12]. While the exact physiological role of Ergo remains unclear, it has demonstrated antioxidant and cytoprotective properties in vitro, although evidence of these effects in vivo is limited [5]. Given its broad-spectrum antioxidant capabilities, the global demand for Ergo has risen. However, limited information is available regarding its biosynthesis via submerged fermentation.

Ergo synthesis primarily originates from amino acid precursors such as His, Met, and Cys [35,36]. In our study, the combined application of Met, Cys, and His at a concentration of 1 mM significantly increased Ergo levels compared to other treatments. These findings emphasize the crucial role of these amino acids in the metabolic pathways involved in the synthesis of secondary metabolites in fungi. Specifically, Met and Cys, being sulfur-containing amino acids, serve as precursors for the biosynthesis of antioxidant compounds, including Ergo [35,36]. At the molecular level, Ergo biosynthesis in fungi is orchestrated by a series of enzymatic reactions that convert precursor amino acids into the final product. In Flammulina velutipes, three key genes—Fvegt1, Fvegt2, and Fvegt3—encode enzymes that catalyze the transformation of His and Cys into Ergo. Among these, Fvegt1 functions as a methyltransferase, whereas Fvegt2 and Fvegt3 serve as cysteine desulfurases, facilitating sulfur transfer during the biosynthetic process [37]. Similarly, in Schizosaccharomyces pombe, the biosynthetic pathway is initiated by egt1, while egt2 catalyzes the terminal step via a pyridoxal phosphate (PLP)-dependent C–S lyase reaction [38,39]. These enzymatic processes are critical in modulating Ergo accumulation and metabolic flux within fungal cells, underscoring their regulatory significance in secondary metabolite biosynthesis.

A sufficient supply of precursor amino acids can enhance fungal enzymatic activity, facilitating the synthesis of bioactive compounds. However, amino acid supplementation aimed at increasing Ergo production was associated with a reduction in fungal growth rate. This suggests a metabolic trade-off in which energy and essential resources are redirected from primary metabolism to secondary metabolite biosynthesis, thereby limiting mycelial proliferation. Moreover, elevated Ergo levels may trigger feedback inhibition mechanisms, further constraining growth—a phenomenon previously observed in other fungal species [24]. The regulation of Ergo biosynthesis is intricately linked to the availability of precursor amino acids, such as methionine, histidine, and cysteine. While these amino acids are essential for Ergo production, their excessive concentrations may exacerbate growth limitations [35]. Several factors may contribute to this growth suppression. First, the biosynthesis of Ergo requires substantial metabolic energy (ATP, NADPH) and precursor molecules, reducing their availability for cellular proliferation. Second, feedback inhibition mechanisms may downregulate key metabolic pathways, restricting further growth. Additionally, amino acid supplementation could disrupt oxidative stress homeostasis by altering the glutathione (GSH/GSSG) balance, potentially impacting cellular redox regulation [24]. Furthermore, the observed reduction in growth may be linked to the modulation of the TOR signaling pathway, which integrates amino acid availability with fungal growth regulation. Alterations in the carbon-to-nitrogen (C/N) ratio due to amino acid supplementation may also induce adaptive metabolic shifts, prioritizing secondary metabolite biosynthesis over biomass accumulation [6].

From a metabolic perspective, the trade-off between growth rate and Ergo biosynthesis likely arises due to competition for intracellular resources. High growth rates demand the extensive allocation of carbon and nitrogen sources toward biomass expansion, potentially restricting flux through the biosynthetic pathway of Ergo. Studies have shown that metabolic bottlenecks can emerge at key enzymatic steps, such as methyltransferase and sulfoxide synthase activity, where substrate availability and cofactor supply dictate pathway efficiency [40]. Moreover, recent metabolic engineering efforts demonstrated that optimizing nitrogen metabolism in Saccharomyces cerevisiae alleviated resource competition, thereby improving Ergo yield [41]. This highlights that the observed inverse correlation between fungal growth and Ergo accumulation may stem from regulatory constraints within central metabolic pathways. Optimizing culture conditions, including fed-batch fermentation strategies and precise nitrogen modulation, may provide a viable approach to mitigating this trade-off. Controlled substrate feeding has been shown to sustain moderate growth while maintaining sufficient precursor pools for secondary metabolite synthesis [42]. Therefore, our findings corroborate the hypothesis that rapid growth diverts resources away from Ergo synthesis, but they also underscore the potential of strategic metabolic regulation to minimize this trade-off and enhance production efficiency.

The observed increase in antioxidant activity and total phenolic content in response to amino acid supplementation can be attributed to the role of these amino acids as precursors or stimulators in the biosynthetic pathways of bioactive compounds [43]. Met and Cys, sulfur-containing amino acids, act as precursors for glutathione, a key antioxidant molecule that scavenges ROS and regenerates oxidized phenolic compounds, thereby enhancing antioxidant capacity [44]. Moreover, His, due to its imidazole side chain, has radical scavenging properties, which likely contribute to its ability to enhance antioxidant activity when supplemented with Met and Cys [27,45] The observed increase in antioxidant activity across all strains treated with Met, Cys, and His is likely a result of the enhanced biosynthesis of low-molecular-weight phenolics and other antioxidant compounds. Phenolic compounds neutralize free radicals by donating electrons or hydrogen atoms, as confirmed by the significant findings in this study [25,46]. These results align with previous studies, such as Ul Islam et al. [26], which demonstrated that sulfur-rich amino acids in the growth medium enhanced phenolic, flavonoid, and antioxidant enzyme production in Helianthus annuus. Additionally, Met and Cys serve as precursors for S-adenosylmethionine (SAM), which acts as a methyl donor in the biosynthesis of phenolic compounds, while His may function as a cofactor for specific enzymes in this pathway [21].

The composition of carbon and nitrogen sources in the growth medium is a critical determinant of fungal growth and metabolism, significantly influencing the production of both primary and secondary metabolites in submerged fermentation systems [47]. Our study demonstrates that the composition of the growth medium influences Ergo production and growth rates across all tested mushroom species. The yeast + peptone treatment, applied at a concentration of 2 + 2 g/L, consistently resulted in the highest Ergo content and growth rates, highlighting the importance of optimal nutrient supplementation. The enhanced Ergo production under this treatment can be attributed to the balanced and bioavailable supply of carbon and nitrogen sources. Yeast extract, abundant in amino acids, peptides, vitamins, and nucleotides, serves as a complex nitrogen and carbon source that supports fungal metabolism by providing essential precursors and cofactors for enzymatic pathways involved in secondary metabolite biosynthesis, including Ergo [48]. Beyond its role as a nutrient source, yeast extract contains growth-promoting factors, such as polyamines, nucleotides, and B vitamins (e.g., biotin and riboflavin), which have been shown to enhance fungal growth and direct metabolic flux toward the production of bioactive compounds [49]. Likewise, peptone provides an additional nitrogen source in the form of short-chain peptides and free amino acids, which are readily assimilated to support protein synthesis and metabolic activity. Recent studies indicate that yeast-derived growth factors can significantly upregulate secondary metabolite production in various fungal species by modulating enzymatic activity and influencing the transcriptional regulation of biosynthetic gene clusters [50].

In our study, the concurrent enhancement of both growth rates and Ergo production with the yeast + peptone treatment emphasizes the dual role of this medium in supporting biomass accumulation and metabolite production. The availability of essential nutrients not only supports cellular division but also meets the energy demands of biosynthetic pathways [51]. This balance is crucial, as secondary metabolite production, such as Ergo synthesis, often competes with primary metabolic processes for cellular resources [48]. The yeast + peptone treatment appears to alleviate this competition, allowing both processes to proceed efficiently. In contrast, the control group exhibited the lowest Ergo content and growth rates, suggesting that basal media were insufficient to meet the metabolic demands of the fungal species. The lack of supplemental carbon and nitrogen likely limits the availability of precursors and cofactors essential for Ergo biosynthesis, as well as the energy needed for growth and maintenance. The nitrogen-rich medium facilitates the assimilation of amino acids such as Met and Cys, essential sulfur precursors for Ergo biosynthesis [52]. The presence of yeast extract and peptone also provides trace elements and vitamins that act as cofactors for enzymes involved in Ergo biosynthesis, improving synthetic efficiency. Vitamins such as thiamine, riboflavin, and B6 have been shown to enhance metabolic pathways, potentially increasing the production of bioactive compounds like erythromycin and Ergo [53]. Optimization of the growth medium, including yeast extract and peptone, has been shown to significantly enhance Ergo production in engineered strains of S. cerevisiae, achieving up to 598 mg/L [45,48].

This study underscores the crucial role of yeast extract and peptone in modulating the antioxidant potential of the Agaricus species. These findings align with previous studies suggesting that organic nitrogen sources enhance the biosynthesis of antioxidant compounds in fungi by providing essential precursors for secondary metabolite production [54,55]. Yeast extract also acts as a biotic elicitor, enhancing flavonoid accumulation in various plant cell cultures. Studies on Oplopanax elatus and Echinacea purpurea have demonstrated that yeast extract supplementation effectively increases flavonoid content, emphasizing its role in secondary metabolite induction [56,57]. Yeast extract has also been shown to stimulate the synthesis of bioactive compounds by upregulating key genes involved in flavonoid biosynthesis, such as isoflavone synthase and phenylalanine ammonia-lyase [58,59]. Additionally, metabolic engineering studies in S. cerevisiae have demonstrated that yeast extract can optimize precursor availability, redirecting metabolic flux toward secondary metabolite production [60]. These findings highlight the broader biotechnological potential of yeast-derived components in enhancing the functional properties of medicinal fungi and plants.

Among the four Agaricus species tested, A. bitorquis exhibited the highest Ergo content, growth rate, total phenol concentration, and antioxidant activity. Differences in Ergo accumulation and metabolic responsiveness between A. bisporus (white) and A. bitorquis stem from distinct genetic and metabolic regulatory mechanisms. Transcriptomic analyses reveal that A. bitorquis has an inherently higher expression of key sulfur assimilation and ergothioneine biosynthetic genes, particularly Egt1 and Egt2, which facilitate histidine and cysteine conversion into Ergo [39]. This suggests an intrinsically active biosynthetic pathway, likely an evolutionary adaptation to stress-prone environments where enhanced antioxidant production provides a selective advantage. Conversely, A. bisporus has lower basal Ergo levels but exhibits a stronger adaptive response under optimized nutritional conditions. This heightened responsiveness is attributed to greater regulatory plasticity in sulfur metabolism and nutrient-sensing pathways [40]. Notably, the transcriptional upregulation of biosynthetic genes in response to exogenous methionine and cysteine significantly enhances Ergo accumulation, surpassing that of A. bitorquis. Metabolically, A. bitorquis demonstrates greater thermotolerance and stress resilience, maintaining a stable yet less flexible secondary metabolism. In contrast, A. bisporus exhibits higher metabolic adaptability, efficiently reallocating resources to secondary metabolite production under favorable conditions [41]. Species-specific variations in methyltransferase and sulfurtransferase activity further influence Ergo biosynthesis. While A. bitorquis is a naturally high Ergo producer, A. bisporus benefits from superior metabolic responsiveness, making it an optimal candidate for large-scale cultivation with controlled nutritional enhancements. Enhancing Ergo biosynthesis through exogenous supplementation offers a promising strategy for improving the nutritional and pharmaceutical value of cultivated mushrooms, addressing the growing global demand for this antioxidant. These findings underscore the critical role of environmental modulation in optimizing fungal bioactive compound production, and they open new avenues for enhancing metabolite yields in commercially relevant strains.

5. Conclusions

This study provides novel insights into the biosynthesis of Ergo and the intricate interplay of genetic, environmental, and nutritional factors that influence its production and fungal growth in the Agaricus species. Our findings reveal that the high concentrations of amino acids significantly reduced growth rates, despite an increase in Ergo content and antioxidant activity. Conversely, supplementation with lower concentrations of amino acids enhanced Ergo production without notably inhibiting fungal growth. However, the trade-off between enhanced Ergo synthesis and reduced growth rate highlights the metabolic cost of secondary metabolite production, emphasizing the need for carefully optimized culture conditions that balance both the growth and yield of bioactive compounds. From an industrial standpoint, these findings suggest that fine-tuning amino acid supplementation can enhance Ergo production in commercial mushroom cultivation while minimizing adverse effects on biomass yield. The most effective formulation—comprising 0.5 mM of methionine, cysteine, and histidine, along with 2 g/L of yeast extract and 2 g/L of peptone—demonstrates potential for large-scale bioprocessing, improving the nutritional and pharmaceutical value of cultivated mushrooms. While metabolic engineering and CRISPR-based genome editing offer promising avenues for enhancing Ergo biosynthesis, their application in the Agaricus species warrants careful consideration due to possible trade-offs in metabolic flux and growth efficiency. Although genetic modifications targeting sulfur metabolism and antioxidant pathways have been explored in other fungi, the direct genetic enhancement of Ergo biosynthesis remains largely unexplored in Agaricus. Future research should prioritize elucidating the molecular pathways underlying Ergo production while critically assessing the feasibility of targeted genetic interventions.

In addition to enhancing Ergo biosynthesis, amino acid supplementation significantly improved the antioxidant capacity and polyphenolic content of the Agaricus species. The highest antioxidant activity, observed with 1 mM Met + Cys + His, highlights a strong link between amino acid metabolism and fungal defense mechanisms. Likewise, optimized supplementation increased total phenolic content, particularly in A. bitorquis and A. bisporus (white). These findings underscore the potential of metabolic modulation to enhance both Ergo production and the bioactive properties of cultivated mushrooms for functional food applications. The observed variations in Ergo content and growth dynamics across different strains further highlight the metabolic diversity shaped by genetic composition. A. bitorquis emerged as a promising candidate for Ergo production due to its rich genetic potential and metabolic efficiency. Nevertheless, the strong responsiveness of A. bisporus (white) to optimized culture conditions presents a viable alternative to naturally high Ergo-producing species like A. bitorquis, which may require more complex and costly cultivation strategies. Importantly, A. bisporus (white) holds promise as a biotechnological model for large-scale Ergo production, offering an economically feasible option for industrial applications.