Optimizing Ge Enrichment in Lyophyllum decastes Fermentation for Enhanced Biological Activity

Abstract

This study enhanced germanium (Ge) enrichment in the liquid fermentation of the edible fungus Lyophyllum decastes in order to boost its biological activity, particularly its antioxidant and immunomodulatory properties. Through the use of single-factor and Plackett–Burman designs, the experiments revealed critical parameters affecting Ge enrichment, including Ge oxide concentration, potato powder concentration, and peptone levels. The optimization of the Box–Behnken response surface methodology resulted in a Ge concentration of 3.61 mg/L, significantly enhancing the biomass, protein, polysaccharide, and flavonoid content in the mycelium. In contrast to traditional fermentation, Ge-rich fermentation enhanced the mycelial biomass by 30.97% and elevated the organic Ge content 50.19-fold. An analysis of the antioxidants revealed that the Ge-enriched mycelial water extract exhibited heightened activity, augmenting TNF-α production in RAW264.7 cells by 73.29% at a concentration of 200 μg/g. These findings indicate that the Ge-enriched fermentation of L. decastes holds promise for functional applications in health-supportive products due to its robust antioxidant and immune-enhancing capabilities.

1. Introduction

Mushrooms, long esteemed in traditional medicine, have recently attracted significant scientific interest. Researchers have associated the bioactive components of mushrooms, especially polysaccharides, with many health advantages, including immunomodulation, anticancer effects, antioxidant properties, and anti-inflammatory actions [1,2]. Lyophyllum decastes, also known as the fried chicken mushroom, belongs to the family Lyophyllaceae of the order Agaricales. It is a rare edible fungus with valuable medicinal properties [3,4,5,6]. L. decastes, an edible mushroom of significant nutritional value, is abundant in protein, various amino acids, vitamins, and polysaccharides. It possesses numerous physiological properties, including antioxidant, anti-aging, anti-tumor, anti-inflammatory, hypoglycemic, hypolipidemic, anti-thrombotic, radiation protection, blood sugar- and lipid-reducing, anti-obesity, and immune system-enhancing capabilities [4,7,8,9,10,11].

Nevertheless, the human body is incapable of absorbing Ge, and increased concentrations of this element may adversely affect human health. Biotransformation or in vitro modification can convert inorganic Ge into low-toxicity organic compounds that are easily assimilated by the human body [12]. Numerous herbal remedies incorporate germanium [13,14]. The ingestion of organic Ge may provide several beneficial effects, encompassing antioxidant, anti-aging, anti-tumor, anti-mutagenic, and immune-enhancing properties [15,16,17]. The scientific community has extensively studied the transformation of inorganic Ge into organic Ge [17,18]. The body absorbs organic Ge acquired by biological enrichment more readily, contributing positively to health.

Research indicates that the mycelia of edible fungi exhibit significant tolerance to and enrichment capabilities for inorganic mineral elements in the environment. They can convert inorganic metal elements into organic forms, thereby mitigating or eliminating the detrimental effects of inorganic metal ions on the body and enhancing the concentration of beneficial compounds in the mycelium [17,19,20,21]. Nonetheless, the majority of research on mushrooms has concentrated on their biological traits, artificial cultivation, component analysis, and bioactivity assessment. Researchers have mostly investigated selenium-enriched mushrooms for trace element enrichment [22,23,24,25], but some have examined both Ge and Se to assess their impacts on growth and biomass production [12,26]. Most fortified foods often utilize organic Ge in the form of Ge-132, a favored option among consumers due to its stability and high water solubility. The advancement of diverse Ge-rich functional foods and dietary supplements has emerged as a significant trend in the health care sector [27,28]. The use of L. decastes as a carrier for Ge-rich mineral element supplements is advantageous due to its numerous pharmacological qualities and its capacity to manage and alter various inorganic trace elements.

This research utilized response surface methods to enhance the liquid fermentation process of L. decastes and evaluate its quality. The study performed a comparison and investigation of the antioxidant and immunomodulatory activities of a mycelium water extract in vitro. Based on an analysis of the effect of Ge enrichment on the mycelium yield and quality of the liquid fermentation of L. decastes, the optimal carbon and nitrogen sources were then screened. Next, Plackett–Burman and Box–Behnken experimental designs were used to optimize the liquid fermentation process of L. decastes enriched with Ge, and the optimization effect was evaluated. Finally, the effects of Ge enrichment on the antioxidant, anti-tumor, and immunomodulatory activities of the water extract of L. decastes were further explored.

2. Materials and Methods

Lyophyllum decastes samples were preserved and provided by the Department of Forest Protection at Northeast Forestry University.

2.1. Experimental Reagent Preparation

To generate a 0.01 mg/mL Ge standard solution, 0.2000 g of Ge powder (purity ≥ 99%; provided by the Department of Forest Protection at Northeast Forestry University) was dissolved in a succession of chemicals, including ammonium hydroxide (NH₄OH, ≥99%) and hydrogen peroxide (H₂O₂, 30% w/w), and then heated and diluted. In addition, a 0.04% phenoxyethanol solution was prepared using anhydrous ethanol and hydrochloric acid. The investigation comprised activating different L. decastes strains on potato dextrose agar (PDA) slopes for optimal growth before transferring them to PDA plates and incubating them at 25 °C for one week. Finally, seed solutions were created by cutting fungal cakes from activated colonies and culturing them in a conical flask for seven days.

2.2. Ge Enrichment

The mycelium yield, organic Ge content, and protein and polysaccharide contents obtained from the liquid fermentation of L. decastes were investigated when the added amount of GeO₂ was 50 mg/L, 100 mg/L, 200 mg/L, 300 mg/L, and 400 mg/L. The benzophenone spectrophotometric method was used to analyze the Ge content.

2.3. Carbon and Nitrogen Source Screening

A Ge-rich L. decastes fermentation culture medium was explored using mycelial biomass, and its carbon and nitrogen sources were thoroughly examined. For the experiment, we used glucose, sucrose, and soluble starch as carbon sources and peptone, yeast extract powder, and soybean powder as nitrogen sources.

2.4. Plackett–Burman Experimental Design

A Plackett–Burman experimental design [29,30,31] with N = 12 was chosen to assess the significance of ten variables that could have influenced the Ge-rich fermentation of L. decastes (

Table 1. Plackett–Burman test design factors, coding, and levels.

Factor	Coding	Level
		+1	−1
Ge addition amount (mg/kg)	X1	250	150
Potato powder concentration (g/L)	X2	30	20
Peptone concentration (g/ L)	X3	15	5
Glucose concentration (g/L)	X4	25	18
Potassium dihydrogen phosphate concentration (g/L)	X5	2	1.5
Magnesium sulfate concentration (g/L)	X6	2	1
Inoculation volume (block/bottle)	X7	5	3
Initial pH of culture medium	X8	7	6
Shaker speed (r/min)	X9	150	120
Culture temperature (°C)	X10	25	23

With the Ge-rich amount (Y) taken as the response value, a Plackett–Burman experimental design with N = 12 was selected to evaluate the significance of 10 factors that may affect the Ge-rich fermentation of L. decastes. The two levels of each factor were set based on the results of preliminary experiments and previous studies [33].

). The response value for each variable was the Ge-rich amount (Y). In order to identify the critical factors influencing the Ge-rich fermentation of L. decastes, procedures such as an experimental design, regression model fitting, and significance analysis were employed, as in previous studies, to set the two levels of each factor [29,31,32].

2.5. Box–Behnken Test Design

In the Plackett–Burman test, three parameters were found to have a substantial impact on the Ge-rich liquid fermentation of L. decastes. These were the concentration of potato powder (X2), the added quantity of GeO₂ (X1), and the concentration of peptone (X3). The Box–Behnken model [34,35,36] was employed to design a response surface in order to optimize these factors. In this design, the Ge richness (Y) was considered the crucial reaction value. The levels and the specific test criteria are provided in

Table 2. Box–Behnken test design: factors and levels.

Level	Factor
	Ge Oxide Addition Amount (mg/L)	Potato Extract Powder Concentration (mg/kg)	Peptone Concentration (g/L)
−1	100	10	5
0	150	20	10
+1	200	30	15

. Through the use of this approach, the ideal fermentation parameters that could optimize the Ge-rich liquid fermentation of L. decastes were found.

2.6. Determination of Mycelial Biomass

To conduct L. decastes mushroom mycelium fermentation, we created uniformly sized mushroom balls and stopped the culture. Then, we transferred the fermentation liquid into a centrifuge tube and centrifuged it at 5000 r/min. Subsequently, the supernatant was discarded, distilled water was added, and the procedure was repeated three times. Then the mycelium was placed in an oven at 45 °C and blow-dried, until a constant weight was achieved. We weighed the final product and ground it, and then strained the mycelium through a 100-mesh filter to obtain dry mycelium powder, which was preserved at −20 °C in darkness for future use.

2.7. Determination of Organic Germanium Content

2.7.1. Determination of Total Germanium Content via Sample Digestion

A total of 0.2 g of dried L. decastes mycelium powder was weighed in an analytical balance and placed into a stoppered conical flask. We then added 5 mL of concentrated sulfuric acid and slowly heated the solution on a heating plate under a fume hood until it had almost dried. After carbonization, 12 mL of hydrogen peroxide solution was slowly added, and the solution was slowly heated again until it was transparent. Then, 7 mL of sodium hydroxide solution (1 mol/L) was added, and heated until it had fully dissolved. Sulfuric acid (1 M/L) was used to adjust the pH to 4.0 (transparent), and the resulting solution was diluted to 50 mL with distilled water for subsequent testing.

2.7.2. Sample Preparation for Determination of Inorganic Germanium Content

A total of 0.5 g of dried L. decastes mycelium powder was placed in a stoppered conical flask, 20 mL of distilled water was added, and the mixture was treated at 100 °C for 1.5 h. Then, this solution was filtered with qualitative filter paper and diluted in a 50 mL volumetric flask for testing.

2.7.3. Determination of Germanium Concentration in Samples

The benzophenone spectrophotometric method was used to determine the Ge content. Absorbance was measured at 530 nm using a UV spectrophotometer, and the Ge standard curve was prepared by measuring different concentrations of a standard Ge solution in sequence. For the preparation of the Ge standard curve, a pipette was used to measure out 0.50, 1.00, 1.5, 2.0, 2.5, 3.0, and 3.5 mL of the standard Ge solution in sequence, according to the above method. The regression equation for the standard curve is Y = 0.9845x + 0.1221; R² = 0.9663.

2.7.4. Determination of Organic Ge Content

The formula for the calculation of the organic Ge content is as follows:

$$\mathit{Organic} \mathit{Ge} \mathit{content} (\mathit{mg}/g)=\mathit{total} \mathit{Ge} \mathit{content} (\mathit{mg}/g)-\mathit{inorganic} \mathit{Ge} \mathit{content} (\mathit{mg}/g)$$

(1)

2.8. Determination of Soluble Protein Content

The solid–liquid ratio for soluble protein extraction was 1:60 g/mL (1.000 g of desiccated L. decastes mycelium powder in 60 mL of distilled water). The Coomassie Brilliant Blue method was employed to determine the concentration of soluble protein [8,37,38]; then, absorbance was measured at 595 nm to assess the protein content in the solution. The protein extract was removed via the Sevag reagent deproteinization procedure [39,40,41,42]. Accuracy was ensured through the triple verification of the protein content and the utilization of bovine serum albumin to establish a standard curve for the determination of the soluble protein concentration of the sample [43,44,45,46].

2.9. Determination of Polysaccharide Content

The polysaccharides were extracted and their concentrations measured using the sulfuric acid–phenol method, in which 2 mL of the aqueous solution of L. decastes mycelium polysaccharides was precisely pipetted into a test tube. In the test tube, 1 mL of 6% phenol and 5 mL of concentrated sulfuric acid were added. After comprehensive mixing, the liquid was allowed to cool for five minutes at an ambient temperature. It was assessed at 490 nm following the transfer of 3 mL of the mixture into a sanitized cuvette [47,48,49].

2.10. Preparation of Aqueous Extracts

The mycelia of L. decastes, obtained by means of various culture methods, were collected, dehydrated, and thoroughly crushed, after which distilled water was added at a liquid-to-solid ratio of 1:10 and mixed uniformly. Hot water extraction was conducted with a magnetic stirrer, followed by the centrifugation of the extract. The supernatant was concentrated using rotary evaporation, freeze-dried, and subsequently pulverized to produce a dry powder of L. decastes mycelium extract, which was stored in a dark environment at −20 °C.

2.11. Preparation of the Solution for the Determination of Total Reducing Power

We accurately pipetted 2.5 mL of a phosphate buffer and potassium ferricyanide solution (1%) into an Eppendorf tube. Using an oscillator, we extensively mixed the mixture in order to determine the reducing power [50] and then pipetted 1 mL of L. decastes water extract, at varying concentrations, into the EP tube containing the mixed solution. The sample was thoroughly mixed, and the Eppendorf tube was stored in a 50 °C water pot for 20 min to react. The solution was then carefully mixed again using an oscillator, and 2.5 mL of 10% trichloroacetic acid solution was pipetted into the Eppendorf tube and then centrifuged. After 2.5 mL of the supernatant, each with a different L. decastes extract concentration, was pipetted into a fresh Eppendorf tube, equal amounts of distilled water and ferric chloride solution (0.1%) were added. After being thoroughly mixed using an oscillator, the mixture was left to sit at room temperature for 10 min, and then absorbance was measured at 700 nm with a spectrophotometer [51].

2.12. Preparation of the Solution for the Determination of Hydroxyl Scavenging Ability

A vortex oscillator was used to thoroughly mix 2.5 mL of distilled water and 1.0 mL of hydrogen peroxide solution (1%) into the mixture. Then, 1.5 mL of 5 mmol/L ortho-phenanthroline anhydrous ethanol solution was accurately pipetted into the tube, followed by 4.0 mL of a phosphate buffer (0.75 mol/L, pH = 7.4) and 1.0 mL of ferrous sulfate solution (7.5 mmol/L). The mixture was thoroughly mixed and incubated for 60 min at 37 °C. Absorbance was then measured at 536 nm. The above procedures were followed to extract 2.5 mL samples with various L. decastes mycelium concentrations, which were then combined with 1.0 mL of 1% hydrogen oxide solution and reacted for 60 min in an incubator. The absorbance at 536 nm was measured [52]. An ortho-phenanthroline anhydrous ethanol solution, a phosphate buffer, and a ferrous sulfate solution were the sole ingredients used in the undamaged group. The absorbance at 536 nm was recorded as A536 undamaged. The following formula was used to determine the hydroxyl radical clearance rate:

$$E\left(\%\right)=\left\{{\displaystyle \frac{A536added medicine-A536damage}{A536undamaged-A536damage}}\right\}\times 100$$

(2)

2.13. Preparation of the Solution for the Determination of Superoxide Anion Free Radical Scavenging Ability

Using a pipette, we placed 4.5 mL of a Tris–HCl buffer (0.05 mol/L, pH 8.2) into an Eppendorf tube. Then, we added 0.1 mL of various concentrations of the L. decastes mycelium extract solution and thoroughly mixed the contents of the tube using a vortex oscillator to determine superoxide anion free radical scavenging ability [53,54]. Then, 0.4 mL of a 2.25 mmol/L pyrogallol solution that had been heated to 25 °C was thoroughly mixed for four minutes. The absorbance at 325 nm was measured every 0.5 s, and the absorbance change was computed. The test solution was replaced with 0.1 mL of distilled water to measure the rate of pyrogallol self-oxidation.

2.14. Preparation of the ABTS Stock Solution

A vortex oscillator was used to thoroughly mix 50 mL of ABTS free radical solution (70 mM) and 0.89 mL of potassium persulfate solution (140 mM) in a centrifuge tube. The mixture was then left for 12 h [55,56,57]. Then, 0.1 mL samples of the fungal extracts were added at various strengths to 1.9 mL of the ABTS working solution; these were mixed via vortex shaking and allowed to react for 6 min at room temperature in a light-proof tin box. The absorbance was measured at 734 nm [55,56,58]. The formula used to calculate ABTS+ free radical scavenging ability is as follows:

$$clearing capability\left(\%\right)=\left\{1-{\displaystyle \frac{A1-A2}{A0}}\right\}\times 100$$

(3)

where A0 represents the sample’s absorbance measured using distilled water, and A1 represents the sample’s absorbance measured using the test solution. A2 denotes the absorbance measured using distilled water instead of the ABTS working solution.

2.15. Preparation of the Solution for the Determination of Immunomodulatory Activity

RAW264.7 cells were cultured in full DMEM media supplemented with 10% fetal bovine serum (FBS) in a temperature-controlled incubator (37 °C, 5% CO₂). They were subsequently plated in a 48-well plate at a cell density of 1 × 10⁵. The cells were cultivated for 12 h to assess the immunomodulatory activity of macrophages from the water extract [57,59,60]. To examine the impact of the L. decastes water extract on the cells, concentrations of 12.5, 25, 50, 100, and 200 μg/mL were administered, and they were incubated for 24 h. The positive control group comprised 2 μg/mL LPS, whereas the blank control group consisted of a complete culture medium. Griess colorimetry was employed to quantify the concentration of nitric oxide (NO) in the supernatant [61,62]. Concurrently, an ELISA kit was used to quantify the concentration of tumor necrosis factor-α (TNF-α) released in the supernatant [63].

2.16. Data Statistics and Analysis

The Design Expert 10.0 software was used to complete the design, data, and regression analysis of the Plackett–Burman test and the Box–Behnken test. The SPSS10.0 software was used to perform a one-way analysis of variance on the experimental data, and Duncan’s multiple comparison of means was used.

3. Results

3.1. Effects of Germanium Enrichment on Mycelial Biomass

Figure 1a demonstrates that when the quantity of GeO₂ increased, the mycelial biomass of L. decastes initially exhibited a gradual increase, followed by a swift decline. The treatment group at 150 mg/L exhibited optimal mycelial growth, with biomass reaching 11.66 ± 0.24 g/L, which represents a 29.99% increase compared to the control group (8.97 ± 0.39 mg/L). This indicates that L. decastes demonstrated significant tolerance to inorganic Ge, and the calculated addition of inorganic Ge was able to enhance the growth of mycelial hyphae in L. decastes. Nonetheless, when the concentration of GeO₂ was above 200 mg/L, mycelial biomass decreased rapidly. The mycelial biomass in the 300 mg/L treatment group was 4.75 ± 0.20 mg/L, which is inferior to that found in the 150 mg/L treatment group by 59.26%, indicating significant harmful consequences. Variance analysis results indicated significant differences in biomass between the 150 mg/L treatment group and other groups at p < 0.01, while the 100 mg/L and 200 mg/L treatment groups exhibited significant differences at p < 0.01 and p < 0.05, respectively.

3.2. Effects of Germanium Enrichment on Organic Ge Content

Figure 1b illustrates that when the Ge addition amount ranged from 50 to 300 mg/L, a substantial positive connection existed between the quantity added and the organic Ge content in the fermented mycelium of L. decastes. At an extra dosage of 300 mg/L, the organic Ge content reached 422.06 ± 9.94 μg/g. In the 50 mg/L treatment group, the organic Ge concentration in the mycelium was 103.14 ± 6.67 μg/g, representing a result 14.78 times higher than that of the control group. The analysis of variance revealed significant differences across the treatment groups at the p < 0.01 and p < 0.05 levels. It can be seen that L. decastes exhibits a strong capacity for Ge enrichment.

3.3. Effects of Germanium Enrichment on Soluble Protein Content

Figure 1c indicates that when the Ge addition amount ranged from 50 to 200 mg/L, the soluble protein content of the hyphae of L. decastes increased proportionally to the amount added. With the addition of 200 mg/L of GeO₂, the soluble protein content of the mycelium reached 268.04 ± 4.76 mg/g, representing a 26.46% increase compared to the control group. Subsequently, as the incorporation of Ge persisted, the soluble protein concentration progressively decreased. The concentration in the 300 mg/L treatment group was 245.07 ± 5.88 mg/g, reflecting a 15.62% increase relative to the control group. The analysis of variance results indicated that at the p < 0.05 level, significant differences existed between the 200 mg/L treatment group and all other groups, except for the 150 mg/L treatment group. Additionally, there were no significant differences in soluble protein content among the 150 mg/L, 200 mg/L, and 250 mg/L treatment groups at the p < 0.01 level. These results indicate that the incorporation of Ge markedly enhances the accumulation of soluble protein in the hyphae of L. decastes.

3.4. Effects of Germanium Enrichment on Polysaccharide Content

Ge enrichment can substantially augment the polysaccharide content of the hyphae of L. decastes, as shown in Figure 1d. As the quantity of supplied Ge escalated, the polysaccharide content initially rose and subsequently declined. With the addition of Ge at concentrations of 150 mg/L and 300 mg/L, the polysaccharide contents were 36.37 ± 1.14 mg/g and 28.61 ± 0.90 mg/g, representing increases of 47.53% and 16.04%, respectively, compared to the control group. Variance analysis results indicated no significant difference between the 150 mg/L and 200 mg/L treatment groups at p < 0.01 and p < 0.05. Nevertheless, significant differences were seen between these two treatment groups and the other treatment groups (p < 0.01).

3.5. Screening of Carbon and Nitrogen Sources

In the screening of carbon sources for L. decastes cultures, glucose proved to be the most effective, yielding the highest biomass (11.11 ± 0.20 g/L) and significantly increasing organic Ge content (263.42 ± 6.31 μg/g) compared to sucrose and soluble starch (p < 0.01). This enhanced performance with glucose is likely due to its efficient absorption and utilization by mycelial hyphae, promoting organic Ge accumulation (

Table 3. Effects of different carbon sources on biomass and organic Ge content during Ge-enriched liquid fermentation.

Index	Biomass (g/L)	Organic Ge Content (μg/g)
Glucose	11.11 ± 0.20 Aa	263.42 ± 6.31 Aa
Sucrose	10.12 ± 0.27 Bb	240.00 ± 6.38 Bb
Soluble starch	9.58 ± 0.42 Bb	233.90 ± 8.74 Bb

Uppercase and lowercase letters indicate significance at the p < 0.01 and p < 0.05 levels, respectively. Different letters in the same column indicate significant differences between groups at the corresponding levels.

).

When nitrogen sources for the Ge-rich fermentation of L. decastes were screened, it was found that peptone provided the highest biomass (11.20 ± 0.35 g/L) and organic Ge content (265.83 ± 7.73 μg/g), outperforming yeast extract and soy flour. Although yeast extract showed similar results, peptone was more effective overall, with significant differences observed in biomass and Ge content compared to soy flour (p < 0.01) (

Table 4. Effects of different nitrogen sources on Ge-rich liquid fermentation biomass and Ge content of L. decastes.

Index	Biomass (g/L)	Organic Ge Content (μg/g)
Peptone	11.20 ± 0.35 Aa	265.83 ± 7.73 Aa
Yeast extract powder	10.84 ± 0.20 Aa	250.92 ± 5.45 Abb
Soy flour	10.42 ± 0.29 Bb	248.48 ± 6.31 BC

Uppercase and lowercase letters indicate significance at the p < 0.01 and p < 0.05 levels, respectively. Different letters in the same column indicate significant differences between groups at the corresponding levels.

).

3.6. Plackett–Burman Design (PBD) Analysis

The Plackett–Burman experimental design (PBD) was utilized to determine the critical variables influencing Ge-enriched fermentation in L. decastes, with Ge concentration as the principal response variable. Ten variables were assessed, revealing that Ge addition (X1), potato starch (X2), and peptone content (X3) were significant factors (p < 0.05), with an R² of 0.9982, signifying a robust model fit. Elevated nitrogen sources markedly improved Ge absorption in the mycelium, whereas elevated quantities of glucose and magnesium sulfate adversely affected growth. The ideal conditions comprised 25 g/L glucose, 2 g/L potassium dihydrogen phosphate, and 1 g/L magnesium sulfate, alongside precise pH, inoculation, and temperature parameters to enhance yield, reduce expenses, and maintain process stability.

Table 5. Plackett–Burman experimental design and results.

Serial #	Factor										Response
	X1 mg/L	X2 g/L	X3 g/L	X4 g/L	X5 g/L	X6 g/L	X7	X8	X9 r/min	X10 °C	Y mg/L
1	250	30	5	25	2	2	3	6	120	25	3.12 ± 0.12
2	150	30	15	18	2	2	5	6	120	23	2.95 ± 0.13
3	250	20	15	25	1.5	2	5	7	120	23	3.32 ± 0.08
4	150	30	5	25	2	1	5	7	150	23	2.80 ± 0.15
5	250	20	15	18	2	2	3	7	150	25	3.36 ± 0.13
6	150	20	5	25	1.5	2	5	6	150	25	2.49 ± 0.16
7	250	20	5	18	2	1	5	7	120	25	3.21 ± 0.14
8	150	30	5	18	1.5	2	3	7	150	23	2.59 ± 0.12
9	250	30	15	18	1.5	1	5	6	150	25	3.45 ± 0.13
10	150	30	15	25	1.5	1	3	7	120	25	2.92 ± 0.20
11	250	20	15	25	2	1	3	6	150	23	3.34 ± 0.12
12	150	20	5	18	1.5	1	3	6	120	23	2.49 ± 0.11

presents the design matrix and the associated experimental results.

The findings of the analysis of variance and univariate regression analysis regarding the key factors involved in the fermentation of the Ge-enriched liquid of L. decastes are displayed in Table S1, while Figure 2 illustrates the impact of each variable on the amount of Ge-enriched liquid obtained from L. decastes during fermentation.

3.7. Box–Behnken Design (BBD) Analysis

Ge enrichment (Y) was utilized as an indicator based on the findings of the PBD experiment. In order to determine the ideal liquid culture conditions for L. decastes, a Box–Behnken design (BBD) three-factor two-level experiment was performed using the response surface methodology. Key factors affecting Ge-enriched liquid fermentation, namely, the Ge addition amount (X1), the potato powder concentration (X2), and the peptone concentration (X3), were optimized.

The BBD experimental design matrix and the L. decastes liquid fermentation findings were obtained using Design Expert 10.0 and are displayed in

Table 6. Design and results of BBD experiment on Ge-enriched fermentation.

Serial Number	X1	X2	X3	Y1 (mg/L)
1	200	25	10	3.68 ± 0.18
2	200	20	5	3.25 ± 0.15
3	150	25	5	2.83 ± 0.09
4	200	25	10	3.67 ± 0.08
5	200	25	10	3.60 ± 0.11
6	250	25	5	3.18 ± 0.15
7	200	30	15	3.42 ± 0.12
8	250	30	10	3.42 ± 0.20
9	150	25	15	3.12 ± 0.18
10	250	20	10	3.03 ± 0.12
11	150	20	10	2.96 ± 0.14
12	150	30	10	2.97 ± 0.09
13	250	25	15	3.19 ± 0.15
14	200	20	15	3.52 ± 0.21
15	200	25	10	3.63 ± 0.15
16	200	30	5	3.37 ± 0.21
17	200	25	10	3.63 ± 0.14

.

As illustrated in Tables S2 and S3, there was a significant influence and a non-linear relationship between the increased amount of Ge (p < 0.05) and the model interaction terms (X₁). Moreover, the Ge-rich model’s F-value, at 44.09, was comparatively large, and the findings of the regression equation analysis demonstrate that all of the p-values were less than 0.0001. There was a high degree of fit in the model and a negligible lack of fit, as indicated by the lack-of-fit term p = 0.0784, which is greater than 0.05. Moreover, the R² value of the model was 0.9827, and the values of Pre. R² = 0.8758 and Adj. R² = 0.9604 were relatively similar. At 1.67%, the variation coefficient was relatively low. This model performed better, as evidenced by its precision of 19.419, which is significantly greater than 4. It is reasonable to forecast the Ge-enriched liquid fermentation process using the fitting degree. The quadratic regression equations for the quantity of Ge-rich liquid were determined via polynomial regression analysis using the Design Expert 10.0 software, resulting in the following equation:

Y = 3.64 + 0.12X₁ + 0.05X₂ + 0.08X₃ − 0.09X₁X₂ − 0.07X₁X₃ − 0.05X₂X₃ − 0.43X₁² − 0.12X₂² − 0.13X₃².

3.8. Three-Dimensional Response Surface Analysis of the Interactions of Various Key Factors

This research examines the correlation between variables and the concentration of Ge enriched during the liquid fermentation of L. decastes. A response surface diagram was generated utilizing the BBD test results. The research indicated that including GeO₂ and potato extract powder enhances the formation of mycelial hyphae in L. decastes, hence augmenting their Ge content. An excessive inorganic Ge concentration can be harmful and impede normal growth. The Ge enrichment of L. decastes varies with the incorporation of potato extract powder and the concentration of peptone. The relationship between Ge concentration and peptone is significant, as elevated levels of nitrogen sources such as peptone enhance protein levels in L. decastes, mitigating the detrimental effects of inorganic Ge on mycelial physiology. Nevertheless, elevated nitrogen and Ge concentrations can induce toxicity, impeding mycelium growth and compromising its capacity to enrich and convert Ge. The optimization of the equilibrium between Ge supplementation and peptone concentration is essential for efficient inorganic Ge transformation.

Figure 3 indicates that the concentration of potato extract powder considerably influenced the Ge content in L. decastes when a constant Ge addition of 150 mg/L was maintained. Initially, the Ge content was elevated; however, as the concentration rose, the Ge-rich content diminished. This resulted from an elevation in osmotic pressure within L. decastes hyphae, adversely impacting their growth and diminishing their capacity to process and accumulate organic Ge, leading to a reduction in Ge enrichment.

3.9. Model Validation

The model validation showed that 207 mg/L of Ge, 26 g/L of potato extract, and 11 g/L of peptone were the best conditions for the Ge-enriched fermentation of L. decastes. This resulted in a Ge concentration of 3.61 ± 0.26 mg/L. This closely matched the model’s predicted value of 3.67 mg/L, indicating the model’s reliability and practical applicability in achieving the targeted Ge enrichment.

3.10. Effects on Ferric Reducing Power

The aqueous extracts of L. decastes from the normal culture, in combination with the Ge-rich culture group, exhibited different reducing capacities, as shown in Figure 4a. There was a positive correlation between the concentrations of the two categories of water extracts and their capacities, which decreased. The water extract’s equivalent OD value gradually rose with an increasing concentration, suggesting that its reducing power was improved. When we compared the two culture techniques further, we discovered that throughout the test concentration range, the OD value of the L. decastes water extract produced via the Ge-rich culture method was consistently greater than that found in the traditional culture group. This was verified by the results of the analysis of variance. The overall reducing power of the water extracts obtained using the two culture methods differed significantly (p < 0.01). According to the linear fitting results, the fitting curve for the Ge-rich culture group was y = 0.07x + 0.0872, R² = 0.9994, while the fitting curve for the traditional culture group was y = 0.05x + 0.0227. Thus, the Ge-rich culture group had a higher reduction force at the same concentration, and the trend of an overall reduction in ability was more obvious.

3.11. Effects on •OH Radical Scavenging Rate

Figure 4b demonstrates that the L. decastes water extracts prepared via either Ge-rich liquid culture or standard liquid culture methods all had a strong scavenging effect on •OH free radicals. In terms of the scavenging of •OH free radicals, the water extract from the Ge-rich fermentation group had an IC50 value of 1.28 mg/mL. In comparison, the IC50 value for the traditional fermentation group was 1.62 mg/mL. This indicates that the Ge-rich fermentation group’s water extract had a stronger ability to scavenge hydroxyl radicals, with an improvement of 26.56%, which was statistically significant (p < 0.01).

3.12. Effects on ABTS Free Radical Scavenging Rate

The results of the OH free radical measurements were consistent with the ABTS+ free radical scavenging capabilities of the L. decastes aqueous extracts obtained via the normal culture method and the Ge-rich culture approach, as shown in Figure 4c. The scavenging rate for ABTS+ free radicals rose as the concentration of the water extract in each group progressively increased, within the range of 0.5–5.0 mg/mL of the chosen extract from L. decastes. Furthermore, there was an extremely significant difference (p < 0.01) between the two. With an IC50 value of 1.58 mg/mL, the aqueous extract of L. decastes prepared via standard liquid culture possessed the ability to scavenge free radicals in the ABTS assay. Moreover, with an IC₅₀ value of 1.12 mg/mL, which was 41.07% higher than that of the conventional culture group, the aqueous extract from the Ge-rich liquid culture group performed better in the scavenging of ABTS+ free radicals. In contrast, the aqueous extract cultured with Ge-rich liquid exhibited stronger scavenging abilities.

3.13. Effects on Superoxide Anion Free Radical Scavenging Ability

The aqueous extracts of L. decastes demonstrated the ability to scavenge superoxide anion free radicals under two distinct culture conditions, as illustrated in Figure 4d. With regard to the scavenging of free radicals, including superoxide anions, the water extract produced via the Ge-rich liquid fermentation culture demonstrated a greater impact. The superoxide anion radical scavenging rate of the aqueous extract obtained through the Ge-rich liquid fermentation culture was consistently greater than that of the traditional fermentation group, within the concentration range of 2 to 20 mg/mL. In particular, at 20 mg/mL, the clearance rate of the water extract from the Ge-rich fermentation group increased to 71.04%, as compared to 56.80% for the conventional fermentation group. This difference is extremely significant (p < 0.01) and suggests that the Ge-rich fermentation method improved the L. decastes aqueous extract’s ability to scavenge superoxide anion radicals.

3.14. NO Release in RAW264.7 Cells

Lipopolysaccharides (LPSs) have a major impact on NO release in RAW 264.7 cells, as seen in Figure 5. The addition of LPSs dramatically increased the release of NO from RAW 264.7 cells when compared to the negative control (NC) group, suggesting that the LPS activated or stimulated macrophages, thereby promoting the production of NO [64,65]. Additionally, the NO content in each group’s water extract exhibited an upward trend as the concentration increased, indicating that the aqueous extracts from both groups dose-dependently promoted NO production. When the two groups of water extracts were further compared, it was discovered that the Ge-rich fermentation group consistently generated more NO than the traditional fermentation group at the same concentration. This finding emphasizes the benefits of the Ge-rich fermentation process and suggests that Ge elements might be helpful in advancing this process. Significant differences existed between the two groups (p < 0.01).

3.15. Effects on TNF-α Secretion in RAW264.7 Cells

The release of TNF-α was significantly enhanced by lipopolysaccharide (LPS) treatment when compared to the negative control (NC) group (Figure 6). Additionally, the TNF-α content of the L. decastes aqueous extract displayed a dose-dependent enhancement as the concentration of the water extract increased, suggesting that the aqueous extract of L. decastes could stimulate the secretion of TNF-α by macrophages through a particular mechanism and subsequently play a role in immune regulation. In contrast, the TNF-α content in the Ge-rich fermentation group was consistently higher than that in the conventional fermentation group at the same dose, indicating that Ge could play a positive role in this process. The analysis of variance showed that at each concentration, the difference between the Ge-rich fermentation group and the conventional fermentation group was extremely significant (p < 0.01).

4. Discussion

This study demonstrates the potential of Ge enrichment in Lyophyllum decastes fermentation to enhance mycelial biomass and the synthesis of bioactive compounds, particularly those with antioxidant and anti-inflammatory properties. These findings align with previous research showing that Ge supplementation improves the biological efficiency of edible mushrooms. Specifically, optimal Ge concentrations significantly enhance the synthesis of proteins, polysaccharides, and flavonoids, all of which have antioxidant and immune-modulating effects.

The observed increase in mycelial biomass under Ge supplementation is consistent with earlier studies indicating that Ge can promote growth through changes in metabolic activity and nutrient utilization. Research on Pleurotus ostreatus and Ganoderma lucidum has shown that low to moderate Ge concentrations promote growth, while concentrations above 200 mg/L cause toxicity due to metal accumulation and oxidative stress [12,18]. Our results reflect this dose-dependent response, with growth stimulation at 150 mg/L and growth suppression at higher concentrations, confirming previous reports of Ge-induced toxicity.

A key finding of this study is the conversion of inorganic Ge to its organic form within the mycelium. This transformation improves bioavailability, as organic Ge is more readily absorbed by the body, offering benefits such as immune system support, cancer prevention, and cellular protection [15]. The biotransformation of Ge in L. decastes suggests its potential in the production of functional foods enriched with bioavailable trace elements, a process previously reported in other fungal species [22].

Additionally, we observed an increase in the production of soluble proteins and polysaccharides in the Ge-enriched L. decastes, both of which are known for their antioxidant properties. These compounds scavenge free radicals, helping to reduce oxidative damage and supporting immune function and cancer prevention. Similar effects have been seen in other fungi, in which metal enrichment, including selenium supplementation, stimulates polysaccharide synthesis and enhances bioactivity [66,67]. The increased polysaccharide content at optimal Ge concentrations further supports these findings. The enhanced ferric reducing and radical scavenging activities of Ge-enriched L. decastes underscore its improved antioxidant capacity, reinforcing the role of Ge in enhancing the antioxidant potential of this fungus. These results are consistent with previous research showing that higher levels of bioactive substances, such as flavonoids and polysaccharides, increase radical scavenging activity in Ge-enriched fungi [25].

Furthermore, L. decastes appears to be a promising candidate for nutraceutical applications due to its potent antioxidant properties, which may help mitigate disorders related to oxidative stress [68]. Ge supplementation also enhanced the immunomodulatory properties of L. decastes, as demonstrated by the increased TNF-α production in RAW264.7 macrophages. This suggests that Ge could improve immune system activation and cytokine modulation, supporting previous studies indicating that organic Ge compounds affect immune cell activity and cytokine release [63].

The observed increases in mycelial biomass and bioactive compound synthesis following Ge supplementation are likely due to the activation of key metabolic pathways. Ge is a cofactor in several enzymatic processes that promote protein synthesis, growth, and the production of polysaccharides and flavonoids. Ge may also enhance energy production and cellular respiration, contributing to increased growth rates in fungal mycelia. Additionally, Ge has been shown to regulate the expression of genes involved in antioxidant defense systems, which may explain the elevated synthesis of bioactive molecules with immune-modulating properties in this study.

The liquid fermentation of L. decastes offers several advantages, including improved growth conditions, better nutrient bioavailability, and increased production efficiency. This method could replace conventional solid-state systems for the synthesis of bioactive fungal compounds. Moreover, the ability of L. decastes to be enriched with Ge enhances its antioxidant and immunomodulatory properties. The optimization of L. decastes for use in functional foods and medicinal applications depends on an understanding of the metal transport mechanisms involved in Ge enrichment and their impact on mycelial growth and bioactivity.

5. Conclusions

The Lyophyllum decastes fermentation procedure significantly improved the Ge enrichment of this fungus, enhancing its bioactive properties, including its immunomodulatory and antioxidant activities. The findings show that controlled Ge supplementation greatly increased mycelial biomass and bioactive components such as proteins and polysaccharides, which was good for the fungus’ health. Overly high amounts of Ge were found to be harmful, which stresses the need for well-balanced supplementation. Future research should largely look at the molecular routes by which Ge influences L. decastes, especially those involving gene expression and metabolic processes that generate bioactive molecules. Furthermore, thorough long-term studies on the potential toxicity at various dosages and the health impact of Ge-enriched L. decastes are crucial. Research into the effects of Ge enrichment on many types of fungi will help in the better application of Ge-enriched functional foods in nutrition and medicine.