Optimization of Fermentation and Transcriptomic Analysis: The Impact of Aspartic Acid on the Antioxidant Activity of Termitomyces

Abstract

Termitomyces, a rare edible fungus with both nutritional and medicinal value, has garnered significant attention for its antioxidant properties. This study aims to elucidate the effects of various nutritional components on the antioxidant activity of Termitomyces. Through assays including FRAP, DPPH, ABTS, and •OH scavenging activity, strain XNQL025, which exhibits high antioxidant activity, was identified. Subsequent optimization of culture medium components using single-factor experiments and response surface methodology revealed that aspartic acid (Asp) significantly enhances the antioxidant capacity of this strain. Transcriptomic analysis showed that Asp activates key pathways, including glycolysis/gluconeogenesis, propanoate metabolism, amino sugar and nucleotide sugar metabolism, valine–leucine–isoleucine biosynthesis, and tryptophan metabolism, along with modulating the peroxisome and mitogen-activated protein kinase (MAPK) signaling pathways. These regulatory actions promote the synthesis of antioxidant compounds and establish a multi-layered antioxidant defense system comprising enzymatic (catalase) and non-enzymatic (leucine/chitooligosaccharides) components. The synergistic interaction between these systems significantly strengthens the antioxidant defense capacity of Termitomyces. This study is the first to elucidate the molecular network by which Asp enhances the antioxidant activity of Termitomyces, thereby providing a foundation for its development as a natural antioxidant.

1. Introduction

Reactive Oxygen Species (ROS) are unavoidable byproducts of aerobic respiration and metabolic processes in living organisms; while they play a crucial role in various physiological processes such as cell signaling, immune responses, and cell proliferation in appropriate amounts, an imbalance between their production and clearance within an organism can lead to oxidative stress [1,2]. This oxidative stress can potentially damage critical cellular macromolecules, such as proteins, lipids, and DNA; accelerate cellular aging; and contribute to the onset of neurodegenerative diseases and other health complications [3,4]. To mitigate this problem, antioxidants are frequently utilized to neutralize excess ROS and maintain the body’s redox equilibrium. However, the long-term use of synthetic antioxidants such as BHA and BHT may pose health risks, including cellular toxicity [5]. Consequently, the development of natural and safe antioxidants is of significant importance.

Edible fungi are an important source of natural antioxidants, and their antioxidant activities are closely associated with a variety of biological functions. Studies have shown that extracts from Ganoderma lucidumcan delay aging by scavenging superoxide radicals [6], while Agaricus bisporus exerts antigenotoxic effects by reducing oxidative stress-induced DNA damage [7]. Edible fungal extracts can also synergistically enhance the endogenous antioxidant enzyme system, thereby reducing the risk of hypertension, cancer, and other oxidative stress-related diseases [8]. More importantly, fungal polyphenols can activate the Nrf/ARE signaling cascade by regulating cellular redox homeostasis, playing a key role in antitumor and anti-infection processes [9,10].

Termitomyces, a rare edible fungus that is symbiotic with termites, has been shown to possess significant antioxidant activity [11], and is rich in bioactive components such as polyphenols, polysaccharides, dietary fiber, saponins, and cerebrosides [12,13]. Modern pharmacological studies have demonstrated that Termitomyces mycelium extracts exhibit antioxidant activity through multiple mechanisms. Polysaccharides enhance the activity of endogenous antioxidant enzymes by activating the HO-1/Nrf2 pathway [14], while polyphenols directly neutralize ROS and also exert anti-inflammatory effects by inhibiting inflammatory pathways [15]. Beyond its antioxidant properties, the diverse bioactive components of Termitomyces construct a rich network of pharmacological effects. Dietary fiber lowers serum total cholesterol and triglyceride levels by modulating gut microbiota or inhibiting cholesterol absorption [16]. The hot water extract of Termitomyces exhibits inhibitory effects on the quorum sensing system of the Pseudomonas aeruginosa CV026 strain [17]. These characteristics position Termitomyces as an excellent source of natural antioxidants and medicinal compounds, presenting immense potential for applications in the health sector. However, the scarcity of its fruiting bodies and the immaturity of liquid fermentation technology have limited in-depth research [18]. The existing optimization of culture media mainly focuses on increasing biomass, while strategies for the targeted regulation of antioxidant activity have not yet been systematically explored.

To address these challenges, this study focuses on enhancing the antioxidant activity of Termitomyces through the optimization of culture media. Glucose and yeast extract have been identified as the key factors influencing the antioxidant capacity of fungi [19,20], and adjustments in inoculum size and fermentation time can significantly enhance the free radical scavenging capacity of ABTS [21]. In other edible fungi such as Ganoderma lucidum, the addition of glycine and methionine has been proven to significantly enhance antioxidant capacity [22,23]. However, the potential of specific amino acids to enhance the antioxidant capacity of Termitomyces has not been fully explored. Moreover, considering the complex symbiotic relationship between Termitomyces and termites, which involves many enzymatic and oxidative reactions in the process of plant biomass transformation, the potential for enhancing antioxidant activity through culture strategies may not have been fully tapped. Therefore, filling this research gap is of great significance.

This research first identified strains of Termitomyces with significant antioxidant activity and then optimized the liquid culture medium by systematically evaluating the carbon and nitrogen sources, as well as the amino acid composition, through single-factor experiments and response surface methodology to determine the optimal growth conditions. Subsequently, transcriptome sequencing was employed to delve into the impact of the optimized medium on the antioxidant activity of Termitomyces, initially elucidating its underlying mechanisms. This research not only provides a scientific foundation for the further development and application of Termitomyces but also expands new avenues for studying the antioxidant activity and functional product development of other edible fungi.

2. Materials and Methods

2.1. Materials and Chemicals

In this study, 12 strains of Termitomyces were collected from the Qianxinan region in Guizhou Province, southwest China, isolated from wild fruiting bodies, cultured on PDA (potato dextrose agar) slants for seven days, identified via ITS sequencing, and subsequently stored at South China Agricultural University.

The PDA medium was prepared with 200 g of peeled potatoes, 20 g of glucose, 3 g of yeast extract, 1 g of magnesium sulfate, 0.46 g of potassium dihydrogen phosphate, 1 g of dipotassium hydrogen phosphate, and 20 g of agar; adjusted to a final volume of 1 L with distilled water; and the pH was set to 6 using hydrochloric acid. The medium was then sterilized at 121 °C for 20 min. PDB (potato dextrose broth) was composed of the same ingredients as PDA but without agar.

2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), thiamine (VB1), riboflavin (VB2), pyridoxine (VB6), folic acid (VB9), cobalamin (VB12), aspartic acid (Asp), proline, glutamic acid, serine, and histidine were all obtained from Macklin Company. Other chemicals and reagents used in this study were of analytical grade and purchased from local suppliers. The total antioxidant capacity (T-AOC) assay kit was acquired from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

2.2. Method

2.2.1. Strain Culture and Liquid Inoculation

After culturing the mycelium on PDA for seven days, it was transferred to 100 mL of sterile water and homogenized to prepare a spawn suspension. The suspension was then inoculated into 100 mL of PDB at a volume ratio of 5% and cultivated at 25 °C and 120 rpm for 14 days. Samples were taken every 24 h, and the mycelium was filtered, washed with distilled water, dried at 50 °C until constant weight, and then weighed to determine biomass. The filtrate was centrifuged at 4000 rpm for 20 min and stored at 4 °C for the subsequent pH and antioxidant activity measurements.

2.2.2. Antioxidant Activity Assay

DPPH Scavenging Activity

The DPPH scavenging activity of the samples was determined according to the method of Begum et al. [24] with slight modifications. A total of 100 μL sample solution was mixed with 100 μL 0.2 μM DPPH solution (prepared in absolute ethanol) and left at room temperature for 30 min in the dark. The absorbance was measured at 517 nm using an enzyme-labeling instrument. The scavenging activity was calculated by Equation (1).

$$\mathrm{Free} \mathrm{radical} \mathrm{scavenging} \mathrm{activity} (\%)=\left(1-{\displaystyle \frac{{\mathrm{A}}_{\mathrm{x}}-{\mathrm{A}}_{\mathrm{x}0}}{{\mathrm{A}}_0}}\right)\times 100\%$$

(1)

where A_x denotes the absorbance measured for the sample solution in the presence of the radical solution (DPPH, ABTS, or •OH radical); A_x0 represents the absorbance of the sample solution when mixed with the solvent; and A₀ corresponds to the absorbance of the radical solution (DPPH, ABTS, or •OH radical) in combination with the solvent.

ABTS Scavenging Activity

This assay was performed by adapting the method of Xie et al. [25]. The ABTS stock solution was prepared by mixing 7 mM ABTS solution (dissolved in deionized water) with 2.45 mM potassium persulfate (in deionized water); after storing in the dark at room temperature for 12–16 h, the ABTS working solution was obtained by diluting it with ethanol to an absorbance of about 0.70 ± 0.02. The 40 μL test solution was mixed with 160 μL ABTS working solution and allowed to stand for 6 min. The absorbance was measured at 734 nm under dark conditions. The ABTS scavenging activity was evaluated by Equation (1).

Hydroxyl Radical (•OH) Scavenging Activity

The •OH scavenging activity method was determined using the Fenton system, as described by Prasad Liu and Malathi et al. [26,27]. An amount of 9 mM salicylic acid (in ethanol) and 9 mM FeSO₄ (in deionized water) was mixed with the test solution in equal proportion. After shaking and mixing, the same volume of 8.8 mM H₂O₂ (in deionized water) was added to start the reaction. The reaction was incubated at 37 °C for 30 min, and the absorbance was measured at 510 nm. The •OH scavenging activity was evaluated by Equation (1).

Total Antioxidant Capacity Assay

The total antioxidant capacity assay of 12 strains of Termitomyces was determined by the T-AOC kit of Nanjing Jiancheng Bioengineering Institute. The ferric reducing antioxidant power (FRAP) ferrous sulfate standard curve is y = 0.0522x + 0.2691, R² = 0.9904.

2.2.3. Comprehensive Evaluation of Antioxidant Activity

Membership Function Method for Strain Screening

The indexes such as mycelial biomass, DPPH, ABTS, •OH, and T-AOC of different strains of Termitomyces were screened by the membership function method, and the strains with the strongest antioxidant capacity were screened [28]. The membership function formula is as follows:

$$\mathrm{U}\left({\mathrm{X}}_{\mathrm{j}}\right)={\displaystyle \frac{\mathrm{X}-{\mathrm{X}}_{\mathrm{m}\mathrm{i}\mathrm{n}}}{{\mathrm{X}}_{\mathrm{m}\mathrm{a}\mathrm{x}}-{\mathrm{X}}_{\mathrm{m}\mathrm{i}\mathrm{n}}}}$$

(2)

$${\mathrm{W}}_{\mathrm{j}}={\displaystyle \frac{{\mathrm{P}}_{\mathrm{j}}}{{\sum }_{\mathrm{j}=1}^{\mathrm{n}}{\mathrm{P}}_{\mathrm{j}}}}, \mathrm{j} = 1, 2, \dots , \mathrm{n}$$

(3)

$$\mathrm{D}={\sum }_{\mathrm{j}=1}^{\mathrm{n}}\left[\mathrm{U}\left({\mathrm{X}}_{\mathrm{j}}\right)\times {\mathrm{W}}_{\mathrm{j}}\right], \mathrm{j} = 1, 2, \dots \dots , \mathrm{n}.$$

(4)

where X is the antioxidant coefficient of a certain index of the test strain; U (X_j) is the membership function value of the j index of the test strain; X_max and X_min are the maximum and minimum values of the antioxidant coefficient of a certain index of the test strain. W_j represents the importance of the jth indicator in all indicators, that is, weight; and P_j is the coefficient of variation in the jth index of each strain. The D value is a subordinate function value for comprehensive evaluation of the antioxidant activity of the strain.

Antioxidant Potency Composite

Antioxidant Potency Composite (APC) is an index used to comprehensively evaluate the total antioxidant capacity of samples [29]. It is achieved by combining the results of antioxidant activity tests of multiple different principles into a single exponential value. The APC index is calculated by the average of the test values. The calculation formula is as follows:

$$\mathrm{Antioxidant} \mathrm{index} \mathrm{score}={\displaystyle \frac{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e} \mathrm{s}\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{e}}{\mathrm{b}\mathrm{e}\mathrm{s}\mathrm{t} \mathrm{s}\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{e}}}\times 100$$

(5)

2.2.4. Optimization of Fermentation Conditions

Single-Factor Experiment on Medium Components

The carbon sources were varied while all the other conditions were held constant, with a control group lacking any carbon source. The tested carbon sources were added at a concentration of 20 g/L, including maltose, sucrose, corn starch, galactose, and soluble starch. A single-factor experimental design was employed for nitrogen sources (3 g/L, comprising beef extract, yeast extract, bran, soybean meal, and peptone), amino acids (0.15 g/L, including proline, serine, glutamic acid, histidine, and aspartic acid), and vitamins (0.15 g/L, comprising VB1, VB2, VB6, VB9, and VB12), mirroring the design used for carbon sources. After identifying the most effective medium components, further investigations were conducted to optimize the concentrations of carbon sources (15~40 g/L), nitrogen sources (1~3.5 g/L), amino acids (0.1~0.3 g/L), and vitamins (0.1~0.3 g/L).

Response Surface Methodology (RSM) for Optimization

Employing a Box–Behnken experimental design, maltose, beef extract, and aspartic acid were selected as the variable factors, each set at three levels (−1, 0, and 1), with each level replicated three times, and the APC index serving as the response indicator (Table S1). Utilizing the Design-Expert 13 software, the optimal cultivation conditions were predicted and subsequently validated through experimental verification.

2.2.5. Transcriptome Analysis

The mycelia cultured in liquid medium for 6 days were taken out and placed in a sterile centrifuge tube, preserved in dry ice, and sent to Biomarker Biotechnology Co. (Beijing, China) for RNA extraction, cDNA library construction, and transcriptome analysis. Each sample was meticulously prepared with three biological replicates to ensure data reliability. Specifically, the control group (CK group) was cultured in the absence of aspartic acid, while the Asp group was cultured in its presence. To discern significant differences in gene expression between these two groups, the DESeq2 1.30.1, set a stringent threshold of a Fold Change of ≥1.5 and a p-value of <0.05 to identify differentially expressed genes (DEGs). Following their identification, these DEGs were subjected to a dual-layered enrichment analysis, leveraging both the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases.

2.2.6. Data Analysis

The results of the data analysis are presented as the mean ± standard deviation of three replicates. All the statistical analyses were completed using SPSS 26.0, with p < 0.05 considered statistically significant. The graphs and charts were created using the Origin 2021 and GraphPad prism 9.5.1 software. The DESeq2 1.30.1 software was utilized to analyze the DEGs between the two groups, and enrichment analysis of these genes was carried out on the BMKCloud Platform (https://www.biocloud.net/, accessed on 1 November 2024).

3. Results

3.1. Screening of High Antioxidant Activity Strains

As illustrated in Figure 1, all 12 strains of Termitomyces were able to grow in PDB, yet there were significant differences in biomass among the strains. Under identical cultivation conditions, XNYL006 exhibited the highest biomass at 10.76 g/L, while XNWM039 had the lowest at 1.0 g/L, a discrepancy potentially attributable to the varying nutritional requirements and utilization preferences of each strain. Recognizing the limitations of relying on a single antioxidant measurement index, it is essential to employ a comprehensive evaluation of their antioxidant capabilities by integrating multiple antioxidant indices and utilizing the membership function method [30].

Table 1. A comprehensive evaluation of antioxidant activity in Termitomyces strains. Different lowercase letters (a–g) indicate significant differences (p < 0.05).

Termitomyces Strains	Antioxidant Activity Assay				D Value
	ABTS/%	•OH/%	FRAP/(mM FeSO4)	DPPH/%
XNYL003	97.23 ± 2 a	92.23 ± 4 a	5.58 ± 0.29 a	63.40 ± 5 d	0.69
XNXY006	81.04 ± 1 bc	77.95 ± 5 cd	2.41 ± 0.37 bc	57.66 ± 3 d	0.55
XNXY008	42.80 ± 23 e	77.74 ± 8 cd	5.91 ± 0.24 a	55.49 ± 1 d	0.32
XNXY009	42.96 ± 19 e	69.15 ± 10 d	5.71 ± 0.36 a	79.42 ± 5 bc	0.40
LPSPZ011	68.65 ± 23 d	93.38 ± 1 a	5.88 ± 0.04 a	71.85 ± 4 c	0.61
XNXR019	82.28 ± 4 bc	87.19 ± 1 abc	5.63 ± 0.61 a	38.93 ± 3 e	0.48
XNAL022	78.48 ± 10 c	89.13 ± 2 ab	2.35 ± 0.31 bc	58.06 ± 1 d	0.63
XNXY023	84.92 ± 5 bc	86.14 ± 5 abc	1.99 ± 0.09 c	87.90 ± 7 a	0.75
XNQL025	87.53 ± 8 bc	96.25 ± 1 a	5.43 ± 0.07 a	86.43 ± 1 ab	0.80
XNWM037	37.61 ± 36 f	81.64 ± 2 bc	5.68 ± 0.15 a	26.72 ± 8 f	0.20
XNWM038	88.45 ± 9 b	91.64 ± 6 ab	2.68 ± 0.24 b	78.25 ± 1 bc	0.78
XNWM039	39.38 ± 47 f	91.71 ± 7 ab	5.96 ± 0.12 a	15.89 ± 3 g	0.21
XNYL003	97.23 ± 2 a	92.23 ± 4 a	5.58 ± 0.29 a	63.40 ± 5 d	0.69
XNXY006	81.04 ± 1 bc	77.95 ± 5 cd	2.41 ± 0.37 bc	57.66 ± 3 d	0.55

reveals that among the 12 Termitomyces strains, XNYL003, XNWM038, XNQL025, XNXY023, and XNXR019 demonstrated relatively superior ABTS scavenging abilities; the DPPH radical scavenging activity ranged from 16% to 88%, with XNQL025 and XNXY023 achieving clearance rates of approximately 85%; the FRAP values indicated that the total antioxidant capacity of most strains was equivalent to 1.99 to 5.96 mM FeSO₄; and the •OH scavenging activity varied from 78% to 96%, with XNQL025 exhibiting the highest activity. These results indicate that different strains of Termitomyces exhibit significantly diverse antioxidant activities, and may be associated with variations in growth environments and metabolic products generated during mycelial fermentation. Consequently, a comprehensive assessment based on biomass, DPPH, FRAP, ABTS, and •OH scavenging rate indices, employing the membership function method, revealed that XNQL025 possessed the highest overall score, thereby demonstrating the most potent antioxidant activity.

3.2. Optimization of Fermentation Medium Components for XNQL025

3.2.1. Judgment of Fermentation Endpoint of XNQL025

The biomass and pH changes in XNQL025 were monitored over 14 days (Figure 2), revealing that the logarithmic growth phase of Termitomyces occurred between days 3 and 10. During this phase, the mycelia exhibited high metabolic activity, rapid proliferation, and a strong capacity to adapt to their environment. Furthermore, the optimal pH for the growth of Termitomyces was determined to be 6.0, leading to the conclusion that the fermentation period for XNQL025 should be set at 6 days.

3.2.2. Impact of Different Medium Components on XNQL025 Growth and Antioxidant Activity

Previous studies have demonstrated that the composition of the culture medium significantly influences the growth and activity of edible fungi [31]. In this research, single-factor experiments and response surface methodology were employed to optimize the culture medium for Termitomyces, prioritizing antioxidant activity as the primary evaluation metric while considering biomass as a secondary indicator.

The results revealed that the addition of carbon sources markedly increased mycelial biomass, with each carbon source exhibiting distinct effects on antioxidant activity (Table S2): sucrose showed the most pronounced enhancement in ABTS scavenging efficiency; galactose yielded the best results in DPPH scavenging; and maltose demonstrated superior performance in •OH clearance, ultimately possessing the highest APC value when both biomass and antioxidant effects were taken into account, leading to its selection as the carbon source.

As illustrated in Figure 3 and Figure 4, the utilization of beef extract as the nitrogen source and Asp as the amino acid significantly boosted the growth and antioxidant activity of the XNQL025. Consequently, the beef extract and Asp were selected for use in the subsequent experiments. Regarding vitamins, B vitamins exhibited notable efficacy in enhancing antioxidant capacity, with VB1 achieving the highest APC value upon addition, thus being chosen as the vitamin for optimizing the culture medium.

3.2.3. Optimization of Medium Component Concentrations for XNQL025

As illustrated in Figure 5 and Figure 6, within a certain concentration range, the biomass of Termitomyces mycelium increases with the rising concentrations of maltose and beef extract, reaching peak APC values when maltose is at 25 g/L and the beef extract is at 2.0 g/L (Table S3), suggesting that these conditions may facilitate the production of increased antioxidant substances. Regarding the impact of amino acids and vitamins, both Asp and VB1 show an upward trend in mycelial biomass and APC values as their concentrations rise from 0.1 to 0.25 g/L, with the APC values peaking at 0.25 g/L. However, when the concentration of VB1 exceeds 0.25 g/L, antioxidant activity begins to decline, potentially due to the inhibition of antioxidant enzyme activity caused by high concentrations of VB1, thereby impeding the synthesis or accumulation of antioxidant substances. Consequently, this study has determined that 0.25 g/L is the optimal concentration for both Asp and VB1 to enhance the antioxidant performance of XNQL025.

3.2.4. Response Surface Analysis and Model Validation

In this study, RSM was employed to optimize the cultivation conditions of Termitomyces to enhance its antioxidant activity (

Table 2. The response surface experiment with APC as the experimental value.

Run	Maltose (g/L)	Beef Extract (g/L)	Asp (g/L)	APC
1	25	2.5	0.25	89.58
2	20	3.0	0.25	87.42
3	30	2.0	0.25	92.61
4	20	2.5	0.30	79.03
5	25	2.0	0.20	79.57
6	30	3.0	0.25	88.17
7	25	2.5	0.25	87.87
8	25	2.5	0.25	87.60
9	20	2.5	0.20	71.42
10	25	2.5	0.25	89.75
11	30	2.5	0.20	75.72
12	20	2.0	0.25	92.77
13	30	2.5	0.30	78.68
14	25	2.5	0.25	88.86
15	25	2.0	0.30	78.57
16	25	3.0	0.20	70.19
17	25	3.0	0.30	79.90

). The preliminary single-factor experiments identified maltose, beef extract, and Asp as the pivotal factors influencing the antioxidant capacity of Termitomyces. To quantify the relationship between these factors and the APC value, a quadratic multiple regression analysis was conducted on the experimental data, yielding the following regression model: Y = 0.8873 + 0.0057A–0.0223B + 0.0241C + 0.0023AB – 0.0116AC + 0.0268BC + 0.0033A² + 0.0118B²–0.1285C², where A, B, and C represent maltose, beef extract, and Asp, respectively, while AB, AC, BC, A², B², and C² denote the interaction and quadratic terms. The analysis of variance (ANOVA) results (

Table 3. ANOVA of the fitted polynomial quadratic model.

Source	Sum of Squares	df	Mean Square	F-Value	p-Value
Model	0.0820	9	0.0091	106.2500	<0.0001
A-Maltose (g/L)	0.0003	1	0.0003	3.0200	0.1260
B-Beef Extract (g/L)	0.0040	1	0.0040	46.4500	0.0002
C- Asp (g/L)	0.0046	1	0.0046	54.1000	0.0002
AB	0.0000	1	0.0000	0.2437	0.6366
AC	0.0005	1	0.0005	6.3100	0.0403
BC	0.0029	1	0.0029	33.4700	0.0007
A2	0.0000	1	0.0000	0.5402	0.4863
B2	0.0006	1	0.0006	6.7800	0.0352
C2	0.0696	1	0.0696	810.9600	<0.0001
Residual	0.0006	7	0.0001
Lack of Fit	0.0002	3	0.0001	0.7787	0.5641
Pure Error	0.0004	4	0.0001

) revealed that the model’s p-value was less than 0.0001, signifying its statistical significance. The lack-of-fit F-value of 0.7787 and p-value of 0.5641 indicated that the model fit the experimental data well and could reliably predict outcomes. Further analysis, as depicted in Figure 7 and Table 3 demonstrated that beef extract (B), Asp (C), and their interaction terms (AC, BC) and quadratic terms (B², C²) significantly impacted the antioxidant activity of Termitomyces, with Asp exhibiting the highest F-value, underscoring its crucial role in influencing antioxidant activity.

The model predicted an APC value of 92.8 under the conditions of 29.977 g/L maltose, 2.001 g/L beef extract, and 0.2444 g/L Asp. For practical application, the parameters were adjusted to 30 g/L maltose, 2 g/L beef extract, and 0.25 g/L Asp. Under these optimized conditions, the ABTS, DPPH, and •OH scavenging activities of Termitomyces were 82.50%, 84.08%, and 87.15%, respectively, closely aligning with the predicted value and thereby validating the accuracy of the model (

Table 4. Determination of biomass and antioxidant activity in the Asp group and CK group.

Group	Biomass/g/L	ABTS /%	DPPH/%	•OH/%
Asp	9.80 ± 0.23	82.5 ± 0.82	84.08 ± 3.73	87.15 ± 2.51
CK	7.86 ± 0.26	74.28 ± 6.68	65.82 ± 1.42	75.76 ± 2.43

).

3.3. Transcriptomic Analysis of Aspartic Acid Effects on Antioxidant Activity

3.3.1. Quality Assessment of Transcriptome Data

To thoroughly investigate the impact of Asp on the antioxidant activity of XNQL025, transcriptome sequencing was conducted on mycelia cultured in both the Asp group and CK group conditions. The sequencing results revealed that both groups exhibited GC content exceeding 50% and Q30 values above 95%, indicative of high-quality transcriptome data (Table S4). Utilizing the established criteria of Fold Change ≥ 1.5 and p-value < 0.05, a total of 340 DEGs were identified from the Asp group, with 177 genes upregulated and 163 genes downregulated (Figure 8a,b). Based on these DEGs, GO functional annotation and KEGG enrichment analysis were performed (Figure 8c,d).

3.3.2. Functional Annotation and GO Analysis of DEGs

GO enrichment analysis primarily encompasses the GO terms related to cellular processes, metabolic processes, biological regulation, response to stimulus, catalytic activity, and antioxidant activity. The upregulation of the GO terms associated with cellular processes, metabolic processes, and catalytic activity indicates that the altered composition of the culture medium affects the activity of metabolic processes, which is closely linked to mycelial dynamics. These DEGs, encoding proteins or enzymes, play a crucial role in facilitating the utilization of the culture medium by the mycelium [32]. Concurrently, the upregulation of the DEGs related to response to stimulus and antioxidant activity activates the mechanism for the synthesis of antioxidant compounds in Termitomyces, leading to the increased secretion of antioxidants into the medium and thereby enhancing the scavenging capacities of Termitomyces against DPPH, ABTS, and •OH.

3.3.3. KEGG Pathway Enrichment Analysis of DEGs

The KEGG enrichment analysis demonstrated that relative to the CK group, the Asp group exhibited a significant enrichment of DEGs across a multitude of pivotal pathways and regulatory circuits, which encompass carbohydrate metabolism (encompassing amino sugar and nucleoside sugar synthesis, as well as glycolysis/gluconeogenesis), propanoate metabolism, tryptophan metabolism, valine–leucine–isoleucine biosynthesis, the peroxisome signaling pathway, and the mitogen-activated protein kinase (MAPK) signaling pathway (

Table 5. RNA-seq analysis of DEGs in the Asp group and CK group.

KEGG	Gene ID	Function	log2 (Fold Change Asp/CK)
Glycolysis/Gluconeogenesis	TRINITY_DN1172_c0_g1	adh	1.35
	TRINITY_DN8757_c0_g1	adhp	−0.74
	TRINITY_DN1413_c2_g2	pdc	1.22
	TRINITY_DN255_c0_g1	pdc	−1.63
Propanoate metabolism	TRINITY_DN5447_c0_g1	prpB	1.19
	TRINITY_DN2667_c0_g1	GRE2	1.47
	TRINITY_DN8350_c0_g1	GRE2	−0.90
amino sugar and nucleoside sugar synthesis	TRINITY_DN3396_c0_g1	chitinase	1.19
	TRINITY_DN34_c0_g1;	chitinase	0.80
	TRINITY_DN5903_c0_g1	chitinase	0.88
	TRINITY_DN3754_c0_g1	chitin synthase	−0.92
	TRINITY_DN3754_c0_g1	N-acetylglucosamine-6-phosphate deacetylase	−0.92
MAPK signaling pathway	TRINITY_DN959_c0_g1	Fus3	0.60
	TRINITY_DN959_c0_g1	Kss1	0.60
	TRINITY_DN2315_c0_g1	Ctt1	0.90
	TRINITY_DN2667_c0_g1	Gre1	1.47
	TRINITY_DN8350_c0_g1	Gre1	−0.90
peroxisome	TRINITY_DN4239_c0_g2	AMACR	0.91
	TRINITY_DN2315_c0_g1	catalase	0.90
tryptophan metabolism	TRINITY_DN3484_c0_g1	CYP1A1	1.81
	TRINITY_DN2315_c0_g1	catalase	0.90
valine, leucine and isoleucine biosynthesis	TRINITY_DN5827_c0_g2	3-isopropylmalate dehydrogenase	0.62

). These observations suggest that Asp exerts its effects by modulating energy metabolism and substance synthesis, consequently activating the antioxidant defense system and augmenting the antioxidant capacity and adaptability of mycelia, thus facilitating enhanced mycelial growth and substance synthesis.

4. Discussion

4.1. Impact of Medium Components on Termitomyces Antioxidant Activity

Alterations in carbon and nitrogen sources significantly affect the biomass and antioxidant capacity of edible fungi [33,34]. In the present study, the incorporation of 25 g/L maltose resulted in optimal biomass and APC values. However, excessive maltose concentrations led to an increase in the osmotic pressure of the culture medium, which inhibited the accumulation of antioxidant compounds within the mycelium and diminished its resistance to oxidative stress. Additionally, this research has shown that nitrogenous compounds in beef extract aid in the synthesis of proteins by the fungus [35,36], which may explain why XNQL025 exhibited enhanced antioxidant activity in the beef extract medium, likely due to an increase in the protein content of Termitomyces.

In this investigation, the addition of amino acids resulted in a marked increase in both biomass and antioxidant activity of the XNQL025 when compared to the CK group, with Asp exhibiting the most significant impact. The results of the RSM identified Asp as a key factor influencing the antioxidant activity of XNQL025. This finding is consistent with Tepwon’s study, which demonstrated that the addition of Asp can significantly enhance resistance to oxidative stress [37]. Previous studies have demonstrated that amino acid metabolic pathways are not only closely associated with cellular signaling pathways but also serve as precursors for the synthesis of amino acids such as leucine [38,39]. Notably, the KEGG pathway enrichment analysis revealed that Asp treatment significantly activated the valine, leucine, and isoleucine biosynthesis pathway (Rich factor = 3, q < 0.05). Within this pathway, the upregulation of the leuB gene, which encodes 3-isopropylmalate dehydrogenase, directly promotes the synthesis of leucine [40]. Leucine, as a precursor of antioxidant peptides, can enhance cellular tolerance to oxidative stress by activating the mTOR signaling pathway [41]. It suggests that Asp may strengthen the non-enzymatic antioxidant system through a dual mechanism involving precursor supply and signal regulation.

4.2. Asp Regulates Termitomyces Metabolism and Signaling Pathways to Synergistically Enhance Antioxidant Capacity

Glycolysis/gluconeogenesis is a key pathway in the energy metabolism of living organisms, and inhibition of glycolysis significantly suppresses the growth of mycelium in edible fungi. The KEGG analysis further revealed that Asp enhances glycolysis and propionate metabolism in a coordinated manner, forming an efficient energy metabolism network. In the XNQL025, the glycolysis/gluconeogenesis pathway was significantly activated, with the upregulation of the genes encoding adh and pdc, while the expression of its adhp was downregulated The enzyme ADH oxidizes ethanol to acetaldehyde [42]., which is further oxidized to acetate that enters the tricarboxylic acid cycle (TCA cycle) to ATP support for the growth and substance synthesis of Termitomyces. Moreover, in propanoate metabolism, the upregulation of the prpB gene, which encodes methylisocitrate lyase, promotes the production of succinate. Upon entering the TCA cycle, succinate reduces mitochondrial ROS leakage through the electron transport chain. The coordinated regulation of the electron transport chain by these two pathways significantly increases ATP production while reducing ROS generation, thereby achieving the dual optimization of energy supply and oxidative defense. This mechanism provides the core driving force for the accumulation of mycelial biomass and the enhancement of antioxidant activity.

Further analysis revealed that Asp not only modulates energy metabolism through metabolic pathways but also enhances antioxidant capacity by synthesizing antioxidant compounds. Gong reported that active carbohydrate metabolism not only supplies energy for the body but also participates in the synthesis of polysaccharides in edible fungi [43]. In the Asp group, the genes related to chitinase in the amino sugar and nucleotide sugar metabolism pathway were upregulated. Chitinase hydrolyzes chitin in the fungal cell wall to produce chitooligosaccharides, which have been proven to possess significant •OH scavenging capacity. Moreover, Asp also exerts a significant regulatory effect on the functionality of the antioxidant enzyme system. The experimental data show that in the Asp group, the expression level of catalase (CAT) was upregulated within the peroxisome signaling pathway and tryptophan metabolism pathway. This upregulation efficiently catalyzed the decomposition of H₂O₂ into H₂O and O₂ [44], thereby significantly reducing the accumulation of ROS within cells [45].

Further analysis revealed a significant upregulation of the key genes Fus3, Kss1, and Ctt1 in the MAPK signaling pathway. Kss1 and its downstream factors play a pivotal role in regulating mycelial growth and maintaining cell viability. The interaction between Gre1 and Fus3 is essential for signal transduction, while Ctt1 is involved in preserving intracellular redox balance [46]. The upregulation of these genes may enhance the expression of anti-oxidative stress-related genes, activate downstream transcription factors, and further upregulate CAT expression.

Combining the KEGG enrichment results, it is hypothesized that Asp triggers a cascade of signals by binding to membrane receptors, thereby activating the cross-regulation of the MAPK signaling pathway, peroxisome pathway, and tryptophan metabolism pathway. This activation inhibits the production of mitochondrial peroxides, increases CAT activity, and ultimately forms a multi-layered antioxidant defense system through the synergistic action of the enzyme system (CAT) and non-enzyme system (leucine/chitooligosaccharides). This mechanism provides new molecular evidence for the regulation of redox homeostasis in edible fungi by Asp.

4.3. Limitations and Future Directions

This study successfully established a fermentation protocol with high antioxidant activity and elucidated the molecular mechanisms by which Asp affects antioxidant activity in Termitomyces. However, the analysis of secondary metabolites responsible for antioxidant activity remains inadequate, and changes in the protein and metabolite levels were not investigated. Future work should prioritize the following areas: future studies should combine proteomics with targeted metabolomics to clarify the translational changes in differentially expressed genes and the accumulation of key antioxidant components. Supplemental Studies with Leucine: supplementing the basal medium with leucine and comparing their antioxidant activities with those of Asp groups could confirm the efficiency of precursor conversion and further elucidate the mechanisms underlying antioxidant activity. Despite these limitations, this study is the first to construct a molecular framework for the regulation of antioxidant activity in Termitomyces by Asp, providing a theoretical basis for the development of functional fermentation technologies in edible fungi.

5. Conclusions

In this study, the high antioxidant activity strains of Termitomyces were initially selected, and the liquid fermentation scheme was optimized through single-factor experiments and RSM to enhance their antioxidant activity. The results indicated that Asp significantly improved the antioxidant capacity of Termitomyces. Further analysis revealed that Asp modulated multiple metabolic pathways, including glycolysis/gluconeogenesis, propanoate metabolism, amino sugar, and nucleotide metabolism, the valine–leucine–isoleucine pathway, tryptophan metabolism, the MAPK signaling pathway, and the peroxisome signaling pathway. These regulatory actions led to the upregulation of antioxidant enzyme gene expression and increased the synthesis of antioxidant compounds, thereby synergistically strengthening the antioxidant defense system and enhancing the organism’s ability to scavenge free radicals. These findings not only confirm the substantial potential of Termitomyces in the development of natural antioxidants but also provide a robust theoretical foundation and practical guidance for the development of its functional products, expanding new horizons for future research and applications.