Use of ergothioneine in the preparation of a product for improving growth performance, immune capacity, antioxidant capacity or gut health in fish

Abstract

The application discloses application of ergothioneine in preparation of products for improving growth performance, immunity, antioxidant capacity or intestinal health of fish, and belongs to the technical field of biotechnology.It is found by experiments that ergothioneine can promote growth of large yellow croaker larvae and improve muscle quality; ergothioneine can enhance immunity and antioxidant defense capacity and improve liver and intestinal health; meanwhile, ergothioneine can improve liver tissue vacuolization and intestinal villus structure and improve intestinal digestive enzyme activity; ergothioneine can regulate intestinal flora structure and intestinal metabolism; and ergothioneine can also induce systematic remodeling of liver metabolism group and transcription group.In summary, ergothioneine is a natural functional feed additive with wide application prospect, which can synergistically promote fish growth, health and quality improvement by enhancing antioxidant defense, improving liver metabolic homeostasis, maintaining intestinal structure and optimizing intestinal microecology.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of ergothionein in the preparation of products that improve the growth performance, immunity, antioxidant capacity or intestinal health of fish. Background Technology

[0002] Large yellow croaker Larimichthys crocea It belongs to the order Perciformes, family Sciaenidae, and genus *Cichlidae*. Larimichthys The large yellow croaker (Croatia spp.) is a typical nearshore warm-temperate migratory fish. Its flesh is tender and highly nutritious, making it a long-standing important target fish for fishing and consumption in coastal areas. Artificially farmed large yellow croaker accounts for 95% of total production, and the industry's development is heavily reliant on high-density cage culture. High-density cage culture not only leads to a decline in the water exchange capacity of local sea areas and the accumulation of uneaten feed and excrement, resulting in eutrophication and ecological degradation, but also significantly enhances the fish's physiological stress response and reduces their immunity, thus increasing the risk of disease outbreaks. This is a major factor limiting the profitability of large yellow croaker farming. In actual production, due to a lack of standardized drug management and low drug administration efficiency in the water, the irrational use of antibiotics and other chemical drugs persists, leading to increased risks of drug residues and environmental pollution.

[0003] In terms of feed utilization, large yellow croaker farming has long employed a parallel feeding model of fresh feed and formulated feed. Fresh feed mainly comes from low-value miscellaneous fish and shrimp. Although it has good palatability, it suffers from unstable nutrient composition, low utilization rate, and significant water pollution. In contrast, industrialized formulated feed has advantages such as balanced nutrition, high utilization efficiency, and strong controllability, which can significantly reduce nitrogen and phosphorus emissions and lower environmental burden. With the deepening research on the nutritional requirements of large yellow croaker, its nutritional requirements for nutrients such as protein, amino acids, lipids, carbohydrates, vitamins, and minerals have been clarified. However, formulated feed still has certain limitations in terms of palatability, digestibility, and functionality, and its complete substitution in large yellow croaker farming has not yet been fully achieved.

[0004] With the gradual promotion of compound feed in commercial production, issues such as optimizing protein sources, regulating fatty acid composition, and replacing fishmeal with plant protein have received widespread attention. These adjustments not only affect fish growth performance but also their antioxidant status and metabolic health. For example, a high-plant-protein diet can lead to differences in the transcriptome between the gut and liver transcriptomes of large yellow croaker. Furthermore, by optimizing feed formulations, such as adjusting the type and ratio of carbohydrates, studies have shown that feed composition has a significant impact on glucose metabolism and liver function in large yellow croaker, providing nutritional and physiological data for the refined design of compound feeds.

[0005] In the research on the application of functional additives, common additives include palatability enhancers and immunomodulatory factors. For example, complex nucleotides are used in juvenile fish feed to improve palatability and feeding behavior, while significantly improving survival rate, growth performance, digestive enzyme activity, antioxidant capacity, and immune indicators. These effects demonstrate their potential as functional additives to promote healthy growth in juvenile fish. Similarly, organic natural compounds such as allicin have been shown to improve the growth and survival rate of juvenile large yellow croaker by promoting intestinal development, alleviating inflammation, and enhancing appetite, suggesting that plant-derived functional factors have broad application prospects in formulated feeds. In addition, other functional components such as peptides and probiotics have also attracted attention in large yellow croaker feed research. For example, adding peptides and probiotics to formulated feed can significantly affect fish growth performance and body composition, indicating that the synergistic regulation of microecology and nutrition can improve nutrient utilization efficiency and enhance stress resistance.

[0006] Ergothioneine (EGT) is a sulfur-containing amino acid derivative naturally found in various fungi, actinomycetes, and some plant and animal tissues. It possesses strong and stable antioxidant and anti-inflammatory activities. Existing aquatic research mainly focuses on the effects of crude ergothioneine extracts on the oxidative stability and quality maintenance of fish meat. Its absorption and distribution as a single functional nutrient in fish, its systemic physiological effects, and its influence on liver function, intestinal homeostasis, and metabolic regulatory networks are still poorly elucidated, especially in large yellow croaker, where a relatively complete research system has not yet been established. Summary of the Invention

[0007] The purpose of this invention is to provide the application of ergothionein in the preparation of products that improve the growth performance, immunity, antioxidant capacity and intestinal health of fish, so as to solve the problems existing in the prior art. Through verification, this invention proves that ergothionein is a natural functional feed additive with broad application prospects. It mainly promotes the growth, health and quality improvement of fish by enhancing antioxidant defense, improving liver metabolic homeostasis, maintaining intestinal structure and optimizing intestinal microecology.

[0008] To achieve the above objectives, the present invention provides the following solution: This invention provides the application of ergothionein in the preparation of products that improve the growth performance of fish.

[0009] Optionally, the product is used to increase fish weight, weight gain rate, specific growth rate, or feed efficiency.

[0010] This invention also provides the use of ergothionein in the preparation of products that enhance the immune and / or antioxidant capacity of fish.

[0011] Optionally, the product is used to increase the levels of complement C3 and complement C4 in fish, and / or to increase the activity of lysozyme, alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase.

[0012] Optionally, the product is used to increase the activity of superoxide dismutase, catalase, and glutathione peroxidase in fish, and to reduce the content of malondialdehyde.

[0013] This invention also provides the use of ergothionein in the preparation of products that improve the gut health of fish.

[0014] Optionally, the product is used to enhance the activity of intestinal lipase and trypsin in fish; The product is used to improve the morphology of intestinal villi in fish.

[0015] Optionally, the product is used to improve the structure and composition of the gut microbiota in fish; The product is used to improve intestinal metabolites in fish.

[0016] Optionally, the fish species include juvenile large yellow croaker.

[0017] The present invention discloses the following technical effects: (1) Ergothioneine can promote the growth of juvenile large yellow croaker and improve muscle quality. Appropriate levels of ergothioneine increased final body weight, weight gain, specific growth rate and feed utilization efficiency, and improved muscle tissue microstructure, indicating that it has a dual effect of promoting growth and improving meat quality.

[0018] (2) Ergothioneine can enhance immune and antioxidant defense capabilities and improve liver and intestinal health. Ergothioneine increases the activity of serum and liver antioxidant enzymes, reduces the level of oxidative damage, and enhances the body's immune defense to a certain extent. At the same time, ergothioneine improves liver tissue vacuolation and intestinal villus structure, and enhances the activity of intestinal digestive enzymes, indicating that it has a significant protective effect on the liver and intestines.

[0019] (3) Ergothioneine can regulate the structure of intestinal flora and intestinal metabolism. Ergothioneine increases the richness and evenness of intestinal flora, transforming the community from a single dominant type to a multi-group coexistence type, and causes changes in a variety of intestinal metabolites and related metabolic pathways, indicating that it can promote host health by improving the intestinal microecology and metabolic environment.

[0020] (4) Ergothioneine can induce systemic remodeling of the liver metabolome and transcriptome. Differential metabolites and differentially expressed genes point to key processes such as lipid metabolism, nucleotide metabolism, redox homeostasis, immune response and signal transduction, indicating that ergothioneine participates in the maintenance of liver physiological function through multi-level regulation.

[0021] In summary, ergothioneine is a promising natural functional feed additive that primarily promotes the growth, health, and quality of juvenile large yellow croaker by enhancing antioxidant defense, improving liver metabolic homeostasis, maintaining intestinal structure, and optimizing the gut microbiota. Its potential mechanisms can be summarized as enhanced antioxidant activity, liver metabolic reprogramming, gut microbiota optimization, and synergistic regulation of the hepatobiliary axis. Attached Figure Description

[0022] Figure 1 The effects of ergothionein on the growth performance of juvenile large yellow croaker; A: initial body weight; B: final body weight; C: survival rate; D: weight gain rate; E: specific growth rate; F: feed efficiency. Figure 2 The effect of ergothionein on the muscle tissue structure of juvenile large yellow croaker; A: E0 group; B: E50 group; C: E100 group; D: E200 group; Figure 3 Ergothionein content in the whole blood of juvenile large yellow croaker; Figure 4 The effects of ergothioneine on serum biochemical parameters of juvenile large yellow croaker; A: TG; B: T-CHO; C: HDL-C; D: LDL-C; E: Glu; Figure 5 Effects of ergothioneine on serum immune markers in juvenile large yellow croaker; A: LZM; B: C3; C: C4; D: ALT; E: AST; F: ACP; G: AKP; Figure 6 The effect of ergothioneine on serum antioxidant indices in juvenile large yellow croaker; A: MDA; B: SOD; C: CAT; D: GSH-Px; Figure 7 The effect of ergothioneine on antioxidant indices in the muscle of juvenile large yellow croaker; A: MDA; B: SOD; C: CAT; D: GSH-Px; Figure 8 The effect of ergothioneine on antioxidant indices in the muscle of juvenile large yellow croaker; A: MDA; B: SOD; C: CAT; D: GSH-Px; Figure 9 The effect of ergothioneine on the activity of intestinal digestive enzymes in juvenile large yellow croaker; A: Tryps; B: LPS; C: AMS; Figure 10 Effects of ergothionein on liver tissue morphology in juvenile large yellow croaker; A and E: E0 group; B and F: E50 group; C and G: E100 group; D and H: E200 group; Figure 11 The effect of ergothionein on the intestinal tissue morphology of juvenile large yellow croaker; A: E0 group; B: E50 group; C: E100 group; D: E200 group; Figure 12 UpSet plot of ASVs distribution; Figure 13 The alpha diversity index of gut microbiota in juvenile large yellow croaker is determined by ergothionein. A: Chao index; B: Ace index; C: Shannon index; D: Simpson index. Figure 14 The Beta diversity index of gut microbiota in juvenile large yellow croaker is shown in Figure 1. Note: A: PCA plot; B: NMDS plot. Figure 15 This refers to the community composition of gut microbiota at the genus level. Figure 16 The effects of ergothionein on the intestinal metabolome of juvenile large yellow croaker; Figure 17 PLC / OPLS-DA score plots of gut metabolites between groups; A: PCA plot; B: PLS-DA plot; Figure 18 Analysis of differential gut metabolites between groups (E50 vs E0, E100 vs E0, and E200 vs E0); the left panel is a volcano plot; the right panel is a box plot of representative metabolites. Figure 19 KEGG enrichment analysis of differentially expressed gut metabolites in E50 vs E0; Figure 20 KEGG enrichment analysis of differentially expressed gut metabolites in E100 vs E0; Figure 21 KEGG enrichment analysis of differentially expressed gut metabolites in E200 vs E0; Figure 22 The graphs show the intergroup liver metabolite PCA / OPLS-DA scores for E50 vs E0, E100 vs E0, and E200 vs E0; the left graph is the PCA graph; the right graph is the PLS-DA graph. Figure 23 Differential liver metabolite analysis between groups E50 vs E0, E100 vs E0, and E200 vs E0; the left panel is a volcano plot; the right panel is a box plot of representative metabolites; Figure 24 KEGG enrichment analysis of differential liver metabolism between E50 vs E0, E100 vs E0 and E200 vs E0 groups; Figure 25 Statistical analysis of differences in liver gene expression levels between groups; A: Number of genes upregulated / downregulated in each group; B: E50 vs E0 volcano plot; C: E100 vs E0 volcano plot; D: E200 vs E0 volcano plot; Figure 26KEGG annotation analysis of differentially expressed liver genes between groups; A: E50 vs E0; B: E100 vs E0; C: E200 vs E0; Figure 27 KEGG enrichment analysis of differentially expressed genes in the liver (top 20); A: E50 vs E0; B: E100 vs E0; C: E200 vs E0. Detailed Implementation

[0023] The ergothioneine used in the following examples was purchased from Shanghai Maclean Biotechnology Co., Ltd.

[0024] Example 1: Effects of ergothioneine on growth performance and muscle quality of juvenile large yellow croaker 1. Preparation of experimental feed The experiment used the basic compound feed as the control group (E0 group). Based on this, ergothioneine groups were set up with 50 mg / kg (E50 group, 50 mg of ergothioneine per kg of basic compound feed), 100 mg / kg (E100 group), and 200 mg / kg (E200 group). The composition and nutritional level of the experimental feed are shown in Table 1.

[0025] Table 1. Composition and nutrient levels of the experimental diet (%, dry weight) 2. Laboratory animals and breeding conditions Large yellow croakers were provided by the Zhoushan Fisheries Research Institute, and the aquaculture experiment was conducted at the institute's research base. Before the formal aquaculture experiment began, juvenile large yellow croakers were temporarily held in indoor cement tanks for 14 days to acclimatize to the experimental conditions. After the temporary holding period, 6000 healthy, uninjured, and uniformly sized juvenile large yellow croakers (1.84 ± 0.02 g) were selected and randomly assigned to rearing tanks (0.5 m²). 3 The fish were randomly divided into four groups (E0, E50, E100, and E200), with three replicates per group and 500 fish per replicate. The rearing period was 60 days. Each group was fed the corresponding experimental diet twice daily at full capacity (8:00 and 17:00), and the amount of food fed was recorded. Natural seawater was used during the period, with 80% of the water replaced daily. The seawater temperature was 21.2–30.5℃, the salinity was 24–28‰, the dissolved oxygen content was 6.2–7.5 mg / L, the nitrite content was <0.005 g / mL, and the ammonia nitrogen content was <0.2 g / mL.

[0026] 3. Experimental Methods 3.1 Sample Collection After the aquaculture experiment, the large yellow croakers were starved for 24 hours. After anesthetizing with 60 mg / L eugenol solution, the fish in each tank were counted and weighed to calculate survival and weight gain rates. Three fish from each tank were randomly selected, and their dorsal muscle tissue was placed in a 25 mL specimen bottle containing 4% paraformaldehyde solution for tissue fixation. This tissue was then sectioned for observation of the tissue structure.

[0027] 3.2 Index Measurement 3.2.1 Calculation of growth indicators for large yellow croaker Calculate the survival rate, weight gain rate, specific growth rate, and feed efficiency for each group using the following formulas.

[0028] Survival rate (%) = 100 × Nt / N0; Weight gain rate (%) = 100 × (Wt - W0) / W0; Specific growth rate (% / d) = 100 × (lnWt - lnW0) / t; Feed efficiency = (Wt - W0) / Wf; Where N0 is the initial number of digits; Nt is the final number of digits; W0 is the initial body weight (g); Wt is the final body weight (g); Wf is the average food intake (g); and t is the number of days in the experiment (d).

[0029] 3.2.2 Sections of muscle tissue from large yellow croaker After the muscle tissue was fixed, it was sent to Hangzhou Haoke Biotechnology Co., Ltd. After the tissue was fixed for 24 hours and the fixation status was good, the tissue was trimmed, dehydrated, embedded, sectioned, stained, mounted and finally examined under a microscope to obtain qualified samples.

[0030] 3.3 Data Analysis Experimental data were analyzed using SPSS 26.0 software through one-way ANOVA. After significant differences were identified, Tukey multiple comparisons were performed, with a significance level set at [value missing]. P <0.05, the test results are expressed as mean ± standard deviation.

[0031] 4. Results and Analysis 4.1 Effects of ergothionein on the growth performance of juvenile large yellow croaker After a 60-day aquaculture experiment, feeding juvenile large yellow croaker with ergothioneine significantly improved their growth performance. Figure 1Compared with the control group, the initial weight of large yellow croaker in each experimental group did not differ significantly, indicating that the initial size of the experimental fish was basically the same and they were comparable. At the end of the rearing period, the final weight, survival rate, weight gain rate, specific growth rate (SGR), and feed efficiency of the experimental group supplemented with ergothioneine were significantly higher than those of the control group, while the differences between the experimental groups were not significant. The survival rate of large yellow croaker did not differ significantly among the groups.

[0032] 4.2 Effects of ergothionein on the muscle tissue structure of juvenile large yellow croaker like Figure 2 As shown, HE-stained sections in group E0 showed relatively large interfiber gaps and a loose arrangement, with some muscle fibers not being uniform in size. In group E50, the muscle fibers were arranged more neatly than in group E0, the interfiber gaps were reduced, and the tissue integrity was improved. In group E100, the muscle fibers were arranged more tightly, the muscle bundle boundaries were clearer, the gaps between muscle fibers were fewer, and the overall tissue structure was more intact. Group E100 also showed relatively tight muscle fiber arrangement and good tissue structure integrity, similar to group E200. In some areas, the muscle fiber arrangement was more regular. The results indicate that the addition of ergothionein helps to improve the muscle microstructure of juvenile large yellow croaker and improve the density and integrity of muscle fiber arrangement.

[0033] In summary, this embodiment systematically evaluated the effects of ergothioneine on the growth performance and muscle tissue structure of juvenile large yellow croaker. The results showed that appropriate levels of ergothioneine could increase the final body weight, weight gain rate, and specific growth rate of juvenile fish, and improve feed utilization efficiency. At the histological level, ergothioneine improved the muscle tissue microstructure, resulting in denser muscle fiber arrangement and better tissue integrity. This embodiment provides a basis for its use as a functional feed additive in the high-quality aquaculture of large yellow croaker.

[0034] Example 2: Effects of ergothionein on the immunity, antioxidant capacity, and digestive capacity of juvenile large yellow croaker 1. Experimental Methods The experimental animals and breeding conditions were the same as in Example 1.

[0035] 1.1 Sample Collection Five fish were randomly selected from each tank. After anesthetizing with 60 mg / L eugenol solution, blood was collected from the tail vein using a disposable blood collection device. The blood was transferred to a blood collection tube and placed on ice for 4 hours, followed by centrifugation at 3000 r / min for 10 min. The supernatant was collected and stored at -80℃ for the determination of serum biochemical, immunological, and antioxidant indicators. After blood collection, the fish were dissected, and the liver, intestine, and dorsal muscle were collected and stored at -80℃ for the determination of antioxidant enzyme activity in the liver and muscle, and digestive enzyme activity in the intestine. Whole blood was collected from another five fish for the determination of ergothioneine content. After blood collection, the fish were dissected, and liver and intestinal samples were fixed in specimen bottles containing 25 mL of 4% paraformaldehyde solution for the preparation of tissue sections to observe their tissue structure.

[0036] 1.2 Index Measurement 1.2.1 Determination of Ergothioneine Content in Whole Blood Take 1.5 mL of whole blood, add 4 mL of distilled water, and heat in a 90 °C water bath for 12 min, shaking every 3 min. Then cool the sample on ice for 3 min, centrifuge at 4 °C and 3000 g for 15 min, and collect the supernatant. Add 12 mL of anhydrous ethanol to the supernatant, mix well, incubate on ice for 30 min, and centrifuge again at 4 °C and 3000 g for 15 min. Collect the supernatant, dry it under vacuum using a rotary evaporator, and redissolve the residue in 4 mL of distilled water. Finally, take 10 μL of the sample for liquid chromatography analysis.

[0037] 1.2.2 Determination of serum biochemical and immune indicators Serum biochemical indicators, including triglyceride (TG), total cholesterol (T-CHO), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and glucose (GLU), as well as serum immune indicators, including complement C3 (C3) and complement C4 (C4), and the activities of lysozyme (LZM), acid phosphatase (ACP), alkaline phosphatase (AKP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), were all measured using a kit. The tissue protein content required for enzyme activity determination was measured using a protein concentration assay kit. The assay procedures were strictly performed according to the kit instructions.

[0038] 1.2.3 Determination of serum and liver antioxidant markers The levels of malondialdehyde (MDA), an antioxidant marker in serum and liver, and the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were all measured using kits. The protein content of the tissues requiring enzyme activity determination was measured using a protein concentration assay kit. All assays were performed strictly according to the kit instructions.

[0039] 1.2.4 Determination of intestinal digestive enzyme activity The activities of intestinal digestive enzymes α-amylase (AMS), lipase (LPS), and trypsin were all determined using kits. The protein content of the tissue to be measured was determined using a protein concentration assay kit to calculate enzyme activity. The assay procedures were strictly performed according to the kit instructions.

[0040] 1.2.5 Liver and intestinal tissue sections Fixed liver and intestinal tissues were sent to Hangzhou Haoke Biotechnology Co., Ltd. After fixation, intestinal samples were dehydrated sequentially with graded ethanol, followed by paraffin embedding and sectioning. Sections were stained with hematoxylin and eosin, dehydrated, mounted, and observed and photographed under an ECLIPSE-E100 microscope. Image-Pro Plus software was used to measure villus length, villus width, and muscle layer thickness in the sections, and the average values ​​of each indicator were calculated.

[0041] 2. Results and Analysis 2.1 Determination of Ergothionein Content like Figure 3 As shown, with the increase of ergothionein addition level in feed, the ergothionein content in whole blood of juvenile large yellow croaker showed a significant increasing trend, reaching the highest in group E200 and the lowest in control group, indicating that exogenous ergothionein can be effectively absorbed by the body and enter the blood circulation, and has a good dose-response relationship within the range of this experiment.

[0042] 2.2 Effects of ergothionein on serum biochemical parameters of juvenile large yellow croaker like Figure 4 As shown, different amounts of ergothioneine had a significant effect on the serum biochemical parameters of juvenile large yellow croaker. Serum triglycerides (TG) were highest in the E50 and E200 groups, significantly higher than those in the E0 and E100 groups. Total cholesterol (T-CHO) showed a similar trend, with the highest in the E50 group, the lowest in the E100 group, and intermediate levels in the E200 and E0 groups. HDL-C was higher in the E50 and E100 groups and lowest in the E200 group. Low-density lipoprotein cholesterol (LDL-C) was highest in the E50 group and lowest in the E100 group. Glucose (Glu) showed a significant fluctuation trend with the addition level, with the highest in the E50 group, followed by the E0, E100, and E200 groups.

[0043] In summary, the E100 group showed lower levels of TG, T-CHO, and LDL-C, while maintaining a higher HDL-C, indicating that 100 mg / kg ergothioneine was more beneficial in improving the serum lipid metabolism of juvenile large yellow croaker. The E50 group had higher levels of TG, T-CHO, LDL-C, and Glu, indicating a relatively high metabolic burden on the body at this dose. While T-CHO and Glu further decreased in the E200 group, TG remained at a high level, and HDL-C reached its lowest point, indicating that excessively high levels did not further improve the overall metabolic status. This suggests that appropriate amounts of ergothioneine may help promote lipid utilization and alleviate dyslipidemia; it also has a positive regulatory effect on serum glucose and lipid metabolism in juvenile large yellow croaker, with 100 mg / kg being the most suitable addition level under the conditions of this experiment.

[0044] 2.3 Effects of ergothionein on serum immune markers in juvenile large yellow croaker Depend on Figure 5 It was found that with the increase of ergothioneine supplementation level in the diet, lysozyme (LZM) showed a significant upward trend, with the E100 and E200 groups significantly higher than the E50 and E0 groups. Complement C3 and complement C4 also increased overall, with the highest level in the E200 group. AKP was significantly higher in the E100 and E200 groups than in the E0 and E50 groups. This indicates that the promoting effect of ergothioneine on the innate immunity of juvenile large yellow croaker is mainly reflected in enhancing lysozyme activity, increasing complement levels, and increasing alkaline phosphatase (AKP) activity, with the effects being more pronounced in the E100 and E200 groups. At the same time, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) showed a downward trend with the increase of ergothioneine supplementation, suggesting that while enhancing immunity, ergothioneine may also help reduce the metabolic burden of the body or improve liver-related physiological states. In summary, the E200 group showed the most significant improvement in complement and lysozyme levels, while the E100 group demonstrated a better balance between higher immune levels and lower ALT levels, indicating that ergothioneine has a good immune-enhancing effect under the conditions of this experiment.

[0045] 2.4 Effects of ergothionein on the activity of antioxidant enzymes in the serum, liver, and muscle of juvenile large yellow croaker like Figure 6As shown, ergothioneine significantly improved the antioxidant status of serum in juvenile large yellow croaker. The serum malondialdehyde (MDA) content in the E200 group was significantly lower than that in the E0, E50, and E100 groups. The superoxide dismutase (SOD) activity in serum reached its highest level in the E100 group. The catalase (CAT) activities in both the E100 and E200 groups were significantly higher than those in the E0 and E50 groups. The glutathione peroxidase (GSH-Px) activity was highest in the E200 group, significantly higher than in the other groups. This indicates that ergothioneine has a significant promoting effect on the serum antioxidant system, with the E100 group being more beneficial in increasing SOD activity, while the E200 group was more beneficial in enhancing GSH-Px activity and reducing MDA levels.

[0046] Through liver antioxidant results ( Figure 7 Analysis showed that ergothioneine at a concentration of 50-100 mg / kg exhibited the best performance. The MDA content in the liver of the E50 group was significantly lower than that of the E0, E100, and E200 groups, with the highest content observed in the E0 group. The CAT activity in the liver of the E50 group was significantly higher than that of the other groups. There was no significant difference in liver GSH-Px levels among the groups. In summary, the E50 group exhibited the characteristics of the lowest MDA and highest CAT levels in the liver, indicating that 50 mg / kg ergothioneine was most effective in reducing hepatic lipid peroxidation and enhancing hydrogen peroxide clearance.

[0047] Through muscle antioxidant results ( Figure 8 Analysis showed that ergothionein's regulation of muscle activity was mainly reflected in MDA, SOD, and GSH-Px. MDA in the E100 group was significantly lower than that in the E0 group, while SOD was highest in the E50 group. There was no significant difference in muscle CAT levels among the groups. GSH-Px in the E100 group was significantly higher than that in the E0 and E50 groups.

[0048] In summary, ergothioneine can improve the antioxidant status of juvenile large yellow croaker, but its effects exhibit significant tissue variability and dose-dependency. In serum, the high-dose group showed the most significant overall antioxidant effect, with the lowest MDA and the highest GSH-Px. In the liver, the low-dose group was more effective in reducing MDA and increasing CAT activity. In muscle, the E100 group showed a better overall effect, reducing both MDA and increasing GSH-Px, while the E50 group was more effective in increasing SOD. This indicates that ergothioneine does not simply increase all antioxidant enzymes simultaneously, but is more likely to regulate the body's redox balance through tissue-specific and enzyme-specific mechanisms. Considering the tissue-accumulating and antioxidant activity of ergothioneine, it can be inferred that its protective effect on juvenile large yellow croaker is mainly related to reducing lipid peroxidation damage, improving peroxide scavenging capacity, and enhancing the enzymatic antioxidant system.

[0049] 2.5 Effects of ergothionein on the activity of intestinal digestive enzymes in juvenile large yellow croaker like Figure 9 As shown, ergothioneine has a significant regulatory effect on intestinal digestive enzymes in juvenile large yellow croaker, but the degree of response varies among different enzyme systems. Trypsin activity in the E50 group was significantly higher than in other groups, indicating that ergothioneine addition significantly improves intestinal protein digestion. Lipase (LPS) activity in the E200 group was significantly higher than in the E50 group, indicating that higher levels of ergothioneine are more beneficial for lipid digestion. The results show that ergothioneine significantly increased trypsin activity and had a certain promoting effect on lipase, but had no significant effect on amylase (AMS), suggesting that its regulatory effect is more biased towards protein and lipid digestion, rather than comprehensively enhancing the activity of all digestive enzymes. This indicates that the intestinal digestive enzymes of large yellow croaker exhibit significant enzyme system selectivity in response to exogenous nutrients, and not all additives cause a simultaneous increase in all three enzyme types.

[0050] 2.6 Effects of ergothionein on the liver and intestinal tissue structure of juvenile large yellow croaker like Figure 10 As shown, different amounts of ergothioneine had a certain impact on the morphology of liver tissue in juvenile large yellow croaker. Under low magnification (×10), the liver tissue in group E0 was relatively loose overall, with many vacuolar structures visible in the hepatocyte cytoplasm. The liver tissue in group E50 was more compact, with significantly reduced vacuolation compared to group E0, and the overall structure was more intact. The liver tissue in group E100 also had a relatively intact structure, with hepatocytes arranged more regularly. The liver tissue in group E200 still had a relatively intact overall structure, with hepatocytes arranged more evenly. Under high magnification (×40), a large number of round vacuoles of varying sizes were visible in the hepatocytes of group E0, some of which were larger, and the cell boundaries were relatively unclear. The number of vacuoles was significantly reduced in group E100, with smaller vacuoles predominating, and the hepatocyte cytoplasm was more compact. Groups E50 and E200 still showed many vacuoles, but the overall vacuole size was more uniform than that in group E0, and the tissue structure was more intact. The results indicate that an appropriate amount of ergothioneine may help reduce lipid deposition or vacuolar damage in the liver of large yellow croaker. In this embodiment, the reduced vacuolation and tighter tissue arrangement in the liver tissue of the E50 group may be related to the fact that an appropriate amount of ergothioneine improves the liver's redox state, reduces lipid deposition, and maintains cell structural stability.

[0051] Table 2 shows that the villus height in groups E50, E100, and E200 was significantly higher than that in group E0. The villus width was highest in group E50, significantly higher than the other groups. Regarding muscle layer thickness, group E50 was significantly thicker than the other groups. Figure 11As shown, the intestinal villi in group E0 were generally shorter and thicker, with some villi arranged irregularly and relatively large inter-villi spacing, and the integrity of the mucosal surface was generally poor. In group E50, the villi were significantly longer, more complete in shape, and more neatly arranged, with relatively more uniform inter-villi spacing. In group E100, the hindgut villi were also longer, and were generally thinner, denser, and more regularly arranged, with a more complete mucosal structure. Group E200 also showed some improvement over group E0, with more complete villi, but it was slightly inferior to groups E50 and E100 in terms of the uniformity of villi thickness and the regularity of arrangement in some areas.

[0052] Table 2. Effects of ergothionein on intestinal tissue structure of large yellow croaker. Note: Different letters indicate significant differences. P <0.05); identical letters or no letters indicate no significant difference ( P >0.05).

[0053] Based on the above results, this embodiment mainly studied the effects of ergothioneine on the immunity, antioxidant capacity, and digestive capacity of juvenile large yellow croaker. The results showed that ergothioneine can promote the accumulation of ergothioneine in whole blood, increase the activity of some serum immune-related indicators and antioxidant enzymes, and reduce the level of lipid peroxidation damage, indicating that it can enhance the body's non-specific immunity and antioxidant defense capabilities. Simultaneously, ergothioneine also increased the activity of intestinal trypsin and lipase, indicating that it helps improve intestinal digestive function. Histological observation further showed that ergothioneine can alleviate liver tissue vacuolation and improve intestinal villus morphology, suggesting that it has a protective effect on liver and intestinal health. In conclusion, ergothioneine can promote the health status of juvenile large yellow croaker by enhancing antioxidant and immune defense, maintaining the integrity of the hepato-intestinal structure, and improving digestive function.

[0054] Example 3: Effects of ergothioneine on intestinal flora and metabolome of juvenile large yellow croaker 1. Experimental Methods The experimental animals and breeding conditions were the same as in Example 1.

[0055] 1.1 Sample Collection Ten fish were randomly selected from each group. After anesthetizing the fish with 60 mg / L eugenol solution, their intestines were dissected, placed in cryovials, and stored in liquid nitrogen for the purpose of measuring intestinal flora and intestinal metabolites.

[0056] 1.2 Intestinal flora assay Intestinal samples were sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. under dry ice conditions, and then DNA extraction, library construction, sequencing and bacterial diversity analysis were performed.

[0057] 1.3 Determination of intestinal metabolites The intestinal samples of large yellow croaker were sent to Shanghai Meiji Biomedical Technology Co., Ltd. under dry ice conditions for non-targeted metabolomics testing.

[0058] 2. Results and Analysis 2.1 Effects of ergothionein on gut microbiota diversity in juvenile large yellow croaker Depend on Figure 12 The results showed significant differences in gut microbiota composition under different ergothioneine addition levels. The total number of ASVs in each group was 305 in group E0, 532 in group E50, 745 in group E100, and 629 in group E200, indicating that the number of detectable ASVs in the gut microbiota increased overall after ergothioneine addition, with the most significant increase observed at the 100 mg / kg addition level. This suggests that appropriate levels of ergothioneine may help improve gut microbiota richness and promote the formation of more specific bacterial communities. Only 28 ASVs were shared across the four groups, indicating that while there were some core shared bacterial communities among the groups, their numbers were relatively small, suggesting that ergothioneine has a strong remodeling effect on gut microbiota structure. Furthermore, each treatment group developed its own specific microbiota composition, with group E100 having the highest number of specific ASVs, indicating that this treatment group had the most significant effect on the selection and enrichment of gut microbes. In summary, ergothionein not only increased the total number of ASVs in the gut microbiota of juvenile large yellow croaker, but also significantly altered the shared and unique distribution patterns among communities, with the E100 group showing the most significant impact on gut microbiota structure.

[0059] like Figure 13 As shown, different levels of ergothionein had some effect on the α-diversity of gut microbiota in juvenile large yellow croaker, but the differences were not significant.

[0060] Depend on Figure 14 As shown in Figure A, there were significant differences in the gut microbiota composition between the control group and the groups after EGT supplementation. The gut microbiota structure after treatment with medium and high doses of ergothioneine showed some similarity, but there were still differences between the groups. Figure 14 The results in Figure B show that the samples from each treatment group also exhibited a relatively clear separation pattern, indicating that different levels of EGT significantly altered the composition of the gut microbiota in juvenile large yellow croaker. Overall, the results indicate that ergothioneine addition has a significant remodeling effect on the gut microbiota structure of juvenile large yellow croaker, with the control group showing the most significant separation from each added group, suggesting that exogenous ergothioneine can significantly alter the gut microbiota composition.

[0061] 2.2 Effects of ergothioneine on the intestinal flora structure of juvenile large yellow croaker like Figure 15As shown, the composition of the intestinal flora of juvenile large yellow croaker changed significantly at the phylum level after treatment with different amounts of ergothioneine. In the control group E0, Fusobacteriota was the absolutely dominant phylum, while Pseudomonadota and other low-abundance phyla accounted for a relatively low proportion. After the addition of ergothioneine, Fusobacteriota decreased significantly in the E50 group, while Pseudomonadota and Cyanobacteriota increased relatively. In the E100 and E200 groups, Pseudomonadota became the dominant phylum. The E100 group also had a certain level of Bacteroidota, while the E200 group maintained the dominance of Pseudomonadota, while Fusobacteriota still accounted for a certain proportion, with a slight increase in Bacillota and Actinomycetota. Overall, after the addition of ergothioneine, the gut microbiota gradually changed from a structure dominated by Fusobacterium in the control group to a community pattern dominated by Pseudomonas and accompanied by a variety of low-abundance phyla, indicating that ergothioneine has a significant remodeling effect on the phylum-level composition of the gut microbiota of juvenile large yellow croaker.

[0062] like Figure 16 As shown, the composition of the gut microbiota at the genus level in juvenile large yellow croaker changed significantly after treatment with different amounts of ergothioneine. In the control group (E0), the gut microbiota of large yellow croaker was dominated by *Cetacea* genus (…). Cetobacterium The dominant genus *Cetacea* was *Pycnodon*, with a relatively concentrated community structure. After the addition of ergothioneine, the composition of the gut microbiota changed significantly in each treatment group. The absolute dominance of *Cetacea* weakened, and the gut microbiota gradually shifted from a single dominant type to a multi-gene coexistence type. In group E50, *Pycnodon* was the dominant genus. Rhodopirellula Synechocybe ( Synechococcus ), genus Rugellium ( Ruegeria The proportion of bacteria of genera such as ergothionein increased, and the distribution of the gut microbiota was more balanced than in the E0 group. This trend was further enhanced in the E100 group, with multiple genera sharing a high proportion, indicating that the evenness and complexity of the gut microbiota were further improved with the addition of 100 mg / kg ergothionein. The gut microbiota structure in the E200 group still maintained a high degree of diversity, and no longer showed absolute dominance of a single genera. Overall, the gut microbiota structure exhibited by the E50 and E100 groups was more balanced, which may be more conducive to the stability of the gut microbiota.

[0063] 2.3 Effects of ergothionein on the intestinal metabolome of juvenile large yellow croaker Depend on Figure 17 It is evident that the addition of ergothionein significantly reshaped the overall metabolic profile of the large yellow croaker samples, and the differences between the E50 and E100 groups and the control group were significant.

[0064] like Figure 18As shown, the addition of ergothioneine significantly altered the metabolic profile of large yellow croaker, and this change exhibited a dose-dependent effect. The E50 group showed a relatively small number of differentially regulated metabolites, indicating that the addition of 50 mg / kg ergothioneine primarily caused mild metabolic regulation. The E100 group showed the highest number of upregulated metabolites, suggesting that the addition of 100 mg / kg ergothioneine tended to promote the accumulation and enhanced metabolic activity of some metabolites. The E200 group showed a significant increase in the number of downregulated metabolites, indicating that while the addition of 200 mg / kg ergothioneine promoted the increase of some key metabolites, it may also have led to the inhibition or redistribution of more metabolic pathways, manifesting as a deeper level of metabolic remodeling.

[0065] From the perspective of differentially metabolite types, the most significantly affected molecules in this embodiment were lipid metabolism-related molecules, including LPA, LPC, PC, PE, and Linoleoyl Ethanolamide. Simultaneously, the abundance of bile acid-related metabolites such as 5β-Cyprinol Sulfate increased, indicating that ergothioneine treatment may further affect lipid absorption, bile acid cycling, and digestive metabolism. On the other hand, peptide metabolites such as Gly-Pro-Arg-Pro-Lys were continuously upregulated in the E100 and E200 groups, suggesting that amino acid transport and peptide metabolism may also be important mechanisms for ergothioneine's effects. Correspondingly, some flavonoid, sugar, and saponin metabolites were downregulated at higher doses, indicating that the addition of 200 mg / kg ergothioneine may cause the body's metabolism to gradually shift from adaptive enhancement to rebalancing regulation. Overall, ergothioneine can significantly reshape the metabolic profile of large yellow croaker, with its regulatory effects mainly concentrated in lipid metabolism, bile acid-related metabolism, and amino acid / peptide metabolism. Among them, 100 mg / kg ergothioneine showed a stronger metabolic activation effect, while 200 mg / kg ergothioneine exhibited a more complex bidirectional regulatory characteristic. This result suggests that adding 100 mg / kg ergothioneine may be more beneficial in improving the body's metabolic state, while 200 mg / kg ergothioneine may induce a more extensive metabolic network remodeling.

[0066] 2.4 Effect of ergothionein on the enrichment analysis of KEGG, a differential metabolite in the intestine of juvenile large yellow croaker like Figure 19 As shown, low-dose ergothioneine can induce metabolic responses in lipid absorption and utilization, nucleotide turnover, and some signal transduction processes; however, overall regulation is still mainly based on basal metabolic remodeling. Figure 20 As shown, in addition to continuing to affect lipid metabolism and transmembrane transport, medium-dose ergothioneine also further influences energy metabolism, redox homeostasis, glycan synthesis, and immune-related metabolic networks. Figure 21As shown, compared with the E50 group, the E200 group showed a significant increase in lipid signaling, immune recognition, and stress regulation-related pathways, indicating that the effect of high-dose ergothioneine on the body's metabolic network has been further extended from the regulation of basic metabolism to the synergistic response of immune-stress-signal transduction.

[0067] The combined results from the three groups indicate that ergothioneine's effects on the metabolic network of large yellow croaker are dose-dependent. Low doses primarily affect lipid absorption and transport, as well as basal metabolic remodeling. Medium doses begin to extend effects on energy metabolism and redox homeostasis, while high doses further involve more complex biological processes such as immune recognition, cellular stress, and signal transduction. The regulation of the metabolic network of large yellow croaker by ergothioneine exhibits a clear dose-progressive characteristic.

[0068] Based on the above results, this embodiment explored the regulatory effects of ergothioneine on the intestinal microecology of juvenile large yellow croaker from two levels: gut microbiota and intestinal metabolites. The results showed that ergothioneine can increase the richness, evenness, and overall diversity of the gut microbiota, and transform the community structure from a relatively singular dominant type to a multi-group coexistence type. At both the phylum and genus levels, ergothioneine significantly altered the composition of the gut microbiota and optimized the microecological structure under normal conditions. Intestinal metabolomics analysis showed that ergothioneine can induce changes in various differential metabolites, mainly involving lipid metabolism, amino acid metabolism, nucleotide metabolism, and related signaling pathways. In summary, ergothioneine can improve the intestinal microecological environment by reshaping the gut microbiota structure and regulating metabolite composition, providing favorable conditions for host nutrient absorption and health maintenance.

[0069] Example 4: Effects of ergothionein on the liver metabolome and transcriptome of juvenile large yellow croaker 1. Experimental Methods The experimental animals and breeding conditions were the same as in Example 1.

[0070] 1.1 Sample Collection Ten fish were randomly selected from each group. After anesthetizing the fish with 60 mg / L eugenol solution, their livers were dissected, placed in cryovials, and stored in liquid nitrogen for subsequent liver metabolomics and transcriptomics experiments.

[0071] 1.2 Liver metabolomics assay The liver samples of large yellow croaker were sent to Shanghai Meiji Biomedical Technology Co., Ltd. under dry ice conditions for non-targeted metabolomics testing.

[0072] 1.3 Liver transcriptome analysis Liver tissue samples from large yellow croakers in each experimental group were immediately frozen after being placed in RNase-free cryopreservation tubes and then sent to Shanghai Meiji Biotechnology Co., Ltd. under dry ice conditions for subsequent transcriptome sequencing analysis.

[0073] 2. Results and Analysis 2.1 Effects of ergothionein on the hepatic metabolome of juvenile large yellow croaker As shown in Figure 22, the three treatment groups and the control group all showed varying degrees of separation trends in PCA and PLS-DA, indicating that the liver metabolic characteristics were altered after different dose treatments. The intergroup separation in the PLS-DA figure was clearer than that in the PCA figure, suggesting that the metabolic differences between groups were further amplified after the introduction of group information. Combining the overall distribution characteristics of PCA and PLS-DA, it can be seen that different dose treatments can lead to the reconstruction of the liver metabolic profile of large yellow croaker. The E50 group showed a relatively clear metabolic shift, the E100 group showed greater intragroup fluctuations, and the separation between the E200 group and the control group was the clearest, indicating that high-dose treatment had the most significant impact on the liver metabolic state.

[0074] like Figure 23 As shown, significant changes in liver metabolites were observed in all treatment groups compared to the control group, but the magnitude and direction of these changes differed according to dosage. This indicates that the abundance of several representative metabolites differed significantly between the treatment groups and the E0 group, with most reaching significant or highly significant levels, demonstrating that the screened metabolites have good inter-group discrimination ability.

[0075] In the E50 and E100 groups, 3-hydroxytetradecanoyl carnitine was continuously elevated, indicating that low-to-medium dose treatment was more likely to promote hepatic fatty acid transport and oxidative metabolism. In the E200 group, although some key metabolites were still elevated, the overall characteristic was an increase in downregulated metabolites, suggesting that under high-dose conditions, hepatic metabolism may shift from adaptive regulation to a deeper level of rebalancing or inhibitory remodeling.

[0076] Depend on Figure 24The KEGG enrichment analysis showed that, compared with the control group E0, the enrichment patterns of differential metabolites in the liver of large yellow croaker exhibited a significant dose-progressive characteristic. In the E50 group, differential metabolites were mainly enriched in pathways such as autophagy (animal), alpha-linolenic acid metabolism, mitophagy (animal), glycerophospholipid metabolism, arachidonic acid metabolism, endocytosis, and glycosylphosphatidylinositol (GPI)-anchor biosynthesis, suggesting that low-dose treatment primarily affects membrane lipid remodeling, polyunsaturated fatty acid conversion, and organelle quality control processes. In the E100 group, the number of enriched pathways increased significantly, indicating that medium-dose treatment extended from local lipid responses to the synergistic regulation of lipid utilization, substance transport, and carbon and nitrogen metabolism. In the E200 group, the enrichment pathways extended further into deeper energy metabolism and substrate turnover, indicating that high-dose treatment had caused a systemic reprogramming of liver energy production, nucleic acid precursor turnover, and sulfur-containing amino acid metabolism.

[0077] The enrichment results suggest that the enrichment pattern observed in group E50 may reflect an early, adaptive hepatoprotective response. With increasing treatment levels, group E100 progressed from "membrane lipid remodeling" to a synergistic regulatory stage involving "lipid utilization—substrate allocation—small molecule transport." Group E200 significantly influenced mitochondrial energy supply, nucleic acid precursor regeneration, and sulfur-containing metabolic networks. Overall, this invention demonstrates that the effects of different treatment levels on the liver metabolism of large yellow croaker are not simply linearly enhanced, but rather progress from a low-dose adaptive membrane lipid-autophagy response to a medium-dose regulation of lipid utilization and substance transport, ultimately culminating in a deep metabolic response at high doses, centered on energy metabolism, nucleotide turnover, and redox homeostasis remodeling.

[0078] 2.2 Effects of ergothionein on the liver transcriptome of juvenile large yellow croaker The transcriptome sequencing data obtained in this embodiment are of good quality and have high sample consistency, which can provide a reliable data foundation for subsequent differential expression analysis and functional enrichment analysis.

[0079] like Figure 25 As shown, the expression level difference analysis was performed using DESeq2 differential analysis software. Compared with the control group E0, there were significant differences in the number of differentially expressed genes in different treatment groups.

[0080] like Figure 26 As shown, KEGG annotation results indicate that, compared with the control group E0, differentially expressed genes in groups E50, E100, and E200 can be categorized into six primary functional categories: metabolism, genetic information processing, environmental information processing, cellular processes, organizational systems, and human diseases. This suggests that different treatments can induce changes in the distribution of transcriptional function in the liver of large yellow croaker. Overall, different treatments can affect the metabolic regulation, substance transport, and physiological response functions of the liver in large yellow croaker, with the E100 group exhibiting the broadest range of transcriptional remodeling.

[0081] Notably, the number of annotated genes in most functional categories was significantly higher in the E100 vs E0 group than in the E50 vs E0 and E200 vs E0 groups, indicating that moderate-dose treatment more readily induces extensive hepatic transcriptional remodeling. Combined with the experimental results, it can be inferred that this treatment level may fall within the most sensitive response range of the organism. Low doses primarily trigger initial adaptation, while at high doses, some responses may shift towards relatively concentrated regulation of key pathways, thus reducing the overall number of annotated genes. This phenomenon of better effects from moderate doses is consistent with the aforementioned finding that the E100 group had the most differentially expressed genes. Overall, the KEGG annotation results of this study indicate that different treatments can induce transcriptional responses in the liver of large yellow croaker in metabolic regulation, signal transduction, substance transport, and immunophysiological functions, with the E100 group inducing the most significant functional remodeling, suggesting that moderate-dose treatment may more significantly activate the comprehensive physiological response network of the liver.

[0082] like Figure 27 As shown, KEGG enrichment analysis revealed that the functional pathways involved in differentially expressed genes in different treatment groups compared to the control group E0 exhibited significant stage-specific changes. The E50 group induced transcriptional responses in the liver related to innate immune recognition, cellular stress regulation, and some glucose and lipid metabolism processes. The E100 group, in addition to continuing to affect immune and stress-related pathways, also significantly involved cell proliferation, DNA replication and repair, protein homeostasis, and antioxidant defense processes. The E200 group primarily affected lipid synthesis and transformation, membrane lipid signaling, amino acid metabolism, and immune signaling regulation. Overall, the E50 group mainly showed changes in immune recognition and early stress adaptation pathways, the E100 group further demonstrated widespread activation of cell cycle, replication and repair, and antioxidant defense, while the E200 group primarily reflected changes in lipid metabolism and membrane signaling remodeling.

[0083] From a pathway composition perspective, the E50 group initially exhibited immune recognition-related pathways such as the phagosome, Toll-like receptor signaling pathway, C-type lectin receptor signaling pathway, and intestinal immune network for IgA production, indicating that 50 mg / kg ergothioneine treatment first activated the liver's innate immune surveillance and phagocytic defense responses. Simultaneously, the E50 group also enriched the FoxO signaling pathway, autophagy-animal pathway, apoptosis, and pentosephosphate pathway, suggesting that the body maintains cellular homeostasis and redox balance during the early response process through cell quality control, programmed cell death regulation, and enhanced NADPH supply.

[0084] Compared to the E50 group, the E100 group was further enriched in cell cycle, DNA replication, Fanconi anemia pathway, p53 signaling pathway, protein processing in endoplasmic reticulum, glutathione metabolism, and mismatch repair. This indicates that treatment with 100 mg / kg ergothioneine has progressed beyond early immune recognition and stress regulation to deeper levels of cell repair, protein homeostasis maintenance, and antioxidant defense responses. Therefore, the multi-level enrichment characteristic of "immune recognition-replication repair-protein homeostasis-antioxidant defense" exhibited by the E100 group suggests that 100 mg / kg ergothioneine treatment likely operates within the most sensitive region of liver response, activating both innate immunity and significantly mobilizing the cell repair and defense systems. This is consistent with the characteristic of this group typically exhibiting a large number of differentially expressed genes and a broad range of functional remodeling.

[0085] The E200 group primarily shifted towards steroid biosynthesis, arachidonic acid metabolism, sphingolipid metabolism, retinol metabolism, the phosphatidylinositol signaling system, the apelin signaling pathway, and cysteine ​​and methionine metabolism. This indicates that the effects of 200 mg / kg ergothioneine on the liver have shifted from the initial immune and replication repair responses to lipid synthesis, membrane lipid signaling, and remodeling of bioactive small molecule metabolism. Simultaneously, the Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, and apoptosis clearance were preserved in the E200 group, suggesting that the liver did not detach from immune surveillance under high-dose conditions, but rather, within the context of a sustained immune response, further enhanced the remodeling of membrane lipid composition, lipid mediators, and substrate utilization. Overall, 50 mg / kg ergothioneine is mainly characterized by early adaptation mediated by immune recognition and FoxO-autophagy, 100 mg / kg ergothioneine is characterized by enhanced replication repair, endoplasmic reticulum homeostasis and antioxidant defense, and 200 mg / kg ergothioneine is more prominent in lipid metabolism and membrane signal reprogramming.

[0086] Based on the above results, this embodiment systematically investigated the effects of ergothioneine on liver metabolism and molecular responses in juvenile large yellow croaker using liver metabolomics and transcriptomics analysis. The results showed that ergothioneine can induce significant metabolic remodeling in the liver. Meanwhile, transcriptomics analysis revealed that differentially expressed genes were mainly concentrated in functional categories such as metabolic processes, cellular processes, biological regulation, signal transduction, and immune responses. KEGG enrichment further indicated that ergothioneine participates in liver antioxidant defense, lipid homeostasis maintenance, and stress regulation by regulating pathways such as FoxO, autophagy, Toll-like receptors, glutathione metabolism, steroid biosynthesis, and arachidonic acid metabolism. Overall, ergothioneine can improve the physiological state of the organism through liver metabolic reprogramming and transcriptional regulation, providing molecular mechanistic support for its multiple nutritional functions.

[0087] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of ergothionein in the preparation of products that improve the growth performance of fish.

2. The application as described in claim 1, characterized in that, The product is used to improve fish weight, weight gain rate, specific growth rate, or feed efficiency.

3. Application of ergothionein in the preparation of products that enhance the immune and / or antioxidant capacity of fish.

4. The application as described in claim 3, characterized in that, The product is used to increase the levels of complement C3 and complement C4 in fish, and / or increase the activity of lysozyme, alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase.

5. The application as described in claim 3, characterized in that, The product is used to increase the activity of superoxide dismutase, catalase, and glutathione peroxidase in fish, and to reduce the content of malondialdehyde.

6. Application of ergothionein in the preparation of products that improve intestinal health in fish.

7. The application as described in claim 6, characterized in that, The product is used to enhance the activity of intestinal lipase and trypsin in fish. The product is used to improve the morphology of intestinal villi in fish.

8. The application as described in claim 6, characterized in that, The product is used to improve the structure and composition of the gut microbiota in fish. The product is used to improve intestinal metabolites in fish.

9. The application as described in any one of claims 1-8, characterized in that, The fish mentioned include juvenile large yellow croaker.

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