A total of 190 Chinese herbal medicines (listed in Supplementary Table S1) were all purchased from Shanxi Junkangda Biotechnology Co., Ltd. (China). The herb No. 100, which was selected for subsequent fermentation, is derived from a defined plant species within the Simaroubaceae family. It is known to be rich in characteristic Quassinoids and also contains alkaloids and flavonoids. These constituents collectively underpin its broad-spectrum antibacterial, anti-inflammatory, and immunomodulatory pharmacological activities. N. seriolae strain NS-23 was isolated from diseased sea bass (Lateolabrax japonicus) at the Hezhou North Estuary Fisheries Research Institute (Zhuhai, China) and preserved in our laboratory, which was determined as a natural virulent strain also being sensitive to largemouth bass (M. salmoides). Bacillus licheniformis strain PP789724 (MS-1), B. amyloliquefaciens strain PP789725 (MS-2), and B. velezensis strain PP789726 (MS-3) were isolated from the intestinal tracts of largemouth bass (M. salmoides). B. velezensis strain PP789722 (CA-1) and B. subtilis strain PP789723 (CA-2) were obtained from the intestines of northern snakehead (Channa argus). All strains were cryopreserved at −80 °C in our laboratory. Healthy juvenile largemouth bass (initial body weight: 15.60 ± 0.38 g) were procured from Guangdong Yangyang Fisheries Co., Ltd. (Foshan, China) and acclimatized in aerated freshwater tanks for 7 days prior to experimentation. A commercial basal diet (Qunfeng brand, crude protein: 50%, crude lipid: 10%) for largemouth bass was supplied by Guangdong Wanghai Feed Industry Co., Ltd. (Jiangmen, China). The diet was fed twice daily at 9:00 a.m. and 4:00 p.m., with a daily feeding rate of 5% of the body weight. The experimental procedures of largemouth bass were carried out in accordance with the Ethics Committee of Guangdong Ocean University (protocol code [2019] 1 and date of approval 10 May 2019).

Fish (n = 30/group) were intraperitoneally injected with 100 μl of N. seriolae strain NS-23 suspensions (1 × 10⁴ ~ 1 × 10⁸ CFU/ml in sterile PBS). Mortality was recorded daily for 14 days, and the LD₅₀ value was calculated using Probit analysis (SPSS 23.0). Following the 49-day feeding trial, fish (n = 60/group) were injected with 2.59 × 10⁵ CFU/ml N. seriolae strain NS-23 (LD₅₀ dose). Survival rates and clinical symptoms were monitored for 14 days post-infection.

Survival Rates (%) = (Total number − Number of Deaths)/Total number × 100. Tissues (intestine, liver, spleen, head kidney) from surviving fish (n = 3/group) were separated and fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned (5 μm thickness), and stained with hematoxylin and eosin (H&E) by Servicebio Co., Ltd. (Wuhan, China). Histopathological changes were examined under light microscopy.

Inhibition zone diameters are presented as means ± standard error of the mean (SEM) of triplicate vaccination. Growth performance, enzyme activities, and gene expression data were analyzed by one-way ANOVA followed by Duncan’s multiple range test (SPSS 23.0). Survival curves were compared using the Kaplan–Meier method with log-rank tests. Significance was set at p < 0.05.

The antibacterial effects of different single Chinese herbs against N. seriolae strain NS-23 exhibited significant variations (Table 2). The herbs of Cullen corylifolium, Houpoea officinalis and especially No. 100 displayed obvious bactericidal effect with inhibition zone diameters at 21.81, 29.27, 38.24 mm respectively, and they were classified as extremely sensitive since inhibition zone diameter ≥20 mm. In contrast, other tested herbs showed no obvious antibacterial activity to N. seriolae. Based on their antibacterial efficacy and pharmacological properties, the herb No. 100 was selected as the raw material for subsequent fermentation experiments. The antibacterial activity of five Bacillus strains against N. seriolae strain NS-23 was evaluated (Table 3), and the results showed that the MS-2 (B. amyloliquefaciens, PP789725) isolated from the intestine of largemouth bass (M. salmoides), exhibited the largest inhibition zone diameter at 45.56 mm, significantly outperforming other Bacillus strains MS-1, MS-3, CA-1, and CA-2 (p < 0.05). Consequently, strain MS-2 was selected as the fermentation strain for subsequent experiments.

As illustrated in Figure 1, the viable cell counts of MS-2 (B. amyloliquefaciens strain PP789725) initially increased and then decreased with increasing herb No. 100 liquid concentration (1–8%). The optimal concentration was determined as 4%, yielding a peak viable cell count of 1.81 × 10⁷ CFU/ml, whereas 8% resulted in a significant decline (0.20 × 10⁷ CFU/ml, p < 0.05). Similarly, the viable cell count peaked at 6.20 × 10⁸ CFU/ml with a 4% inoculum of MS-2, declining slightly to 5.90 × 10⁸ CFU/ml at 8% (p < 0.05). Fermentation time and pH optimization revealed peak viable counts at 18 h (12.10 × 10⁸ CFU/ml) and pH 7.5 (14.07 × 10⁸ CFU/ml), respectively (p < 0.05). Thus, the optimal fermentation parameters were determined as follows: 4% herb liquid, 4% MS-2 inoculum, 18 h fermentation time, and pH 7.5.

After 7 weeks of feeding, the fish in group D (PFH) exhibited significantly higher weight gain rate (WGR: 310.51 ± 37.44%) compared to the group A (277.13 ± 29.08%, p < 0.05). No significant differences were observed in condition factor (CF), viscerosomatic index (VSI), or hepatosomatic index (HSI) among groups (p > 0.05). These results indicate that PFH significantly enhances weight gain rate without adverse effects on metabolic efficiency (Table 4).

Serum superoxide dismutase (SOD) and catalase (CAT) activities in the fish of groups B, C and D were significantly higher than those in the fish of group A (p < 0.05, Figure 2). CAT activity followed the order: Group D > Group B > Group C (p < 0.05). Malondialdehyde (MDA) levels decreased in all treatment groups, but differences were not significant (p > 0.05). Non-specific immune enzyme activities (Figure 2) revealed that group D significantly enhanced acid phosphatase (ACP), lysozyme (LZM), and nitric oxide (NO) levels compared to group A (p < 0.05). Alkaline phosphatase (AKP) activity was elevated in all treatment groups (p < 0.05), though intergroup differences were insignificant.

The upregulated expression of several genes related to inflammatory, antioxidant, and humoral immune genes including TNF-α, IL-8, IL-10, SOD, IgM, NF-κB, IFN-γ, CAT, and IL-1β genes across tissues (liver, spleen, head kidney, and intestine) were observed in the fish of group D (Figure 3). The fish of group B were showed significantly elevation in gene expressions of TNF-α, IL-8, IL-10, and CAT in blood, spleen, and head kidney (p < 0.05), but lower IgM and NF-κB expression in liver and spleen compared to the fish of group D. The fish of group C exhibited limitation in immune enhancement, primarily in the intestine and head kidney. In contrast, the fish of group D were demonstrated systemic immune enhancement, significantly elevating IL-8, IL-10, SOD, IgM, NF-κB, and IFN-γ expression in blood, liver, spleen, head kidney, and intestine (p < 0.05).

16S rRNA sequencing revealed distinct gut microbiota profiles among different groups (Figure 4). Unique operational taxonomic units (OTUs) were identified in each group (A: 2589; B: 3474; C: 4866; D: 4938 (p < 0.05)). The relative abundance of Proteobacteria was highest in groups A and B, whereas Firmicutes represented the predominant phylum in both groups C and D. Critically, genus-level analysis demonstrated that PFH supplementation (Group D) significantly increased beneficial genera (e.g., Lactobacillus, Bacillus) while reducing potential pathogens (e.g., Aeromonas). This refined analysis provides a more precise understanding of the microbial restructuring induced by PFH, suggesting that the phylum-level shifts are driven by changes in specific, functionally relevant bacteria.

Gradient-dose challenge tests revealed dose-dependent mortality (Figure 5). The LD₅₀ was calculated as 2.59 × 10⁵ CFU/fish (Table 5). The fermented group exhibited a significantly higher immunoprotection rate of 77% (p < 0.05, Table 6) compared to the control group (52%), with the herb and probiotic groups achieving intermediate values of 65 and 68%. These findings collectively demonstrate the efficacy of all three interventions in enhancing host resistance against pathogens, while highlighting the superior prophylactic potential of PFH through synergistic interactions among its bioactive components.

Histopathological analysis (Figure 6) demonstrated significant N. seriolae multi-organ damage in group A, characterized by severe intestinal mucosal inflammation, hepatic granulomatous lesions, and spleno-renal necrosis, which were substantially mitigated in treatment groups. Group D exhibited superior protective efficacy, evidenced by higher villus height and intestinal wall thickness (p < 0.05, Table 7), and complete prevention of granuloma formation compared to group A. While groups B and C showed intermediate improvements in necrotic area reduction (35–40% vs. group A), only group D maintained near-intact histoarchitecture across all examined organs, suggesting synergistic enhancement of mucosal immunity and systemic anti-inflammatory capacity through PFH intervention strategies.

The superior antibacterial efficacy of herb No. 100 against N. seriolae-demonstrating the largest inhibition zone diameter among all evaluated herbs-dictated its selection as the keystone component in the anti-nocardial formulation. This decision is pharmacologically anchored in the root’s rich repertoire of >30 quinolizidine alkaloids (e.g., matrine, oxymatrine) and >120 flavonoids (e.g., kurarinone, sophoraflavanone G) (20, 21), which concurrently disrupt bacterial membranes via lipid peroxidation, inhibit protein synthesis through 30S ribosomal subunit binding, and suppress virulence gene expression (22). B. amyloliquefaciens has been shown to significantly enhance intestinal health in piglets by activating the Nrf2/Keap1 signaling pathway (23). Its fermentation products exhibit antibacterial, antioxidant, and anti-inflammatory activities, effectively improving intestinal barrier function and reducing oxidative stress levels (14). In aquaculture, B. amyloliquefaciens has demonstrated dual benefits in hybrid sturgeon (16) and Nile tilapia (15). It not only enhances resistance to Aeromonas hydrophila infection by modulating the expression of immune-related genes but also significantly improves growth performance and antioxidant capacity under high-density culture conditions, while reducing stress hormone cortisol levels. As an efficient fermentative strain, B. amyloliquefaciens exhibits remarkable biotransformation capabilities. For instance, its fermentation of soybean meal can reconstruct the metabolite lineage to improve fish quality (17). During the fermentation of traditional Chinese medicinal materials, the contents of astragalus polysaccharide (24) and Isatis indigotica (R, S)-geichan (25) were increased by 39.59 and 7.07% respectively, confirming its significant effect in enhancing the biological activity of the products. MS-2 (B. amyloliquefaciens strain PP789725) used in this study possesses unique advantages, including extreme environmental adaptability (pH 2.0–12.0, bile salt tolerance of 5.0 g/L), excellent surface properties (hydrophobicity 50–93.3%, autoagglutination rate 43.6–69.2%), and abundant metabolites (26). Its inhibition zone against N. seriolae strain NS-23 reached at 45.56 mm (p < 0.05). Combined with its antibiotic sensitivity profile, MS-2 is an ideal candidate for fermentation with both biosafety and functionality, providing a valuable resource for the development of novel feed additives and functional fermented products.

In this study, the optimal conditions for the fermentation of herb No. 100 solution by MS-2 (B. amyloliquefaciens strain PP789725) were established through single-factor optimization. The key fermentation parameters were identified as follows: when the concentration of herb No. 100 solution reached 4%, the viable bacterial count peaked. Excessive substrate concentration led to a 9-fold reduction in viable bacterial count, likely due to inhibitory components such as phenols. An 18-h fermentation cycle effectively avoided the risk of nutrient depletion, aligning with the growth peak of MS-2 (26). A pH at 7.5 was optimal for maintaining the highest enzyme activity (27). Mechanistic analysis revealed that substrate concentration regulated bacterial population density through the synergistic effects of nutritional and inhibitory components, forming a complementary mechanism with pH sensitivity (yield increased by 18.7% in acidic environments) (28). This is similar to the observation that flavonoid content first increased and then decreased with fermentation time in the fermentation process of Sophora chinensis (24). The inoculum size optimization was consistent with the solid-state fermentation process of S. japonica (response surface method increased product yield by 34.5%), confirming the commonality of microbial fermentation population effects (29). Compared with traditional Chinese medicine fermentation processes (e.g., 48 h for astragalus polysaccharide fermentation), the fermentation time was reduced by 62.5%, significantly improving fermentation efficiency. This optimized process provides a solid technical basis for further studies on the efficacy of fermented herb No. 100 against fish nocardiosis. It is important to note that the single-factor optimization method employed here, while effective for establishing initial fermentation parameters, does not account for potential interactions between factors such as substrate concentration, inoculum size, pH, and fermentation time. Future studies utilizing response surface methodology (RSM) would be valuable to model these interactions and further refine the process for maximum metabolite yield.

In this study, the weight gain rate of group D was significantly higher than that of group A (p < 0.05), but it did not exceed the values observed in groups B and C. Additionally, no significant differences were observed in metabolic indices such as condition factor and hepatosomatic ratio, suggesting that the growth-promoting effect of group D was attributed to intestinal microecology and immune regulation rather than enhanced metabolic efficiency. This finding is consistent with previous studies on the fermentation of licorice root, astragalus, and Siberian ginseng herb compound using Clostridium butyricum (no significant change in weight gain) (30) and the co-fermentation of Pleurotus eryngii root waste and soybean meal (slight increase in feed utilization) (31). These results indicate that the growth-promoting effect of a single fermentation substrate is limited. In contrast, systems containing polysaccharides, sulfides, and other active components, such as fermented astragalus (32), fermented garlic powder (19), and mulberry leaf-turmeric (33), have demonstrated significant growth promotion by regulating digestive enzyme activity. The mild effect of fermented herb No. 100 in this study may be due to the metabolic balance formed by the residues of quassinoid-like inhibitory components. The core active substances favored immune activation and antioxidant properties (with significant increases in SOD/CAT activities and serum IgM levels), highlighting its potential for developing disease-resistant feeds.

This study revealed that PFH exhibited unique advantages in antioxidant and immune regulation, suggesting synergistic effects among its bioactive components. The significant increase in CAT activity indicated that the fermentation process enhanced the ability to alleviate oxidative stress damage, potentially by improving the bioavailability or efficacy of active ingredients. This effect appeared to surpass the more limited impact observed with herbs or probiotics alone. In terms of non-specific immunity, the simultaneous enhancement of ACP and LZM activities and NO levels suggests a dual role in both direct bactericidal capacity and potential immunomodulation (34). This effect is closely related to the systematic regulatory characteristics of fermentation products. After fermentation of herb No. 100, partial degradation of complex components may have increased the bioavailability of active compounds, which could subsequently contribute to the observed improvements in intestinal barrier function and microbiota structure (20). This multi-target mode of action echoes similar fermentation systems: astragalus fermentation product (ALB) (32) enhances disease resistance by upregulating resistance genes (Mx, IRF-3, TNF-α, IL-1β, and IL-10) and bacterial diversity. Lentinus edodes fermentation (LEF) (35) improved intestinal amylase activity and CAT levels, decreased liver MDA levels, and inhibited inflammatory factors (TNF-α) to improve metabolic health, and the fermented tea residue (TF) (36) enhanced systemic antioxidant capacity through T-AOC. Although there are significant differences in the action pathways of different fermentation substrates, their commonality lies in the multi-dimensional gain of physiological functions through the reconfiguration of metabolites. While the specific pathways (e.g., MAPK/NF-κB) were not assayed here, the coordinated upregulation of immune and antioxidant genes in our study is consistent with such modulations reported in the literature. The outstanding performance of PFH on the antioxidant-immune axis in this study provides a novel candidate for developing strategies to prevent and control aquatic diseases targeting oxidative stress and immunosuppression.

In this study, PFH exhibited significant upregulation of multiple immune-related genes. This observation suggests that fermentation products may exert a synergistic effect in modulating the host immune response through multiple pathways. The immunomodulatory mechanism of fermentation products is likely closely associated with their complex bioactive components. The fermentation process may activate microbial metabolic pathways, leading to the production of novel bioactive substances capable of regulating the host immune system (37). The upregulation of pro-inflammatory genes in fermentation products does not necessarily induce excessive inflammatory responses but may represent a moderate immune activation that enhances the host’s immune capacity. For instance, pro-inflammatory cytokines such as TNF-α and IL-8 play crucial roles in the early immune response by mobilizing immune cells to the site of infection (36). This differential effect may be attributed to the specific composition of the fermentation product, experimental conditions, and the immune status of the subjects studied. For example, fermented tea residue primarily exerts anti-inflammatory effects by inhibiting inflammatory signaling pathways, such as the NF-κB pathway (36). Zhou et al. reported that fermented Chinese herbal medicine significantly increased the relative mRNA expression levels of intestinal anti-inflammatory factors TGF-β and IL-10, while decreasing the expressions of pro-inflammatory factors IL-8, IL-15, and TNF-α, thereby playing an immune regulatory role. In contrast, the fermentation products in this study may promote the synergistic upregulation of both pro-inflammatory genes (TNF-α, IL-8) and anti-inflammatory genes (IL-10, IFN-γ) by activating multiple signaling pathways in immune cells, such as the TLR4-mediated NF-κB pathway (34). Additionally, PFH may enable more comprehensive immune regulation by enhancing the antioxidant defense system (via NRF2-mediated SOD/CAT expression) while promoting the acquired immune response (IgM secretion). The source of PFH and fermentation conditions can also influence their immunomodulatory effects. Different fermentation strains and fermentation times may lead to distinct metabolite profiles, which in turn may affect the expression of immune genes.

At the phylum level, the relative abundances of Firmicutes and Bacteroidetes were significantly increased in group D, while the proportion of Proteobacteria was relatively low. Firmicutes are closely related to the host’s energy metabolism and nutrient absorption, and their increase can improve the host’s growth performance and health status (18). Bacteroidetes play a vital role in carbohydrate metabolism and the production of short-chain fatty acids (SCFAs), which possess anti-inflammatory and immunomodulatory functions (32). By introducing a wider variety of microorganisms, PFH optimize the intestinal microecology, promoting the proliferation of beneficial bacteria and inhibiting the growth of harmful bacteria. This optimization of the microbiota structure was potentially associated with a significant increase in enzyme activity and immune gene expression in group D. The significant increase in weight gain rate in group D may be related to the increase in Firmicutes and Bacteroidetes, which are microorganisms that secrete a variety of digestive enzymes and promote the breakdown and absorption of nutrients. Meanwhile, the significant upregulation of immune-related genes (such as IL-10, IFN-γ, IgM, etc.) in group D may be closely related to the optimization of the intestinal microbiota. These results suggest that PFH significantly enhance the growth and immune function of largemouth bass by modulating the intestinal microbiota. This approach of improving fish health through microbiota regulation provides a novel direction for the development of natural and efficient aquatic functional feeds.

In the present study, PFH had significant effects on the antioxidant capacity and immunity of largemouth bass, thereby enhancing resistance to pathogens (38). This indicates that PFH was able to significantly delay the onset and progression of the disease (39).

The results showed that the protective effect of PFH on the liver, intestinal tract, spleen, and head kidney was significant. N. seriolae is a pathogen that can cause granulomatous inflammation and necrotic injury in multiple organs of the host. This pathological feature is closely related to the intracellular survival strategy of the pathogen and the imbalance of the host immune response (40). The widely distributed granulomas in the liver of the control group are typical markers of nocardiosis, consistent with previous study in the liver of rainbow trout (44). The pathogen activates the NLRP3 inflammasome of macrophages through the secretion of ESAT-6-like proteins, resulting in excessive release of IL-1β, which drives the formation of granulomas (42). The depletion of intestinal mucosal goblet cells and intestinal villi atrophy indicates that pathogens can directly destroy the mucus barrier and promote bacterial translocation, consistent with the vicious cycle mechanism of “mucus depletion–flora imbalance” in Gram-positive bacterial infection models (43). Hematopoietic tissue necrosis was present in the spleen and head kidney, indicating chronic hemolysis and oxidative stress injury (41). The changes in goblet cell number, villus height (VH), crypt depth (CD), intestinal wall thickness (ITW), and muscularis thickness (MT) in intestinal sections reflect the repair effectiveness of different intervention strategies on the mucosal barrier (40). The number of goblet cells in groups C and D were significantly higher than that in group A, consistent with reports that bacillus products promote the repair of the intestinal mucus barrier by promoting the M2 polarization of intestinal macrophages and restoring the number and function of intestinal epithelial goblet cells via the WNT-ERK1/2 axis (45). The significant increase in VH and ITW in group D indicates that short-chain fatty acids in fermentation products promote the proliferation of crypt stem cells by activating the Wnt/β-catenin pathway (46). Crypt depth is an important index for evaluating intestinal structural changes (47). The significant thickening of MT in group C may enhance intestinal peristaltic function by regulating intestinal glia-smooth muscle signal transmission, thereby accelerating pathogen excretion (40). Granulomas disappeared completely group D, while focal necrosis was still present in group B, highlighting the multi-target advantage of fermentation products and indicating that unfermented plant components are difficult to effectively inhibit the activation of the NLRP3 inflammasome (47). Studies have shown that ellagic acid in fermentation products alleviates oxidative stress by blocking the assembly of inflammasomes and upregulating SOD activity, which may be the key to its high efficiency (48). In summary, considering the immune protection rate, growth performance data, and histological analysis, PFH demonstrates strong safety and efficacy in the prevention and control of fish nocardiosis.