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Bacillus subtilis HGCC-1 improves growth performance and liver health via regulating gut microbiota in golden pompano

Animal Microbiome volume 7, Article number: 7 (2025) Cite this article

Abstract

Probiotics as green inputs have been reported to regulate metabolism and immunity of fish. However, the mechanisms by which probiotics improve growth and health of fish are unclear. Therefore, the aim of this study was to investigate the effect of Bacillus subtilis HGCC-1, an indigenous probiotic isolated from fish, on growth performance, host lipid metabolism, liver inflammation and gut microbiota of golden pompano. 160,000 golden pompanos with the initial body weight of 93.6 ± 5.0 g was randomly assigned to two dietary groups: Control and HGCC-1 (control diet supplemented with 0.3 g/kg Bacillus subtilis HGCC-1 fermentation product), and after three weeks of feeding, 26 golden pompanos were randomly collected from each group for gut microbiome and host phenotype analysis. Dietary supplementation with Bacillus subtilis HGCC-1 significantly promoted growth performance (P < 0.05) and enhanced feed utilization. Besides, HGCC-1 improved liver health and alleviated hepatic steatosis and inflammation. Furthermore, Bacillus subtilis HGCC-1 enhanced intestinal lipid absorption, promoted hepatic utilization of dietary fat by improving hepatic lipid uptake/transport and fatty acid β-oxidation to provide energy, and reduced hepatic TG level (P < 0.05), which may be the potential mechanism of Bacillus subtilis HGCC-1-mediated growth promotion. Finally, Bacillus subtilis HGCC-1 significantly altered the structure and function of gut microbiota (P < 0.05), leading to enrichment of beneficial taxa such as Bacillus (P < 0.0001) and increased of the ratio of “Functional Group 2/Functional Group 1” (P = 0.00092). Interestingly, the ratio of “Functional Group 2/Functional Group 1” was linked to the growth traits (Spearman, P < 0.05), while the intestinal abundance of Bacillus was correlated with serum TG in fish (Spearman, R = 0.47, P = 0.00091), suggesting a role of the intestinal microbiota in HGCC-1 mediated effect on growth and lipid metabolism. In summary, Bacillus subtilis HGCC-1 promotes growth performance, alleviate hepatic steatosis and enhances liver health via regulating gut microbiota in golden pompano, which ultimately showed as beneficial effect of fish growth and health.

Background

Sustainable aquaculture is a major goal in the blue transformation in action and plays an important role in global food security [1]. Aquaculture intensification and high-density have become key measures to improve farm production [2, 3]. Meanwhile, aquaculture feeds tend to contain higher energy (e.g., high-sugar diets) [4] and high plant protein replacement (e.g., non-fishmeal diets) [3, 5] to improve the economic efficiency of farming and achieve sustainable intensification. However, gut microbiota disorders [6], impaired nutrient metabolism [7], and liver diseases [8, 9] are important limiting factors for sustainable development and expanded production.

Antibiotics are a common way of applying aquaculture disease prevention and treatment [10, 11], and are even used to promote the growth of farmed animals [12]. However, the lack of targeting nutritional metabolic diseases and the inevitable leading gut microbiome imbalance [13], as well as the fact that antibiotics lead to microbial resistance threatening the environment and human health [14], have led to the search for alternatives to address the banning of antibiotics. Probiotics, as green inputs, are effective alternatives to antibiotics and have the potential to regulate fish nutrient metabolism and growth, immune function and health, and the gut microbiome [15, 16]. For example, promoting growth in Atlantic salmon [17] and affecting lipid metabolism in zebrafish [18], regulating the expression of immunity genes in common carp [19] and rainbow trout [20], and enhancing gut bacterial adhesion [21], altering the gut bacterial community composition of Chinese perch [22].

Bacillus subtilis, a Gram-positive bacterium, has been widely reported as a classical probiotic in aquaculture animals [23]. Bacillus subtilis was reported to resist pathogens or enhance host immunity [24], such as viral diseases [25, 26], bacterial diseases [27], and immunomodulation [28]. Besides, Bacillus subtilis (e.g., B. cereus and B. subtilis) has been reported to reshape the gut microbiota of Pengze crucian carp (Carassius auratus var. Pengze) on a high plant protein diet [29]. However, there is lack of studies on nutritional metabolic diseases and liver inflammation. In addition, there is a risk that traditional probiotics, such as Lactobacillus from livestock animal sources, may lead to intestinal damage or microbiota dysbiosis [30]. Therefore, the development of indigenous probiotics, such as Bacillus subtilis, is of great value for novel probiotics used in aquaculture. Bacillus subtilis HGCC-1 used in this study is an indigenous bacterium isolated from the fish gut, which was initially studied to alleviate TG accumulation in liver and modulate liver immunity in zebrafish [31]. However, the effects of Bacillus subtilis HGCC-1 on growth performance and gut microbiota of economic fish are not clear, and whether the gut microbiota is involved in the mechanism of regulating host liver health and growth performance has not been reported.

Golden Pompano (Trachinotus ovatus) is widely distributed in China and some other countries [32]. On the one hand, golden pompano is protein-rich and has delicate meat, becoming more and more favored by Chinese consumers [33]. In addition, owing to rapid growth and salinity adaptability, golden pompano has become one of the most important seawater fishes in China. In this study, we investigated the effects of dietary supplementation with Bacillus subtilis HGCC-1 on growth performance, gut microbiota, host lipid absorption and metabolism, and liver health of golden pompano, aiming to explore the potential relationship for probiotics to coordinate host health and growth through crosstalk between gut microbes and host lipid metabolism.

Materials and methods

During the whole experiment period, all efforts were made to reduce the suffering of animals. All experiments and animal care procedures were approved by the Animal Care Committee of Feed Institute, Chinese Academy of Agricultural Sciences (Guarantee No.2022- AF-FRI-CAAS-001).

Bacterium culture and experimental diets

Bacillus subtilis HGCC-1 was isolated from the intestine of tilapia, preserved in the Institute of Feed Research, Chinese Academy of Agricultural Sciences. HGCC-1 was fermented by solid fermentation as previously described [31]. Golden pompano feed production was carried out in Guangdong Yuehai Feed Group Co., Ltd. and the specific operation process was standardized according to the No. 3 feed (Cat#0202020202060001). During the production of feeds, HGCC-1 fermentation product was supplemented at 0.3 g/kg to the final concentration of 108 CFU/g. The selection of the optimum concentration was based on previous studies [31]. The feeds were produced with reference to existing studies [34]. The conventional nutrient content of the two isonitrogenous and isoenergetic feeds was determined to be 51% crude protein and 12% crude fat (Table 1), and they were divided into the Control group and Bacillus subtilis HGCC-1 (referred to as HGCC-1). The moisture content of the diets was determined by drying them at 105 °C according to AOAC method 2001.12. Crude protein content was determined using a fully automated Kjeldahl nitrogen determination system according to AOAC method 2001.11. Crude fat content of the feed was determined using petroleum ether Soxhlet extraction method according to AOAC method 920.39. Ash content was determined by roasting at 550 °C for 10 h in a muffle furnace based on AOAC method 942.05. Subsequently, the amino acid content of the feeds was measured in Table 2 using an automated amino acid analyzer (HITACHI L-8900, Tokyo, Japan). The sample weighing about 0.05 g was placed in a sealed glass tube and hydrolyzed by adding 6 mL of HCl (6 mol/L) at 110 °C for 24 h. The hydrolysate was filtered and diluted to 50 ml with distilled water. HCl was removed from the filtrate in a vacuum dryer for 24 h and then evaporated to dryness at 60 °C. 2 mL of distilled water was added and evaporated for another 24 h. The precipitate was then dehydrated with 8 ml of 0.1 mol/L HCl and filtered through a 0.22 mm Millipore membrane, leaving 1 ml of supernatant for analysis of amino acids.

Table 1 Composition and nutrient levels of the diets (DM basis, g/kg)
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Table 2 Analysis of amino acid profile of diets for golden pompano
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Animals feeding and sample collection

The golden pompano was purchased from Yangxi County Yihui Deep-sea Aquaculture Technology Co., Ltd. The experiment was carried out in deep-sea aquaculture nets (30 m in diameter and 4 m in depth) near Qingzhou Island (21°28’ N,111°28’ E). Subsequently, golden pompano was randomly assigned to four net tanks with 40,000 fish /net tank for 93.6 ± 5.0 g/fish. The fish were fed by satiation feeding at 7:00–8:00 a.m. and 5:00–6:00 p.m. At the end of the 3-week feeding period, 13 golden pompanos were randomly salvaged from the net at 4–8 h postprandial, for a total of 26 golden pompano per group. Blood and gut contents as well as intestinal and liver tissues were subsequently collected from single fish. All samples were rapidly frozen with liquid nitrogen and then transferred to -80 °C for storage until analysis.

Growth performance

Prior to the above sample collection, growth trait related indicators were collected, including: Initial mean body weight (IBW, g/fish), Final body weight (FBW, g/fish), Final body length (FBL, cm/fish), Carcass weight (CW, g/fish) and Carcass ratio (CR, %), Feed mean intake (FI, g/d). Calculation of relevant parameters: Weight gain rate (WGR, %), Specific growth rate (SGR, %/d), Condition factor (CF, g/cm3), Feed conversion ratio (FCR), refer to previous studies [34].

Detection of TGs, ALT and AST content

The collected blood of golden pompano was centrifuged at 1500 rpm for 10 min, and the supernatant was taken with a pipette and the collected serum was placed at -80 °C for the determination. The TGs, ALT and AST assay kits were purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd. This determination method referred to the manufacturer’s instructions.

Real-time quantitative PCR (RT-qPCR)

The RNA extraction from intestinal and liver tissue was based on the Trizol. Concentrations were determined with a NanoDrop 2000; agarose gel electrophoresis was also performed on all extracted samples, and only qualified samples were used for the next study. RNA was reverse into cDNA by using FastKing gDNA Dispelling RT SuperMix (Tiangen, China) kit. Subsequently, qPCR analysis Real-time fluorescence quantitative PCR reaction was performed on 480 system (LichtCycler® 480 Real-Time PCR System, Roche) using AceQ qPCR SYBR Green Master Mix (Vazyme, China). Data were analyzed by 2−ΔΔCT method using β-actin as reference and the amplification primers are shown in Table 3.

Table 3 Primer sequences for qRT-PCR analysis
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DNA extraction, PCR amplification and Illumina sequencing

Total DNA was extracted from intestinal contents using the E.Z.N.A.® soil DNA kit (Omega Bio-tek, Norcross, GA, U.S.). Amplification of the full-length 16 S rRNA used primers 27 F (5′-AGAGTTTGATCMTGGCTCAG − 3′) and 1492R (5′- ACCTTGTTACGACTT − 3′). PCR amplification reference previous study [34]. The amplification products were purified with magnetic beads and the purified products were detected and quantified by Qubit 4.0 (Thermo Fisher Scientific, USA). Library construction, including DNA damage repair, end repair, and junction joining, was performed using SMRTbell. Finally, sequencing was performed using the Pacbio Sequel IIe system and HiFi reads were produced from the sequenced sub-threads via the CCS mode of SMRT-Link v11.0.

Bioinformatics analysis of 16 S rRNA amplicon sequencing

The optimized sequences were noise cancelled by using DADA2 in QIIME2. Draw levelling of all samples according to the lowest sample sequence. Species taxonomic annotation of amplicon sequence variants (ASVs) was performed by the RDP classifier (version 2.11) [35] compared to the NCBI-nt database (2022-10). Alpha diversity indices (ACE, Chao 1, Simpson, and Shannon indices) were calculated using mothur software (version 1.30.1) [36], and the Wilcoxon rank sum test was used to analyze differences between groups. PCoA (Principal Coordinate Analysis) analysis based on bray-Curti’s distance was used to test the similarity of microbial community structure among samples. Spearman correlation analysis and heatmap was performed in R (version 3.3.1) using the ggpubr (version 0.6.0) and corrplot package (version 0.92). The ratio of “Functional Group 2/Functional Group 1” was calculated to our previously published study [37].

Statistical analysis

The statistical analysis was performed by GraphPad Software (Version 9.0). The data were analyzed by Student’s t-test or Mann-Whitney U-test. All data are presented as the mean ± SEM. Differences were defined as significant at P < 0.05.

Results

Dietary supplementation of Bacillus subtilis HGCC-1 promoted the growth performance in golden pompano

With no obvious difference in initial body weight (Table 4), dietary supplementation of HGCC-1 significantly increased the final body weight (P < 0.01), weight gain rate (P < 0.01), and specific growth rate (P < 0.05) of golden pompano (Table 4) compared to control group. In addition, body length was significantly higher (P < 0.01) in the HGCC-1 group than control, but there was no apparent difference in Condition factor (Table 4). Compared to control group, carcass weight was significantly increased (P < 0.01) in HGCC-1 group (Table 4), but there was no significant difference on carcass ratio (Table 4). For feed utilization, HGCC-1 group showed lower Feed conversion ratio (Table 4).

Table 4 Dietary supplementation of Bacillus subtilis HGCC-1 can promote the growth performance in golden pompano
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Bacillus subtilis HGCC-1 improved liver health of golden pompano

Compared with the control group, the HGCC-1 group significantly decreased serum ALT and AST levels (P < 0.0001; P < 0.0001) (Table 5). In addition, the study found that the HGCC-1 group significantly down-regulated the expression of nf-kb, il-1β (P < 0.0001; P < 0.0001) (Fig. 1A, B), and had no significant effect on the expression of tnf-α, il-8 and ifn-γ (all the P > 0.05) (Fig. 1C, D, E). Interestingly, the HGCC-1 group was revealed to significantly enhance the expression of tgf-β and il-10 compared to the control (P < 0.05; P < 0.01) (Fig. 1F, G).

Table 5 Effects of dietary supplementation of Bacillus subtilis HGCC-1 on serum ALT, AST, TG in golden pompano
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Fig. 1
figure 1

Effects of Bacillus subtilis HGCC-1 on liver health of golden pompano. (A) Relative mRNA expression levels of the nuclear factor kappa-b (nf-kb), (B) interleukin 1β (il-1β), and (C) tumor necrosis factor-α (tnf-α). (D) and (E) Relative mRNA expression levels of the interleukin 8 (il-8) and interferon gamma (ifn-γ) gene. (F) and (G) Relative mRNA expression levels of transforming growth factor-β (tgf-β) and the anti-inflammatory-related gene interleukin 10 (il-10). Data represent the means ± SEM of each group (n = 25 or 26). * P < 0.05, ** P < 0.01; **** P < 0.0001; P < 0.05 represents significant difference

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Bacillus subtilis HGCC-1 modulates lipid absorption and metabolism

Peripheral blood TG is essential for the internal circulation of dietary fatty acids after absorption in animals. Compared with controls, HGCC-1 significantly upregulated serum TG (P < 0.001) (Table 5). Furthermore, compared with controls, HGCC-1 significantly upregulated mRNA expression of intestinal cd36, fabp1 and fabp2 genes (Fig. 2A, B, C). Liver lipid deposition is an important factor contributing to hepatic metabolic disorders in fish, and high levels of serum TG often led to abnormal accumulation of hepatic TG [38]. Interestingly, HGCC-1 significantly reduced the level of hepatic TG compared to the control group (P < 0.05) (Table 5). The expression of agpat3 gene was significantly down-regulated in the HGCC-1 group (P < 0.01) (Fig. 2D). In addition, HGCC-1 significantly up-regulated the expression of fans (P < 0.01), acc1 (P < 0.0001) genes compared to control (Fig. 2E, F). In the aspect of fatty acid β-oxidation, the expression of cpt1a (P < 0.0001) and ppar-α (P < 0.001) genes of HGCC-1 was significantly higher than the control group (Fig. 2G, H). Moreover, HGCC-1 had no significant impact on the ppar-γ, lpl and hsl gene expression (Fig. 2I, J, K). In lipid transport, dietary supplementation of Bacillus subtilis HGCC-1 significantly up-regulated the hepatic expression level of cd36 compared to the control group (P < 0.05) (Fig. 2L), with no significant effect on the expression of apoB100 (Fig. 2M).

Fig. 2
figure 2

Bacillus subtilis HGCC-1 modulates lipid absorption and metabolism. The mRNA expression of the fatty acid translocase 36 (cd36) (A), fatty acid binding protein 1 (fabp1) (B) and fatty acid binding protein 2 (fabp2) (C) for gut in golden pompano. mRNA expression of the 1-Acylglycerol-3-Phosphate O-Acyltransferase 3 (agpat3) (D), fatty Acid Synthase (fasn) (E), carnitine palmitoyl-transferase 1 A (cpt1a) (F), acetyl-CoA carboxylase1 (acc1) (G), peroxisome proliferator-activated receptor alpha (ppar-α) (H), peroxisome proliferator-activated receptor gamma (ppar-γ) (I), lipoprotein lipase (lpl) (J) and hormone sensitive lipase (hsl) (K), respectively. The mRNA expression of apolipoprotein B-100 (apoB100) (L) and fatty acid translocase 36 (cd36) (M). Data represent the means ± SEM of each group (n = 25 or 26). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; P < 0.05 represents significant difference

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Dietary supplementation of Bacillus subtilis HGCC-1 modulated the gut microbiota

16 S rDNA sequencing was used to analyse the gut microbiota. Rarefaction curve analysis all samples were found to be saturated (Fig. 3A), indicating that the depth of this sequencing reflects the gut microbiota characteristics. Compared with the control, HGCC-1 significantly increased the observed ASVs (P < 0.05) (Fig. 3B). VENN analysis revealed 892 and 2274 ASVs specific to control and HGCC-1 respectively, while there were 320 ASVs in common (Fig. 3C), implying that a large number of ASVs differing between HGCC-1 and the control existed. Subsequently, PCoA was utilized to analyse the structure of the gut microbiota at the level of phylum and ASVs, increased segregation in the HGCC-1 group compared to control (Fig. 3D). ANOSIM and Adonis analyses found significant differences in community structure (P = 0.0018, P = 0.0132; P = 0.0137, P = 0.0027, respectively). HGCC-1 group increased community richness (P = 0.039) and community diversity (P = 0.086) compared to the control (Fig. 3E). Based on the species composition, the gut microbiota of golden pompano was mainly dominated by Proteobacteria and Firmicutes and the HGCC-1 increased the relative abundance of Firmicutes and decreased Proteobacteria compared to the control group (Fig. 4A). In contrast, they were dominated at the genus level by Bradyrhizobium, Novosphingobium, and Bacillus, and the HGCC-1 increased the relative abundance of Bacillus and decreased Bradyrhizobium (Fig. 4B). LEfSe analysis revealed (LDA > 3.5 as a threshold) that Proteobacteria, Novosphingobium were significant biomarkers in the control group, while Firmicutes and Bacillus were significant biomarkers in the HGCC-1 group (Fig. 4C). At the species level, it was found that HGCC-1 was significantly different from the control for Bacillus subtilis (P = 8.7e-7) and Bacillus amyloliquefaciens (P = 3.0e-6), as well as for the unclassified Bacillus (P = 3.6e-2) (Fig. 4D).

Fig. 3
figure 3

Effects of Bacillus subtilis HGCC-1 on the diversity of gut microbial community in golden pompano. (A) Rarefaction curves plotted between the number of Reads and coverage indexes. (B) Number of ASVs observed in all samples. (C) VENN plot analysis of Control and HGCC-1 for ASVs. (D) Principal coordinate analysis (PCoA) was utilized to analyze the structure of gut microbes at the phylum and ASVs levels based on Bray-Curtis’s distance, respectively; in addition, ANOSIM (Analysis of similarities) was employed for assessing differences in gut microbiota communities and utilize Adonis to explore the degree of variation explained by grouping factors on microbial communities. (E) shows the α-diversity of the gut microbiota, denoted as Chao1 and Shannon indexes. Data represent the means ± SEM of each group (n = 25 or 26). * P < 0.05; P < 0.05 represents significant difference

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Fig. 4
figure 4

Effects of Bacillus subtilis HGCC-1 on the composition of the gut microbiota. (A) and (B) Species composition of the gut microbiota at the phylum and genus levels of top10, respectively. (C) LEfSe (Linear discriminant analysis Effect Size) was used to analyse the difference between control and HGCC-1 for biomarker, where a threshold of LDA greater than 3.7 was demonstrated. (D) Relative abundance of Firmicutes and Bacillus in Control and HGCC-1 diets. (E) Screening for significantly different gut microbiota was performed at the species level, where relative abundance top15 was shown, Wilcoxon rank-sum test were used for analysis of variance, and P-values were shown after correction by FDR (false discovery rate). Data represent the means (± SEM) (n = 25 or 26). *P < 0.05 and ****, P < 0.0001, ns represent no significant difference

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Gut microbiota was strongly linked to the growth performance of golden pompano

Previous work has confirmed the existence of two mutually exclusive functional groups in the fish gut microbiota: Functional Group 1 and Functional Group 2 [37]. In the study, two ecologically competitive Functional Groups of golden pompanos were also found, with relative abundance showing a highly significant negative correlation (R = -0.78, P = 2.2e-16) (Fig. 5A). Subsequently, the ratio of “Functional Group 2/Functional Group 1” was significantly increased in the HGCC-1 group compared with the control (P = 9.2e-4) (Fig. 5B). Correlation analysis between gut microbiota and growth performance revealed highly significant negative correlations between final body weight, final body length, carcass weight, weight gain rate, specific growth rate and Functional Group 1 (all P < 0.05) (Fig. 5C), whereas Functional Group 2 and “Functional Group 2/Functional Group 1” were positively correlated with the above growth traits (all P < 0.05). There was no significant correlation between condition factor and Functional Groups (all P > 0.05) (Fig. 5C). Regression analyses revealed significant linear correlations between the ratio of “Functional Group 2/Functional Group 1” and final body weight, carcass weight, and rate of weight gain (R = 0.4, P = 3.3e-3; R = 0.37, P = 8.1e-3; R = 0.39, P = 5.1e-3) (Fig. 5D, E, F).

Fig. 5
figure 5

Gut microbiota was strongly linked to the growth performance of golden pompano. (A) Spearman’s correlation analysis of Functional Group 1 and Functional Group 2. (B) The ratio of “Functional Group 2/ Functional Group 1” in control and HGCC-1 groups, where relative abundance was normalized by taking log10. (C) The correlation between Functional Groups and growth trait phenotypes. (D), (E) and (F) Analysis of linear correlation between the ratio of “Functional Group 2:/Functional Group 1” and Final body weight (FBW, g/fish), Carcass weight (CW, g/fish) and Weight gain rate (WGR, %), respectively. (G) showed correlation analyses between intestinal Bacillus and host serum TG levels. (H) showed correlation analyses between intestinal Bacillus and Weight gain rate (WGR, %). The data show 25 or 26 biological replicates. *P < 0.05; ** P < 0.01; P < 0.05 represents significant difference

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Gut microbes have been demonstrated to modulate blood TG levels in animals [39]. Interestingly, correlation analyses revealed a significant correlation between Firmicutes and Bacillus and serum TG (R = 0.47, P = 0.00091) (Fig. 5G, S. Figure 1C). Meanwhile, intestinal Bacillus was significantly correlated with the weight gain rate of golden pompano (R = 0.35, P = 0.013) (Fig. 5H). This suggests that intestinal Bacillus contributes to the increased serum TG in fish, which may play a role in the microbiota-mediated growth-promoting effect.

Discussion

Probiotics as green inputs to replace antibiotics will be important for the sustainable intensified aquaculture, promoting animal growth performance, improving nutrient metabolism, regulating immune function and so on [16]. In the present study, the dietary supplementation of Bacillus subtilis HGCC-1 was found to significantly enhance the final body weight, weight gain rate and specific growth rate, which promoted the growth performance of golden pompano. The results are similar to the report by Shawky et al. [40], but different from the report by Wang et al. [31]. Basal diet might be a possible cause of the discrepancy (e.g., high-fat diet). Meanwhile, Bacillus subtilis HGCC-1 significantly enhanced the carcass weight of golden pompano and reduced the feed conversion rate to some extent, indicating the HGCC-1 can improve the economic value of cultured fish.

The gut microbiota plays an important function in host immunity, nutrient metabolism, and health processes, and probiotics are able to modulate the fish gut microbiota [15]. In this study, we found that dietary supplementation of Bacillus subtilis HGCC-1 has significantly elevated the number of ASVs and altered the composition of gut microbiota. A significant increased relative abundance of Firmicutes, as potentially beneficial commensal bacteria, and decreased Proteobacteria, as potential opportunistic pathogens in fish, were observed at the phylum level, similar to studies in other fish species [15]. The analysis of α diversity revealed that Bacillus subtilis HGCC-1 significantly elevated the community richness of the gut microbiota, and its reduction was an important feature of ecological dysbiosis of the microbiome [41]. Meanwhile, Bacillus subtilis HGCC-1 altered the structural characteristics of the intestinal microbiota (both at the level of Phylum and ASVs) compared to the control, which was confirmed by ANOSIM analyses. Thus, the present study confirmed that Bacillus subtilis HGCC-1 altered the composition and structure of gut microbiota, elevating the abundance of beneficial bacteria (e.g., Bacillus) and decreasing the abundance of opportunistic pathogens (e.g., Proteobacteria), which was similar to the results in Crucian carp and Chinese perch [22, 29].

Bacillus has been reported to be involved in fish lipid metabolism, but the mechanism is unclear [42]. The study showed that HGCC-1 significantly reduced liver TG deposition. AGPAT3 is important for the synthesis of TG [43] and it was further found that the expression of agpat3 was down-regulated after HGCC-1 supplementation, implying that hepatic TG synthesis was reduced. Activation of HSL and LPL is an important pathway to reduce triglyceride accumulation [44], and the study found no significant changes in the expression of hsl and lpl between HGCC-1 and control, implying that the alleviation of liver TG deposition was not due to lipolysis. Meanwhile, the expression of ppar-α and cpt1a genes was found to be significantly up-regulated in the HGCC-1 group, suggesting that HGCC-1 promoted hepatic fat oxidation for β-oxidation, which facilitates the use of hepatic fatty acids for the body’s energy supply to alleviate hepatic TG deposition [45]. Meanwhile, hepatic lipogenesis plays an important role in the regulation of lipid metabolism [46, 47]. Thus, Bacillus subtilis HGCC-1 alleviates hepatic TG deposition by promoting hepatic fatty acid β-oxidation and inhibiting TG synthesis pathway, which accelerates fatty acid utilization [48]. Interestingly, dietary supplementation with Bacillus subtilis HGCC-1 resulted in the gut containing higher abundance of B. subtilis (Fig. 4E), which may be a potential factor in alleviating hepatic TG deposition.

Previous studies have reported that hepatic lipid transport is an important way to regulate lipid metabolism in the body (e.g., serum TG). We found that HGCC-1 significantly elevated hepatic cd36 expression with no significant effect on apoB100 expression, suggesting enhanced uptake and transport of TG into the liver. Meanwhile, our study found that HGCC-1 significantly up-regulated the expression of fasn and acc1 genes, and CD36 was reported to regulate the expression of hepatic fatty acid synthesis-related genes [49]. Therefore, we hypothesized that accelerated hepatic lipid uptake/transport may be a potential utilization of blood TG, with subsequent hepatic β-oxidation to yield ATP rather than for TG deposition. In zebrafish, it was confirmed that Firmicutes and metabolites increased the number of intestinal lipid droplets [50]. Lactobacillus rhamnosus increased fatty acid absorption in mice [39]. In this study, dietary HGCC-1 improved intestinal expression of CD36, as well as serum TG. Together, these results suggest that Bacillus may promote growth by increasing the uptake and utilization of dietary fat. In summary, on the one hand, dietary supplementation of Bacillus subtilis HGCC-1 significantly up-regulated the abundance of Bacillus, elevated gut absorption of dietary fat, and up-regulated blood TG levels. On the other hand, Bacillus subtilis HGCC-1 up-regulated ppar-α, cpt1a expression to alleviate fat deposition in the liver and ultimately promote the utilization of dietary fat through fatty acid β-oxidation for energy supply. The above may be the potential mechanism by which Bacillus subtilis HGCC-1 enhances the growth performance of golden pompano, which is linked to the gut microbiota (e.g., Bacillus).

Liver health is an important foundation for maintaining fish growth and immunity [9, 51]. Our study found that HGCC-1 significantly reduced serum ALT and AST, suggesting that Bacillus subtilis HGCC-1 alleviated liver injury in golden pompano. In addition, the study revealed that Bacillus subtilis HGCC-1 significantly reduced the mRNA expression of il-1β, nf-kb, implying that the level of inflammation in the liver was controlled. In addition, HGCC-1 significantly elevated the expression of tgf-β and il-10, suggesting that dietary supplementation with HGCC-1 can enhance the expression of anti-inflammatory cytokines to perform the inflammation-suppressive effect in golden pompano, which was similar to the results in zebrafish [31, 52]. Previous studies have found that the expression of host liver inflammation-related genes is associated with gut commensal microbes [37], and we similarly found that HGCC-1 treatment significantly up-regulated the relative abundance of beneficial bacteria (e.g., Bacillus), which may favor hepatic inflammation alleviation. Furthermore, metabolic disorders (e.g., abnormal TG accumulation) contribute to liver inflammation and injury, whereas HGCC-1 alleviated hepatic TG accumulation. Thus, this study suggested that attenuating TG accumulation and reducing inflammation levels in the liver may be associated with modulation of the gut microbiota (e.g., enrichment of Bacillus).

The ratio of “Functional Group 2/Functional Group 1” has been utilized to describe the structure and function of the gut microbiota in grass carp [37], and to assess the health [53, 54] of fish. In this study, dietary supplementation of Bacillus subtilis HGCC-1 significantly elevated the ratio of “Functional Group 2/Functional Group 1”, supporting gut microbiota to improve liver health. Furthermore, we found that the ratio of “Functional Group 2/Functional Group 1” had a significant positive correlation with final body weight, final length and carcass weight of golden pompano, suggesting that growth performance in cultured fish was linked to the Functional Groups, although there are still gaps in the mechanisms of growth promotion. Interestingly, in the cultured populations of golden pompano, we observed that individuals from HWGR had higher levels of Bacillus including Bacillus subtilis (e.g., ASV23) compared to LWGR (S. Figure 2), which was independent of diet type. “Functional Group 2/Functional Group 1” was higher in the golden pompano population with high weight gain rates, suggesting that the microbial Functional Groups is a potentially important contributor to growth traits in fish, which seems to be supported by studies in common carp [55]. In this study, dietary supplementation of Bacillus subtilis HGCC-1 significantly enhanced the ratio of “Functional Group 2/Functional Group 1”, and the growth performance of golden pompano was also significantly enhanced. Therefore, gut microbiota Functional Groups are the potential pathway mediating the growth performance and liver health of cultured fish.

The mechanisms by which probiotics shape the “Functional Group” of gut microbes remain unclear. Dietary supplementation with HGCC-1 increased the relative abundance of Bacillus especially Bacillus subtilis (Fig. 4E), and we attempted to analyse whether the observed enrichment of Bacillus subtilis in the gut was derived from HGCC-1 ingestion. Unfortunately, all the 16 S rDNA sequence of ASVs belonging to Bacillus subtilis didn’t accord with the sequence of HGCC-1, indicating that the elevated abundance of Bacillus/Firmicutes was not due to exogenous B. subtilis HGCC-1 colonization of the gut. Previous studies have demonstrated that the ratio of “Functional Group 2/Functional Group 1” correlates with both adaptive and intrinsic immunity [37], and B. subtilis has been reported to have immunomodulatory functions [28]. Therefore, HGCC-1 may contribute to the elevation of the ratio “Functional Group 2/Functional Group 1” by regulating the intestinal immune response, which requires more evidence in the future.

Conclusions

In this study, dietary supplementation with Bacillus subtilis HGCC-1 significantly promoted growth performance and enhanced feed utilization of golden pompano. Besides, Bacillus subtilis HGCC-1 enhanced intestinal lipid absorption, promoted hepatic utilization of dietary fat by improving hepatic lipid uptake/transport and fatty acid β-oxidation to provide energy, which may be the potential mechanism of Bacillus subtilis HGCC-1-mediated growth promotion. Meanwhile, Bacillus subtilis HGCC-1 was discovered to alleviate liver inflammation and hepatic steatosis, which improved liver health. Bacillus subtilis HGCC-1 significantly altered the structure of gut microbiota, and enhanced the ratio of “Functional Group 2/Functional Group 1” to alleviate the gut microbial dysbiosis. Finally, the ratio of “Functional Group 2/Functional Group 1” was associated with the growth of golden pompano, while the intestinal abundance of Bacillus was correlated with serum TG and growth performance. In summary, dietary supplementation of Bacillus subtilis HGCC-1 promoted dietary fat absorption while reduced liver lipid deposition, alleviated liver inflammation, and improved gut microbiota homeostasis in golden pompano, which ultimately led to a positive effect on fish growth and health.

Data availability

The original sequence data provided in the study have been deposited in the National Center for Biotechnology Information Sequence Red Archive under accession number PRJNA1083016.

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Acknowledgements

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Funding

This study was funded by National Natural Science Foundation of China (NSFC 32122088, 31925038) and Agriculture Science and Technology Innovation Progam (ASTIP) of the Chinese Academy of Agricultural Sciences (CAAS-IFR-ZDRW202401，CAAS-IFR-ZDRW202405).

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  1. China-Norway Joint Lab on Fish Gastrointestinal Microbiota, Institute of Feed Research, Chinese Academy of Agricultural Sciences, Beijing, 100081, China

    Ming Li, Hui Liang, Jian Zhang, Jie Chen, Shichang Xu, Wenhao Zhou, Qianwen Ding & Zhigang Zhou

  2. Biotechnology of the Ministry of Agriculture and Rural Afairs, Institute of Feed Research, Chinese Academy of Agricultural Sciences, Beijing, 100081, China

    Yalin Yang, Zhen Zhang, Yuanyuan Yao & Chao Ran

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Contributions

Z.Z.G. and R.C. conceived the project. L.M performed the experiments, wrote and revised the manuscript. L.H., Z.J., C.J., X.S.C., and Z.W.H., reviewed the manuscript. D.Q.W., Y.Y.L., Z.Z., Y.Y.Y co-discussed the results. R.C. and Z.Z.G. revised and edited the manuscript. All authors approved the manuscript.

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Li, M., Liang, H., Zhang, J. et al. Bacillus subtilis HGCC-1 improves growth performance and liver health via regulating gut microbiota in golden pompano. anim microbiome 7, 7 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s42523-024-00372-x

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