A multi-omics integrated analysis reveals the effects of adding Bacillus coagulans BC66 to feed on the intestinal health and immune function of Parabramis pekinensis

Highlights

• •Integrated 16S rRNA and serum metabolomics reveal BC66 repairs gut villi, muscle, basement membrane in bream.
• •BC66 reshapes microbiota: Firmicutes 80.8%→92.5%, Proteobacteria 18.9%→7.3%, suppresses Aeromonas.
• •BC66 raises serum AKP, LZM, SOD, CAT, GSH-PX and lowers MDA, TG, TCH, LDH. (91 characters).
• •BC66 upregulates ABC transporters and vitamin absorption; downregulates caffeine and xenobiotic metabolites.
• •Multi-omics links BC66 to enhanced immunity and antioxidant capacity via microbiota-metabolite-immunity axis.

Abstract

With the widespread adoption of high-density, intensive farming systems, environmental stressors in Parabramis pekinensis farming have become increasingly severe, leading to intestinal dysfunction, reduced digestive and absorptive capacity, and frequent outbreaks of disease. This study aimed to investigate the effects of adding Bacillus coagulans BC66 to feed on the intestinal health and immune function of Parabramis pekinensis, as well as the underlying multi-omic mechanisms. Parabramis pekinensis with an initial body weight of 150±5 g were divided into a control group (CK) and a B. coagulans-supplemented group (BC); samples were collected for analysis after 60 days of rearing. The results showed that the BC group exhibited approximately 16.7% greater body length growth and 11.8% greater weight gain, alongside increased intestinal villus height, thickened muscular layers and intact basement membranes. Serum activities of AKP, LZM, SOD, CAT and GSH-PX were significantly elevated, whilst levels of MDA, TG, total TCH and LDH were significantly reduced. Analysis of the intestinal microbiota indicated that the relative abundance of the Firmicutes phylum in the BC group increased from 80.8% to 92.5%, whilst that of the Proteobacteria phylum decreased from 18.9% to 7.3%. The Bacillus reached 92.3%, the Lactococcus was enriched, and opportunistic pathogens such as the Aeromonas were suppressed. Serum metabolomics identified 22 differentially expressed metabolites, among which Trehalose, Riboflavin (Vitamin B2) and Sphingosine 1-phosphate were up-regulated, whilst plant-derived alkaloids, methylxanthines, and steroid hormones and their analogues were down-regulated; ABC transporters and Vitamin digestion and absorption pathways were up-regulated, whilst Caffeine metabolism and other pathways were down-regulated. In summary, B. coagulans BC66 enhances the intestinal health and immunity of Parabramis pekinensis in multiple dimensions by repairing intestinal structure, optimising microbial community composition, enhancing immune and antioxidant capacity, and regulating metabolic networks.

Introduction

As one of the world’s most important economically significant freshwater farmed fish species, Parabramis pekinensis has long played a vital role in ensuring a supply of high-quality animal protein and driving economic development, thanks to its rapid growth, strong adaptability and tender flesh [1]. However, the widespread adoption of high-density, intensive farming systems has exacerbated environmental stress in the rearing of Parabramis pekinensis; this stress can induce gastrointestinal dysfunction, reduced digestive and absorptive capacity, and damage to the intestinal barrier, ultimately leading to frequent outbreaks of disease. As the most critical organ for digestion, absorption and immune defence in fish, the health of the intestine directly influences the organism’s nutritional metabolic efficiency and disease resistance [2]. Research indicates that under high-density farming conditions, the fish intestine frequently exhibits damage to villous structures, increased epithelial cell apoptosis and down-regulation of tight junction protein expression. This, in turn, leads to increased intestinal permeability, a heightened risk of translocation of opportunistic pathogens, and the induction of chronic inflammation and metabolic disorders. These intestinal health issues not only lead to reduced growth performance and feed conversion efficiency in Parabramis pekinensis but also significantly increase their susceptibility to subsequent pathogen invasion, resulting in frequent disease outbreaks that severely constrain the sustainable development of the industry [3,4]. Consequently, focusing on maintaining intestinal health to identify green regulatory strategies capable of alleviating stress from high-density farming, enhancing intestinal barrier function, and optimising the microecological structure has become a key research focus in the field of sustainable Parabramis pekinensis aquaculture.

Probiotic-based green feed additives are finding increasingly widespread application in aquaculture due to their ability to regulate the host’s intestinal microecological balance, boost immunity and promote nutrient absorption [5,6]. In recent years, a substantial body of evidence has accumulated regarding the effects of probiotics on freshwater fish such as Ctenopharyngodon Idella [7,8]. In terms of growth performance, studies have shown that adding appropriate levels of probiotics to feed can significantly improve the weight gain rate and specific growth rate of Ctenopharyngodon Idella, whilst reducing the feed conversion ratio [9,10]. Research by Yang et al. found that adding 2 g/kg of probiotics to the base diet resulted in a significant 8.20% increase in final body weight, a 16.48% increase in weight gain rate, an 11.54% increase in specific growth rate, and a 13.07% reduction in feed conversion ratio for Ctenopharyngodon Idella. The research similarly confirmed that the addition of probiotics significantly enhances the growth performance of Ctenopharyngodon Idella, with an optimal addition level of 2 g/kg [11]. Zhu et al. evaluated the effects of Bacillus subtilis, Clostridium butyricum and Enterococcus faecalis on Ctenopharyngodon Idella finding that all three probiotics significantly increased the final body weight, weight gain rate, specific growth rate and crude protein content of the fish, whilst reducing the feed conversion ratio [12]. With regard to gut health and immune modulation, research by Zhang et al. indicated that a combination of Bacillus subtilis and Lactobacillus effectively improved liver lipid metabolism and gut microbial diversity in Ctenopharyngodon Idella, ultimately enhancing their growth performance [13].

Among these, B. coagulans, as a key probiotic strain within the Bacillus, combines the acid-producing and antibacterial properties of Lactobacillus with the heat and stress tolerance of Bacillus subtilis. By forming spores, it acquires exceptional environmental resilience, enabling it to withstand the high temperatures of feed pelletisation as well as the stresses of gastrointestinal acidity and bile salts [14]. Once effectively colonised in the gut, it secretes proteases, lipases, amylase, directly enhancing the host’s efficiency in breaking down and absorbing nutrients [15]; simultaneously, the lactic acid, short-chain fatty acids and antimicrobial peptides produced by its metabolism selectively inhibit the growth of pathogenic bacteria, promote the proliferation of beneficial bacteria such as Bifidobacterium and Lactobacillus, and exert systemic regulatory effects in areas such as intestinal barrier protection and immune modulation [16,17]. Research by Alavinejad et al. indicates that the addition of B. coagulans (2 × 10⁷ CFU/g) to Cyprinus carpio feed significantly increases intestinal amylase, lipase, protease, trypsin and chymotrypsin activity in Cyprinus carpio feed, whilst simultaneously increasing serum total protein, total immunoglobulin, lysozyme activity and ACH50 levels in the alternative complement pathway, and significantly reducing mortality following Aeromonas hydrophila infection [18]. Currently, B. coagulans has emerged as a key candidate strain for replacing antibiotics and driving the green transition in aquaculture.

The composition and dynamic changes of the gut microbiota in fish are directly linked to the host’s health status and growth performance [19]. Studies have shown that probiotics can regulate the structure of the gut microbiota, inhibit the proliferation of opportunistic pathogens, and promote the colonisation of beneficial bacteria, thereby maintaining the homeostasis of the gut microecology and enhancing intestinal barrier function [20,21]. Research on the intestinal health of Parabramis pekinensis has similarly confirmed the importance of microbiota regulation. Yin et al. conducted qualitative and quantitative analyses of the hindgut microbiota in Parabramis pekinensis and found that the abundance of Bifidobacterium in the hindgut contents and hindgut wall was correlated with the fish’s diet, and that the total bacterial count in the hindgut contents was significantly influenced by the type of feed [22]. Research by Wang et al. on six species of cyprinid fish, including Parabramis pekinensis, in the Nanhai Wetland of Baotou has further revealed the presence of common dominant phyla such as the Pseudobacillales, Firmicutes, and Actinobacteria, and identified species-specific dominant genera such as Bacilli, Rhodobacteraceae and Lactobacillales. These microbial communities are significantly associated with the host’s growth performance and antioxidant capacity [23].

This study focused on B. coagulans BC66. Following in vitro antibacterial experiments that confirmed its inhibitory effect on Aeromonas hydrophila, the study utilised intestinal tissue morphology observations, inflammatory cytokine assays, blood biochemical parameter analyses, high-throughput gut microbiota sequencing and serum metabolomics techniques to systematically investigate the effects of B. coagulans BC66 on the growth performance of Parabramis pekinensis and its underlying mechanisms of action. By examining both the structure of the gut microbiota and host metabolic regulation, this study aims to elucidate the potential mechanisms underlying its ability to promote growth, enhance immunity and maintain intestinal homeostasis. The objective is to provide a theoretical basis for the application of B. coagulans in aquaculture, and to offer scientific support for the development of highly effective and safe probiotic preparations, thereby advancing the development of green and healthy farming models for Parabramis pekinensis.

Materials and methods

Test materials

The B. coagulans BC66 used in this study was supplied by Wecare Probiotics Research Institute. After passing viability testing, the strain was stored in a freezer at −80 °C for future use. The Parabramis pekinensis used in the experiments were supplied by the Benxin Ecological Agriculture Professional Cooperative in Yixing City, Jiangsu Province; the fish were healthy and showed no visible signs of disease.

Experimental design and animal management

This study comprised two experimental groups: the control group (CK) and the group fed B. coagulans BC66 (BC). Each group consisted of three replicate treatments (n = 3), with 50 Parabramis pekinensis stocked in each replicate, totalling 300 fish. All fish were sourced from the same batch, were in good health (Individuals with no external injuries, intact scales and fin rays, and which are active in swimming and feeding), had an initial weight of 150±5 g, and were of uniform size. The aquaculture trials were conducted in an indoor recirculating aquaculture system using concrete tanks, each measuring T4 m × 2.5 m × 0.9 m (length × width × height), with an effective water volume of approximately 9 m³. All concrete tanks were fitted with independent water supply and drainage systems, as well as aeration equipment to maintain an optimal dissolved oxygen environment. Water temperature: maintained at 15–20 °C; pH: controlled at 7.0–7.5; dissolved oxygen: not less than 5.0 mg/L; ammonia-nitrogen concentration controlled below 0.2 mg/L; daily light exposure of approximately 12 h; approximately 30% of the water volume was replaced daily to maintain stable water quality. Prior to the experiment, all Parabramis pekinensis underwent a 3-day acclimatisation period, during which they were uniformly fed a basal diet (See Table 1 for the composition: carbohydrates 39.5%, crude protein 30.0%, fat 6.0%, moisture 9.0%, crude fibre 7.0%, ash 8.5% and energy 14.8 MJ/kg. Manufactured by Zhuhai Hailong Biotechnology Co., Ltd.) and their health status was monitored. During the experiment, all groups were fed the same basal diet; however, the BC group had B. coagulans BC66 added to their feed at a concentration of 200 billion CFU/g, the method of administration involves mixing the probiotics directly into the feed, whilst the control group received no probiotics. However, B. coagulans should be taken every other day. The feeding regimen was as follows: feeding frequency was twice daily (08:00 and 17:00); feeding rate was 3–5% of the fish’s body weight, adjusted as appropriate based on feeding behaviour. The entire experimental cycle lasted 60 days.

Table 1.

Composition of basic feed.

Ingredients	Proximate composition (%)
Fish meal	14.0	Moisture	9.0
Soybean meal	25.0	Crude protein	30.0
Rapeseed meal	18.0	Crude lipid	6.0
Cottonseed meal	6.0	Crude fiber	7.0
Wheat bran	15.0	Ash	8.5
Wheat flour	17.0	Carbohydrate	39.5
Soybean oil	2.0	Energy(MJ/kg)	14.8
Ca(H₂PO₄)₂	1.5	Moisture	9.0
Salt	0.2
Premix*	1.3

*Premix supplied the following minerals (g/kg) and vitamins (IU or mg/kg): CuSO4·5H2O, 2.0 g; FeSO4·7H2O, 20 g; ZnSO4·7H2O, 25 g; MnSO4·4H2O, 10 g; Na2SeO3, 0.04 g; KI, 0.03 g; CoCl2·6H2O, 0.1 g; Vitamin A, 600,000 IU; Vitamin D3, 150,000 IU; Vitamin E, 4000 mg; Vitamin K3, 200 mg; Vitamin B1, 300 mg; Vitamin B2, 1000 mg; Vitamin B6, 2000 mg; Vitamin B12, 1.6 mg; Vitamin C, 5000 mg; Pantothenate, 1000 mg; Folic acid, 150 mg; Choline, 60,000 mg.

Sample collection

After 60 days of rearing, five Parabramis pekinensis from each of the CK and BC groups were dissected; sections of the intestine were fixed in Bouin’s solution for morphological analysis. The remaining intestinal tissue was placed in sterile cryovials and stored at −80 °C for analysis of intestinal microbial diversity and metabolomics. At the same time, catch five Parabramis pekinensis, blood was drawn using a 1 mL syringe and transferred into 1.5 mL centrifuge tubes. These were left to stand at 4 °C for 24 h, after which they were centrifuged for 10 min (12,000 rpm, 4 °C, the diameter of the centrifuge rotor is 65 mm) to separate the serum. The serum was then stored at −80 °C for the determination of biochemical parameters.

Morphological analysis of intestinal tissue

Sections of 0.5 cm each were taken from the hepatopancreas and intestinal tissues from the Bouin’s fixative. These were dehydrated using an automatic tissue dehydrator (KD-TS3A) with alcohol solutions of varying concentrations, followed by paraffin embedding. The solidified paraffin blocks were trimmed and sections were cut using a microtome (model: Leica RM2235, manufactured by Leica, Germany) to a thickness of 6 μm; Following dewaxing, the sections were stained with haematoxylin and eosin (H&E). After mounting, the sections were examined using an upright fluorescence microscope (Leica DM5500B).

Analysis of blood biochemistry parameters

The activities of Alkaline Phosphatase (AKP, A059–2–2), Acid Phosphatase (ACP, A060–2–2X), Lysozyme(LZM, A050–1–1), Superoxide Dismutase (SOD,A001–1–2), Catalase (CAT, A007–2–1), Total Cholesterol (TCH, A111–1–1), Malondialdehyde (MDA, A003–1–2), Glutathione Peroxidase-Peroxidase (GSH-PX, A005–2–1), Triglyceride (TG, A110–1–1) and Lactate Dehydrogenase (LDH, A020–2–2) were all measured using kits purchased from the NanJing JianCheng Bioengineering Institute.

High-throughput sequencing analysis of microbial communities

Purification of sample DNA

gDNA was purified from the samples using the Zymo Research BIOMICS DNA Microprep Kit (Cat# D4301). The integrity of the gDNA was assessed by 0.8% agarose gel electrophoresis, followed by nucleic acid concentration measurement using the Tecan F200 (PicoGreen dye method).

PCR amplification

Based on the sequencing region, specific primer pairs with index sequences were synthesised to amplify the V4 region of the 16S rDNA from the samples. The primer sequences are as follows:

Primers 5′-3′: 515F (5′-GTGYCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′).

PCR enzyme: TOYOBO KOD(Thermococcus kodakaraensis KOD1)-Plus-Neo DNA Polymerase (KOD-401B).

PCR instrument: Applied Biosystems^Ⓡ PCR System 9700.

The PCR reaction conditions are shown in Table 2.

Table 2.

PCR System.

Ingredients	Volume added (μl)	Final concentration
10X PCR Buffer for KOD-Plus-Neo	5	1X
2 mM dNTPS	5	0.2mM
25 mM MgSO4	3	1.5mM
U515F	1.5	0.3μM
U806R	1.5	0.3μM
KOD-Plus-Neo(1U/μl)	1	1U/50μl
Matrice DNA	2	20ng/μl
H2O	31	—

PCR protocol: Pre-denaturation at 94 °C for 1 min, 1 cycle; denaturation at 94 °Cfor 20 s, annealing at 54 °C for 30 s and extension at 72 °C for 30 s, 25–30 cycles; 72 °C for 5 min, 1 cycle; hold at 4 °C. Each sample was subjected to three PCR replicates; the PCR products from the linear phase were mixed in equal volumes and used for subsequent library preparation.

Detection, purification and quantification of PCR products

The PCR products were mixed with six times the volume of loading buffer, followed by electrophoresis on a 2% agarose gel to detect the target fragment. From samples that passed the screening, the target band was recovered using the Zymoclean Gel Recovery Kit (D4008); the amount was quantified using a Qubit^Ⓡ 2.0 Fluorometer (Thermo Scientific); and finally, the products were mixed in equimolar quantities.

Library construction

The library was prepared using the MGIEasy Universal DNA Library Prep Kit (MGI#1000,005,282) from MGI Tech Co., Ltd.

High-throughput sequencing

PE250 sequencing was employed, using the DNBSEQ-G99RS high-throughput sequencing kit from MGI.

Data analysis

The raw sequencing data is filtered using tools such as qcat and NanoFILT to yield high-quality target sequences suitable for subsequent analysis. Subsequent bioinformatics processing is carried out using tools such as Kraken2, whilst statistical analysis and visualisation are primarily performed using R⁴, Python and Java.

Serum metabolomics analysis

Metabolite analysis

After extraction, the metabolites in the sample are introduced into a high-resolution mass spectrometer, where they are analysed in both positive and negative ion modes, generating a large volume of mass spectrometry data. This analysis begins with peak extraction using the XCMS(version 3.14.0) software. The XCMS software identifies specific compounds based on their primary mass-to-charge ratio (m/z) and retention time, and extracts the area of the primary chromatographic peak for that compound as quantitative information. Metabolite identification is performed by matching the primary m/z and secondary fragment m/z of the metabolites against standards in the database.

Mass spectrometry peak extraction and identification

Raw mass spectrometry data are primarily processed as follows:

Data conversion. The data are converted to mzXML format using the official MSConvert(version 3.0.21,125, ProteoWizard) software.

Ion peak extraction. The data are imported into the R package XCMS for peak extraction, yielding information such as the mass-to-charge ratio, retention time and ion area of metabolite peaks. The XCMS workflow primarily comprises the following steps: peak alignment, peak extraction and peak area calculation.

Quality control for mass spectrometry peak extraction

Each compound exhibits a unique mass-to-charge ratio and retention time in mass spectrometry. Quality control of substances extracted by the XCMS software primarily involves:

Summing the intensities of all metabolites detected by mass spectrometry for each sample, plotting a three-dimensional total ion chromatogram (TIC), and using the TIC to monitor the overall mass spectrometry signal intensity of the sample; and analysing the distribution of total ions across the mass-to-charge ratio (M/Z) and retention time dimensions;

Extracting an extracted ion chromatogram (XIC) for each metabolite peak.

Results

The effect of B. coagulans BC66 on the growth and development of parabramis pekinensis

The results of the comparative analysis following the conclusion of the rearing trial are shown in Fig. 1. Compared with the control group (CK), the BC group exhibited an approximate 16.7% increase in length and an approximate 11.8% increase in weight. In terms of appearance, the BC group appeared plumper and broader, with a more lustrous scale sheen. This indicates that, following a period of use, B. coagulans led to improvements in body length and weight in Parabramis pekinensis, thereby enhancing their growth performance. See Table 3 for detailed data.

Fig. 1.

Comparison of the growth patterns of Parabramis pekinensis.

Table 3.

Growth Records for Parabramis pekinensis Farming.

Projects	Group	0d	10d	20d	30d	40d	50d	60d
Total length	CK	22	22.7	23.5	24.5	25.6	26.9	28.3
	BC	22	23.3	24.8	26.5	28.4	30.6	33.0
Body length	CK	20	20.5	21.1	22.0	23.0	24.3	25.7
	BC	20	21.1	22.4	24.0	25.8	27.8	30.0
Height at withers	CK	7.0	7.2	7.5	7.8	8.2	8.6	9.0
	BC	7.0	7.4	7.9	8.5	9.1	9.8	10.5
Weight	CK	150	183	220	260	304	351	402.5
	BC	150	183	223	270	323	383	450
Death toll	CK	0	1	2	2	3	3	3
	BC	0	0	0	0	1	1	1

Morphological analysis of the intestinal tissue of parabramis pekinensis

In Fig. 2, the CK group exhibited generally poor continuity of the intestinal basement membrane, a relatively thin muscular layer, sparse and short intestinal villi, with some villi showing atrophy and damage; the arrangement of intestinal epithelial cells was disordered, and the integrity of the intestinal mucosal barrier structure was poor; Compared with the CK group, the BC group exhibited a more intact and continuous intestinal basement membrane, An 8% increase in muscle layer thickness, and intestinal villi that were neatly and densely arranged with a markedly increased height. The villi were morphologically intact with no damage or shedding, and the number of intestinal epithelial cells had increased with a regular arrangement. The structural integrity of the intestinal mucosal barrier was significantly improved, and the health status of the intestinal tissue was substantially enhanced.

Fig. 2.

Histological structure of the intestinal tissue of Parabramis pekinensis following H.E. staining under different treatment conditions.

Note: Figures A-C show intestinal sections from Parabramis pekinensis sampled at 60 days in the CK group; Figures d-F show intestinal sections from Parabramis pekinensis sampled at 60 days in the BC group. V. Villi; M. Muscular layer; BM. Basement membrane.

Analysis of the activity of immune-related enzymes in parabramis pekinensis serum

As shown in Fig. 3, the activity of enzymes associated with non-specific immunity and antioxidant defence was significantly elevated in the serum of Parabramis pekinensis from Group BC, with upregulation observed in AKP, ACP, LZM, SOD, CAT and GSH-PX. At the same time, markers reflecting oxidative damage and lipid metabolism showed some improvement, with a reduction in MDA levels and decreases in TCH, TG and LDH activity.

Fig. 3.

Effect of B. coagulans BC66 on the activity of serum-associated metabolic enzymes in Parabramis pekinensis.

Note: Definition of Kim’s unit/100 mL: The amount of enzyme required to hydrolyse 1 mg of phenol every 15 min at 37 °Cand pH 10.0.

Effects of B. coagulans BC66 on the structure and composition of the intestinal microbiota in parabramis pekinensis

Analysis of test data quality

The project comprised a total of 10 samples, with the volume of raw sequencing reads ranging from 35,817 to 48,897. To obtain high-quality sequences, quality control was performed; the volume of clean reads obtained after quality control ranged from 32,681 to 44,389, as shown in Table 4. The efficiency rates were all close to 95%, and it is therefore considered that this sequencing reflects the true composition of the gut microbiota.

Table 4.

Summary of sequencing data for each sample.

	Raw_reads	Raw_N50	Raw_Q20	Raw_Q30	Clean_reads	Clean_N50	Clean_Q20	Clean_Q30	Efficiency_rate
CK_1	37,023	1461	90.18	82.83	33,537	1460	90.48	83.29	90.58
CK_2	39,180	1461	90.26	82.88	35,464	1461	90.6	83.4	90.52
CK_3	46,668	1461	90.24	82.84	42,198	1461	90.59	83.36	90.42
CK_4	46,531	1461	90.36	83.05	42,493	1461	90.69	83.55	91.32
CK_5	44,071	1460	90.4	83.14	38,420	1460	90.72	83.63	87.18
BC_1	36,455	1455	90.2	82.79	33,393	1455	90.54	83.3	91.6
BC_2	40,551	1459	90.17	82.81	37,142	1459	90.48	83.28	91.59
BC_3	48,897	1459	90.4	83.1	44,389	1459	90.7	83.55	90.78
BC_4	42,473	1461	90.19	82.83	38,587	1461	90.58	83.42	90.85
BC_5	35,817	1461	90.32	83.05	32,681	1460	90.71	83.62	91.24

Analysis of alpha diversity

The results of the alpha diversity analysis shown in Fig. 4 indicate that, compared with the CK group, the BC group exhibited significantly higher values for the Chao1, Shannon, Simpson and PD indices, suggesting that BC66 enhanced the species richness, diversity and phylogenetic diversity of the gut microbiota in Parabramis pekinensis, with the microbial community structure tending towards a healthier and more stable state. The coverage index for both groups was close to 100%, indicating sufficient sequencing depth.

Fig. 4.

Boxplot of alpha diversity.

Note: The x-axis represents sample names, and the y-axis represents the values of the alpha diversity index. The lower boxline, near the bottom of the x-axis, represents the lower quartile, which is the 25th percentile of all values in the sample when arranged in ascending order. The vertical line within the box represents the median, which is the 50th percentile of the values in the sample when sorted from smallest to largest. The upper box line represents the upper quartile, which is the 75th percentile of the values in the sample when sorted from smallest to largest. The lower and upper ends of the extension lines represent the lower and upper bounds, respectively. The dots represent outliers.

Dilution curve of gut microbiota

As the sequencing depth increased from 0 to 30,000, the number of reads in both groups rose in tandem with the depth. At each depth point, Fig. 5 shows that the number of reads in the BC group was slightly higher than that in the CK group, indicating that the BC group had a slightly larger volume of sequencing data and more comprehensive sample coverage, thereby providing a reliable data foundation for subsequent microbial community analysis.

Fig. 5.

Rarefaction Curve.

Note: The x-axis represents the number of sequencing reads selected at random, whilst the y-axis represents the alpha diversity index calculated based on that number of reads; the curves of different colours represent different samples.

Beta diversity in the gut microbiota

PCoA

Fig. 6 shows that, based on Bray-Curtis distance PCoA analysis, samples from the CK group all had negative values on the PCo1 axis (ranging from −0.099 to −0.004), whilst samples from the BC group, with the exception of BC_4 and BC_5, all had positive values; samples from the two groups were clearly separated along the PCo1 axis. The distribution of samples along the PCo2 and PCo3 axes was relatively dispersed, indicating a degree of intra-group variation; overall, the BC66 intervention significantly altered the gut microbiota structure of the Parabramis pekinensis.

Fig. 6.

PCoA based on unweighted UniFrac.

Note: The horizontal axis represents the first principal coordinate, and the percentage indicates the contribution of the first coordinate axis to the variation among samples; the vertical axis represents the second principal coordinate, and the percentage indicates the contribution of the second coordinate axis to the variation among samples. Each point in the figure represents a sample, with samples from the same group shown in the same colour. The two coordinate axes represent two sets of factors influencing community composition, respectively. The confidence ellipses in the figure indicate the 95 per cent confidence intervals for each group.

NMDS

The NMDS analysis in Fig. 7 shows that the samples in the CK group all have negative values on the MDS1 axis (ranging from −0.33 to −0.08), whilst the BC_1, BC_2 and BC_3 samples in the BC group have positive values (ranging from 0.16 to 0.51); the samples from the two groups are clearly separated along the MDS1 axis. With a stress value of <0.2, the ranking results are reliable.

Fig. 7.

NMDS based on Bray-Curtis with group ellipses.

Analysis of differential species in the gut microbiota

LEfSe analysis

LEfSe analysis was employed to identify microbial taxa exhibiting significant differences in abundance between the two groups. The results shown in Fig. 8, Fig. 9 indicate significant differences in microbial community structure between the BC group and the CK group at multiple taxonomic levels. Taxa significantly enriched in the BC group included Firmicutes, Bacilli, Bacillales, Bacillaceae and Bacillus, which is directly related to the addition of B. coagulans BC66 to the feed. Furthermore, lactic acid bacteria groups such as Lactobacillales, Lactococcus and Streptococcaceae were also significantly enriched in the BC group, indicating that BC66 promoted the proliferation of other beneficial lactic acid bacteria. Concurrently, Azospirillum also showed a trend towards enrichment in the BC group. Conversely, the groups significantly enriched in the CK group were primarily Proteobacteria and its sub-groups, including Alphaproteobacteria, Rhodospirillales and Rhodospirillaceae. The Proteobacteria phylum contains various opportunistic pathogens, and their enrichment in the CK group suggests a potential risk of microecological imbalance in the gut of the control group. The results indicate that B. coagulans BC66 promotes the colonisation of the Firmicutes phylum and associated beneficial bacterial communities, whilst inhibiting the growth of potentially harmful bacterial communities such as the Proteobacteria phylum.

Fig. 8.

LDA score of LEfSe.

Note: The x-axis represents the magnitude of the LDA score; in other words, the longer the bar, the more significant the cluster. The colours represent different groups, indicating that within each group, these clusters exhibit significant enrichment—that is, they have the highest average abundance and show significant differences between groups.

Fig. 9.

Cladogram of LEfSe.

Note: Construct a circular dendrogram of the groups showing significant results based on the hierarchical structure of the categorical data.

Effects of B. coagulans BC66 on the composition of the intestinal microbiota in parabramis pekinensis

As shown in Fig. 10, the gut microbiota of both groups of Parabramis pekinensis was dominated by Firmicutes and Proteobacteria. Compared with the CK group, the average relative abundance of Firmicutes in the BC group increased from 80.8% to 92.5%, whilst that of Proteobacteria decreased from 18.9% to 7.3%. The average abundance of Fusobacteria was 0.10% in the CK group, whereas it was not detected in the BC group. Gemmatimonadetes was detected at only 0.10% in the BC group. The abundances of Actinobacteria, Bacteroidetes and other groups were low in both groups (<0.06%), with little difference between them.

Fig. 10.

Bar chart of relative abundance at phylum level.

Note: The x-axis shows the sample names, the y-axis shows relative abundance, and different colours represent different taxonomic groups. ‘Others’ refers to the sum of the relative abundances of all phyla ranked outside the top 20 and those for which no annotation information is available.

As shown in Fig. 11, the average abundance of Bacillus in the CK group was 78.5%, rising to 92.3% in the BC group. In the BC group, the average abundance of Lactococcus was 1.9%, whilst it was not detected in the CK group; the abundance of Azospirillum in the BC group was 1.9%, which was 0.24% higher than that in the CK group. Conversely, the abundances of Sphingomonas, Pseudomonas, Pantoea, Aeromonas and others were all higher in the CK group than in the BC group. The average abundance of Fusobacterium in the BC group was 0.1%, whilst it was not detected in the CK group. The results indicate that intervention with B. coagulans BC66 promoted the enrichment of beneficial bacteria such as Lactococcus, whilst simultaneously inhibiting the growth of various potential opportunistic pathogens.

Fig. 11.

Bar chart of relative abundance at genus level.

Note: The x-axis shows the sample names, the y-axis shows relative abundance, and different colours represent different taxonomic groups. ‘Others’ refers to the sum of the relative abundances of all genera ranked outside the top 20 and those for which no annotation information is available.

Effects of B. coagulans BC66 on the serum metabolomics of parabramis pekinensis

Raw sample data and principal component analysis

To visually reveal the overall differences in serum metabolic profiles between different treatment groups and their patterns of change, this study employed Principal Component Analysis (PCA) to perform dimensionality reduction on the multidimensional metabolic data, thereby extracting the primary sources of variation within the data. As an unsupervised multivariate statistical method, PCA is capable of objectively demonstrating the natural clustering trends of samples without the need for predefined groupings. As shown in Fig. 12, the samples from each group exhibit a certain degree of separation in the PCA score plot, suggesting that the intervention with B. coagulans BC66 may have induced changes in the serum metabolic profile of Parabramis pekinensis. Building upon the unsupervised analysis, Partial Least Squares Discrimination Analysis (PLS-DA) was further employed to maximise intergroup differences and identify key metabolites. By constructing a classification model, PLS-DA enhanced the separation between groups and reduced the interference of intra-group variation. As shown in Fig. 13, samples from both the CK and BC groups fall within the 95% confidence interval and exhibit clear separation on the score plot, with no overlap. This indicates that there are significant differences in the metabolic profiles between the two groups, providing a reliable basis for subsequent screening of differentially expressed metabolites and pathway analysis.

Fig. 12.

PCA plots for the CK and BC groups.

Note: The x-axis (PC1) represents the explanatory power of the first principal component, whilst the y-axis (PC2) represents the explanatory power of the second principal component. Each point in the graph represents a sample; if the samples are clustered together, this indicates that there is little variation amongst them; conversely, the greater the distance between the samples, the greater the variation amongst them.

Fig. 13.

PLS-DA plots for the CK and BC groups.

Screening of differentially expressed metabolites

In this study, OPLS-DA was combined with t-tests to screen for metabolites that differed between groups. Specifically, the variable importance scores (VIP) from the OPLS-DA model were first used to assess the contribution of each metabolite to the sample classification model, thereby identifying potential key differentially expressed metabolites; these were then validated using t-tests to confirm statistically significant differences between groups. As shown in Figs. 14, Fig. 15, Fig. 16, a total of 284 differentially expressed metabolites were identified between the CK and BC groups, with 47 of these showing statistically significant differences; of these, 25 were up-regulated and 22 were down-regulated.

Fig. 14.

Compound Venn diagram.

Note: A Venn diagram is used to illustrate the results for each pair of groups; here, it shows the number of unique and shared metabolites between the two groups.

Fig. 15.

Statistical chart showing the number of differentially expressed metabolites.

Fig. 16.

Compound volcano plot.

Note: The x-axis shows the log2(FC) values for the comparison between the two groups, and the y-axis shows the -log10(p-value) values. In the figure, red and blue dots represent metabolites showing significant differences after screening, whilst grey dots represent metabolites showing no significant differences. The closer a dot is to either side of the plot, the greater the difference in expression between the two groups; the closer a dot is to the top of the plot, the more significant the difference between the two groups.

Metabolites showing statistically significant differences

To illustrate more clearly the differences in the expression of gut samples and metabolites across different samples, this study performed hierarchical clustering on the expression levels of all significantly differentially expressed metabolites ranked in the top 20 based on VIP scores. The results are shown in Fig. 17. Metabolites significantly up-regulated in the two-group comparison included Trehalose, Riboflavin (Vitamin B2) and Sphingosine 1-phosphate; downregulated metabolites included Gramine, 1-Naphthylacetic acid, Carbofuran, Caffeine, Estrone, Chikusetsusaponin IV, Norethindrone, Cholest-4-en-3-one, Curcumin, Anethole, Harman, 1,7-Dimethyixanthine, Trioxsalen, Gibberellin A53, theobromine, androstenedione, decanedioic acid, palmitoylethanolamide.

Fig. 17.

Compound STAMP plot.

Note: STAMP differential analysis plot; the colours at the top represent different groups. The y-axis shows the names of different metabolites, whilst the left x-axis (Mean proportion %) indicates the average relative abundance of the metabolite in each group (left panel). The right-hand x-axis represents the average difference in abundance between the two groups; points to the left of 0 indicate CK > BC, points to the right of 0 indicate BC > CK, and points close to 0 indicate that the difference in metabolite abundance between the two groups is not significant. The horizontal lines on either side of the points represent the 95% confidence interval. The asterisks on the far right indicate the results of statistical tests for differences in metabolite levels between the two groups. * indicates P < 0.05 (statistically significant difference); ** indicates P < 0.01 (highly significant difference); *** indicates P < 0.001 (extremely significant difference).

Metabolic pathway enrichment analysis

In this study, LC-MS metabolomics was employed to analyse two groups of intestinal samples, yielding statistically significant differences; based on annotations from the KEGG metabolic database, metabolically active pathways exhibiting significant disruption were identified, as shown in Fig. 18, Fig. 19. Enrichment analysis of the differentially expressed metabolites revealed that they were primarily concentrated in the following pathways: ABC transporters, caffeine metabolism, vitamin digestion and absorption, the phosphotransferase system (PTS), taste transduction, and biosynthesis of various secondary metabolites-part 2.

Fig. 18.

Compound Enrichment Bar Chart.

Note: The vertical axis represents the KEGG Level 3 category, whilst the horizontal axis shows the number of metabolites enriched at the KEGG Level 3 category. The colour of the bars indicates the level of significance, with darker shades representing higher levels of significance.

Fig. 19.

Compound Enrichment Dot Plot.

Note: The vertical axis represents the KEGG Level 3 category; the horizontal axis represents the ratio of the number of differentially expressed metabolites annotated to the KEGG Level 3 category to the total number of differentially expressed metabolites; the size of the dots represents the number of metabolites annotated to the KEGG Level 3 category; and the shade of colour indicates the level of enrichment significance.

Discussion

Effects of B. coagulans BC66 on the growth performance and intestinal absorption of parabramis pekinensis

The addition of B. coagulans BC66 to the feed resulted in Parabramis pekinensis exhibiting superior body length, weight and physical appearance compared to the control group; the fish appeared plumper and broader, and their scales were more lustrous. These findings are consistent with previous reports on the growth-promoting effects of probiotics in fish [24]. As a facultative anaerobic probiotic, B. coagulans is able to colonise the gut and secrete various digestive enzymes, thereby improving the digestibility and utilisation of nutrients in the feed. At the same time, the short-chain fatty acids it produces lower the intestinal pH and inhibit the growth of harmful bacteria, thereby creating a healthier intestinal microenvironment for Parabramis pekinensis and indirectly promoting growth [25]. Morphological analysis of the intestinal tissue revealed that the basement membrane of the Parabramis pekinensis intestine was intact and continuous, the thickness of the muscular layer had increased significantly, the intestinal villi were arranged in a neat and dense pattern with a marked increase in height, the villi were intact with no damage, and the number of intestinal epithelial cells had increased with a regular arrangement. These morphological improvements directly enhance the intestinal absorption surface area and barrier function. The increased height of the intestinal villi expands the sites of contact and absorption for nutrients, whilst the thickened muscular layer aids intestinal peristalsis and the propulsion of chyme. The integrity of the basement membrane maintains the epithelial barrier function, reducing the invasion of pathogens and toxins [26,27].

Effect of B. coagulans BC66 on the antioxidant capacity of parabramis pekinensis serum

Changes in serum biochemical parameters provide a comprehensive reflection of fish nutritional metabolism, immune function and health status. SOD, CAT and GSH-PX are key components of the body’s antioxidant enzyme system; they work synergistically to scavenge ROS generated during metabolism and protect cells from oxidative damage [28]. Research by Gao et al. indicates that B. coagulans T242 can significantly upregulate the activity of SOD, CAT and GSH-PX in HT-29 cells, activate the Nrf2/Keap1 signalling pathway, and enhance cellular antioxidant capacity [29]. Research by Alavinejad et al. on Cyprinus carpio similarly confirmed that the addition of 2 × 10⁷ CFU/g of B. coagulans to the diet significantly upregulates the expression levels of the SOD, CAT and GPx genes in liver tissue [18]. The results of this experiment indicate that the addition of B. coagulans BC66 to the diet significantly increases the SOD, CAT and GSH-PX activities in the serum of Parabramis pekinensis, consistent with the findings of the aforementioned studies, suggesting that B. coagulans possesses the ability to enhance the body’s antioxidant defence capacity.

MDA is the primary end product of lipid peroxidation, and its concentration directly reflects the extent of oxidative damage in the body [30]. Research by El-Bab et al. on Oreochromis niloticus found that the addition of B. coagulans to the diet significantly reduced serum MDA levels whilst simultaneously increasing CAT activity [31]. A study by Yu et al. on juvenile Carassius auratus gibelio also indicated that B. coagulans, administered via different feeding regimens, effectively reduced liver MDA content and enhanced antioxidant enzyme activity [32]. The results of this study show that serum MDA levels in the BC group of Parabramis pekinensis were significantly lower than those in the control group, exhibiting a synergistic trend with the increased SOD, CAT and GSH-PX activities. This indicates that probiotic intervention effectively mitigated lipid peroxidation damage in the body and maintained the integrity of cell membrane structure and function.

AKP and ACP are marker enzymes of lysosomes and play a key role in the process by which phagocytes eliminate pathogens [33]. Zhang’s research on Megalobrama amblycephala that, following the addition of 0.5% B. coagulans to the feed for 42 days, ALP activity in the test group was significantly higher than at the 14-day mark [34]. Research by Zhu et al. similarly demonstrated that the addition of different species of Bacillus to the diet significantly increased AKP activity in Litopenaeus vannamei (P < 0.05) [35]. LZM is an important effector molecule in the non-specific immune response of fish; it hydrolyses the cell walls of Gram-positive bacteria, thereby exerting an antibacterial defence function. Research by Yang et al. on Carassius auratus confirmed that combined immunisation with B. coagulans and recombinant Lactococcus lactis expressing the mshB gene significantly increased serum LZM, ACP and AKP activities [36]. The results of this study are consistent with the aforementioned reports, with LZM, ACP and AKP activities in the BC group being significantly higher than those in the CK group.

LDH, as a marker enzyme of cellular damage, is released into the bloodstream in large quantities when the permeability of tissue cell membranes increases or when cells undergo necrosis. In a study on Cyprinus carpio, Chang et al. found that B. coagulans SCC-19 significantly reduced the cadmium-induced elevation in serum ALT and AST activity, thereby alleviating liver damage [37]. In the present study, serum LDH activity in the BC group of Parabramis pekinensis was significantly lower than that in the CK group, indicating that B. coagulans helps to maintain cell membrane integrity and reduce tissue damage.

In terms of lipid metabolism, TG and TCH are key indicators of the body’s lipid metabolic status [38]. Studies on Coilia nasus have shown that supplementation can significantly reduce the increases in liver TG and CHO concentrations induced by a high-fat diet. The mechanism of action may be related to the secretion of bile salt hydrolases by probiotics, which promote the conversion of cholesterol into bile acids for excretion, as well as the activation of the PPAR signalling pathway to promote fatty acid β-oxidation [39]. Serum TG and TCH levels in the BC group of Parabramis pekinensis were significantly lower than those in the control group, indicating that B. coagulans helps to improve lipid metabolism and reduce the risk of hepatic fat deposition.

Effects of B. coagulans BC66 on the structure and composition of the intestinal microbiota in parabramis pekinensis

The gut microbiota serves as a vital bridge connecting the host to the external environment, playing a central role in physiological processes such as nutrient metabolism, immune regulation and defence against pathogens. Extensive research indicates that the composition and dynamic changes in the gut microbiota of fish are closely linked to the host’s health status and growth performance [19]. When the microbial community is in a state of stable equilibrium, it helps maintain intestinal barrier function, promotes nutrient absorption and inhibits the colonisation of pathogenic bacteria; however, once the microbial balance is disrupted, it may lead to intestinal inflammation, metabolic disorders and even the onset of disease [40]. As a green regulatory approach, probiotics can, by modulating the structure of the gut microbiota, inhibit the excessive proliferation of opportunistic pathogens whilst promoting the colonisation and growth of beneficial bacteria. This restores the homeostasis of the gut microecology, enhances intestinal barrier function, and ultimately promotes host health [41,42]. In this study, high-throughput 16S rRNA sequencing was employed to systematically analyse changes in the composition of the intestinal microbiota of Parabramis pekinensis at the phylum and genus levels following intervention with B. coagulans BC66, thereby elucidating the mechanism by which BC66 restores the intestinal microecological balance.

At the phylum level, Firmicutes and Proteobacteria were the dominant phyla in the gut microbiota of both groups of Parabramis pekinensis. Compared with the CK group, the average relative abundance of Firmicutes in the BC group increased significantly from 80.8% to 92.5%, whilst that of Proteobacteria decreased significantly from 18.9% to 7.3%. The Firmicutes are a core beneficial phylum of the gut microbiota; their principal members, such as the Bacillales and Clostridiales, participate in the fermentation and metabolism of dietary fibre, producing short-chain fatty acids such as butyrate, which provide an energy source for intestinal epithelial cells [43]. They also play a role in maintaining intestinal barrier function, regulating immune responses and inhibiting the colonisation of pathogenic bacteria. An increase in the abundance of the Firmicutes phylum is generally regarded as an indicator of a healthy and stable gut microbiota [44]. Conversely, the Bacteroidetes comprises various opportunistic pathogens, such as the genera Aeromonas, Escherichia and Pseudomonas; an increase in their abundance is often closely associated with intestinal inflammation, dysbiosis and disease states [45,46]. The significant decrease in Proteobacteria abundance observed in the BC group in this study indicates that the B. coagulans BC66 intervention effectively inhibited the growth of potential pathogens, thereby reducing the risk of intestinal inflammation. The average abundance of Fusobacteria was 0.10% in the CK group, whereas it was not detected in the BC group. The Fusobacteria comprises common commensal bacteria in the fish gut but possesses opportunistic pathogenic potential under certain conditions; its downregulation further corroborates the optimising effect of BC66 on the gut microbiota [47]. Gemmatimonadetes was detected at 0.10% in the BC group, whilst Actinobacteria, Bacteroidetes and others exhibited low abundances in both groups with minimal intergroup differences. These changes at the phylum level indicate that the B. coagulans intervention significantly reshaped the macrostructure of the Parabramis pekinensis gut microbiota, promoting enrichment of the Firmicutes phylum and suppression of the Bacteroidetes phylum, thereby shifting the overall structure towards a healthier and more stable state.

At the genus level, Bacillus was the dominant genus in the gut microbiota of both groups, with an average abundance of 78.5% in the CK group and 92.3% in the treatment group. Bacillus is a representative genus of the Firmicutes phylum and is widely used as a probiotic in aquaculture. It promotes the absorption of nutrients by secreting various digestive enzymes, such as proteases, lipases and amylases, whilst simultaneously producing bacteriocins and organic acids to inhibit the growth of pathogenic bacteria [48]. B. coagulans BC66, which was added in this study, belongs to this genus. Its high abundance in the BC group indicates that the exogenous probiotic has successfully colonised or passed through the gut and occupied a certain ecological niche. The average abundance of Lactococcus reached 1.9% in the BC group, whereas it was not detected at all in the CK group. Lactococcus is an important member of the lactic acid bacteria, widely used in food fermentation and probiotic preparations. It lowers intestinal pH by producing lactic acid, thereby inhibiting the growth of pathogenic bacteria, whilst also possessing immunomodulatory activity that enhances the host’s non-specific immune function [49]. The specific enrichment of Lactococcus in the BC group provides direct evidence that BC66 promotes the colonisation of beneficial bacteria. The abundance of Azospirillum in the BC group was 1.9%, significantly higher than the 0.24% in the control group. Although this genus is renowned as a plant rhizosphere-associated growth-promoting bacterium, its role in the fish gut is gradually attracting attention; it may be involved in the regulation of nitrogen metabolism or the production of growth-promoting substances [50,51]. Conversely, the abundance of several genera within the Proteobacteria class was significantly higher in the CK group than in the BC group. The average abundance of Sphingomonas in the CK group was 1.1%, higher than the 0.68% in the BC group; Pseudomonas was 2.1% in the CK group and 1.9% in the BC group; Pantoea was 2.7% in the CK group and 1.8% in the BC group; Aeromonas was 0.53% in the CK group and 0.006% in the BC group. Aeromonas is one of the most common opportunistic pathogens in freshwater fish and is closely associated with bacterial haemorrhagic disease and enteritis; its abundance was almost completely suppressed in the BC group, further confirming the anti-pathogenic effect of BC66 [52]. The average abundance of Fusobacterium was 0.1% in the BC group, whilst it was not detected in the CK group. This genus plays a dual role in the fish gut; its low abundance may reflect an increase in microbial diversity. Potential pathogens such as Escherichia, Shigella and Klebsiella were present at low abundances in both groups, with no significant differences between them [53,54]. This remodelling of the microbial community structure is highly consistent with the results observed in this study, including upregulation of serum immune enzyme activity and antioxidant enzymes, and improvement in intestinal tissue morphology.

Regulation of intestinal metabolites and pathways in parabramis pekinensis by B. coagulans BC66

This study utilised non-targeted metabolomics techniques to analyse changes in the metabolic profile of Parabramis pekinensis gut contents following intervention with B. coagulans BC66. Combined with metabolic pathway enrichment analysis, the study systematically elucidated the molecular mechanisms by which B. coagulans regulates intestinal metabolism in crucian carp. The results showed that, compared with the CK group, a total of 22 significantly differentially expressed metabolites were identified in the BC group, including three up-regulated metabolites: trehalose, riboflavin (vitamin B2) and sphingosine 1-phosphate; 17 downregulated metabolites, including gramine, 1-naphthylacetic acid, carbofuran, caffeine, oestrone, chikusetsusaponin IV, norethindrone, cholest-4-en-3-one, curcumin, anethole, harman, 1,7-dimethylxanthine, trioxsalen, gibberellin A53, Theobromine, Androstenedione, Decanedioic acid, Palmitoylethanolamide. Metabolic pathway enrichment analysis indicated that the ABC transporters, Vitamin digestion and absorption, and Phosphotransferase system (PTS) pathways were significantly up-regulated, whilst the Caffeine metabolism and Biosynthesis of various secondary metabolites – part 2 pathways were significantly down-regulated.

All three metabolites identified are closely associated with intestinal barrier function and immune regulation. Trehalose is a functional disaccharide that stabilises cell membranes and protein structures, thereby protecting intestinal epithelial cells from damage caused by oxidative stress and osmotic pressure fluctuations [55,56]. Research indicates that trehalose acts as a prebiotic, selectively promoting the proliferation of beneficial bacteria such as Bifidobacterium and Lactobacillus, whilst the short-chain fatty acids produced by its metabolism provide an energy source for intestinal epithelial cells [39,57,58]. Riboflavin (Vitamin B2) is a precursor to FAD and FMN and, as a coenzyme for glutathione reductase, plays a role in the antioxidant defence system. Intestinal epithelial cells undergo rapid renewal and have a high demand for riboflavin (vitamin B2); elevated levels of this vitamin indicate an adequate supply in the gut, which helps maintain the structural and functional integrity of intestinal villi [59]. Sphingosine 1-phosphate is a key signalling molecule in sphingolipid metabolism and plays a central role in regulating lymphocyte migration and maintaining intestinal immune homeostasis [60,61]. Bryan et al. found that probiotics can increase S1P levels by upregulating sphingomyelinase activity, thereby alleviating intestinal inflammatory responses. Further confirmed that treatment with the VSL#3 probiotic blend significantly upregulates alkaline sphingomyelinase activity in the intestinal mucosa of Mus musculus with colitis and patients with ulcerative colitis, promoting S1P production whilst reducing disease activity scores. This indicates that the upregulation of S1P is one of the key mechanisms by which probiotics improve gut health [62]. Research by Iloba et al. on B. coagulans JBI-YZ6.3 also revealed that the metabolites of this bacterium possess significant immunomodulatory activity; under normal culture conditions, they enhance the expression of natural killer cell activation markers CD25 and CD69, whilst under inflammatory conditions, they promote the production of the anti-inflammatory cytokine IL-10 [63]. These studies provide strong evidence supporting the ability of BC66 to upregulate S1P and enhance intestinal immune function in Parabramis pekinensis.

In contrast to the up-regulated protective metabolites, the vast majority of the 18 down-regulated metabolites were exogenous substances or endogenous metabolic intermediates that had accumulated abnormally; their down-regulation reflects the improvement in intestinal barrier function and enhanced liver detoxification capacity. Plant-derived alkaloids and secondary metabolites, such as gramine, harman, gibberellin A53 and trioxsalen, are widely present in feed ingredients; excessive intake may interfere with the normal metabolism of intestinal cells [64]. A review by Li et al. indicates that after ingestion by insects, plant secondary metabolites undergo various biochemical modifications within the gut, and their toxicity or anti-nutritional effects are closely related to the metabolic activity of gut microbiota [65]. The downregulation of these substances in this study indicates that, following intervention with BC66, the intestinal barrier integrity of Parabramis pekinensis was improved, leading to reduced absorption of antinutritional factors in the feed, or an enhanced ability of gut microbiota to degrade and transform them. Of particular importance is the significant downregulation of carbofuran, a highly toxic carbamate insecticide, which serves as a clear indicator of enhanced hepatic detoxification function [66]. Recent research by Chen et al. has confirmed that ABCC family transporters are capable of specifically recognising and effluxing pesticide substrates such as carbofuran, expelling them from the cell via ATP-driven transmembrane transport, thereby exerting a detoxifying effect [67]. This is highly consistent with the results of upregulation of ABC transporters observed in this study, indicating that B. coagulans activated the active detoxification system in the Parabramis pekinensis, effectively eliminating environmental pollutants. The downregulation of methylxanthine compounds such as caffeine, 1,7-dimethylxanthine and theobromine similarly suggests reduced intestinal absorption of exogenous alkaloids [18]. Research by Lyte et al. found that high concentrations of caffeine can inhibit the growth of Lactobacillus and the production of its neurotransmitters (GABA and serotonin metabolites), thereby affecting gut-brain axis communication [68]. Through a large-scale cohort study, Manghi et al. confirmed that coffee consumption is significantly associated with the abundance of Lawsonibacter asaccharolyticus in the gut; this bacterium is capable of metabolising chemical components in coffee and influencing host health [69]. The downregulation of caffeine metabolism observed in this study does not imply a decline in the liver’s ability to break down caffeine, but rather a reduction in total caffeine absorption due to enhanced intestinal barrier function.

The downregulation of steroid hormones such as oestrone, androstenedione and norethindrone, and their analogues, reflects the restoration of endocrine and metabolic homeostasis [70]. Cholest-4-en-3-one is an intermediate product of cholesterol metabolism; its excessive accumulation is typically associated with cholesterol metabolism disorders, and its downregulation indicates that lipid metabolic pathways are functioning smoothly [71]. As an intermediate product of mitochondrial fatty acid β-oxidation, a decrease in decanedioic acid levels indicates normal mitochondrial function and that the fatty acid oxidation pathway is not abnormally blocked. Although palmitoylethanolamide (PEA) possesses endogenous anti-inflammatory activity, its downregulation may be related to a reduction in baseline intestinal inflammation following probiotic intervention, with the body reducing PEA synthesis via negative feedback regulation [72].

Metabolic pathway enrichment analysis further revealed the molecular network through which B. coagulans BC66 regulates the intestinal health of Parabramis pekinensis. The upregulation of ABC transporters was found to be directly causally linked to the downregulation of 19 exogenous metabolites. ABC transporters are a class of ATP-driven transmembrane transport systems highly expressed in detoxification organs such as the liver and intestine, responsible for the active efflux of toxins, drugs, and heavy metal-glutathione complexes [73]. Their upregulation indicates a significant enhancement in the ability of Parabramis pekinensis liver and intestinal cells to clear environmental pollutants, representing a core mechanism underlying the improvement in the fish’s health [74]. The upregulation of vitamin digestion and absorption, corroborated by elevated riboflavin (vitamin B2) levels, indicates that following the repair of intestinal villi structure, the efficiency of micronutrient absorption has improved, providing an adequate supply of coenzymes for the antioxidant enzyme system [75]. The phosphotransferase system (PTS) is the primary mechanism for bacterial uptake of sugars; its upregulation reflects enhanced metabolic activity of beneficial microorganisms in the gut [76]. The specific mechanism is shown in Fig. 20. Trehalose, as a substrate for the PTS system, can be utilised by probiotics such as Bifidobacterium to promote their proliferation and optimise the microbial community structure. Conversely, the downregulation of caffeine metabolism, combined with reduced levels of caffeine and its metabolite 1,7-dimethylxanthine, further confirms that the absorption of exogenous substances is reduced following the restoration of intestinal barrier function. The downregulation of ‘Biosynthesis of various secondary metabolites-part 2′ indicates a reduction in the total amount of plant-derived secondary metabolites from the feed entering the bloodstream, thereby reducing the metabolic burden on the body.

Fig. 20.

Map of the immunometabolic pathways in Parabramis pekinensis.

Note: Schematic model illustrating the multi-dimensional mechanisms by which B. coagulans BC66 influences gut health and immunity in Parabramis pekinensis.

Conclusions

The addition of B. coagulans BC66 to the feed in this study significantly improved the growth performance and intestinal health of Parabramis pekinensis. Parabramis pekinensis in the BC group exhibited an increase of approximately 16.7% in body length and approximately 11.8% in body weight; intestinal villi height increased, the muscular layer thickened, the basement membrane remained intact, and the absorptive surface area expanded. Serum levels of AKP, LZM, SOD, CAT and GSH-PX activity increased, whilst MDA, TG, TCH and LDH levels decreased, indicating enhanced non-specific immune and antioxidant capacities. Analysis of the gut microbiota revealed that the relative abundance of the Firmicutes phylum rose from 80.8% to 92.5%, whilst that of the Proteobacteria phylum fell from 18.9% to 7.3%. The Bacillus reached 92.3%, the Lactococcus was enriched, and opportunistic pathogens such as the Aeromonas were suppressed. Serum metabolomics identified 22 differentially expressed metabolites: trehalose, riboflavin (vitamin B2) and sphingosine 1-phosphate were up-regulated, whilst plant-derived alkaloids, methylxanthines, and steroid hormones and their analogues were down-regulated; pathways involving ABC transporters and vitamin digestion and absorption were up-regulated, whilst pathways such as caffeine metabolism were down-regulated. In summary, B. coagulans BC66 enhances the intestinal health and immunity of Parabramis pekinensis in multiple dimensions by repairing intestinal structure, optimising microbial community composition, strengthening immune and antioxidant defences, and regulating metabolic networks.

Author's statement

I solemnly declare that this thesis is the result of my independent research under the guidance of my supervisor. In the process of writing this dissertation, I have strictly abided by the academic norms in good faith, and I have not plagiarized, plagiarized, or infringed upon the intellectual property rights of others, whether in words, data, or ideas. All the information and materials involved in this study have been obtained through legal channels, and have been appropriately cited and labeled in the text. The research results, documents, diagrams, etc. of others have been reasonably cited and acknowledged in accordance with the intellectual property law and relevant regulations. I have carefully reviewed the whole thesis, and respect the labor achievements of others in the process of writing. Other research work and academic results have been appropriately cited and respected. I would like to express my special thanks to all the people and organizations that have helped and supported me in this research.

Finally, I solemnly declare that this thesis does not contain any false facts, and the analysis and conclusions of the research results are true and objective. I will be responsible for any errors or defects found in this thesis, and I am willing to accept the criticisms and corrections from my supervisors and relevant experts.

CRediT authorship contribution statement

Zehui Su: Writing – original draft, Supervision, Project administration, Investigation, Funding acquisition, Data curation. Runze He: Validation. Ping Shen: Project administration. Shuguang Fang: Resources. Fu Gao: Resources. Zhangliang Ma: Methodology. Kai Cai: Supervision. Shasha Liu: Methodology. Yali Wang: Writing – review & editing. Kun Hu: Writing – review & editing.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Zehui Su reports financial support was provided by National Freshwater Genetic Resource Center (FGRC: 18,537). Kun Hu reports a relationship with the projects of China that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

No data was used for the research described in the article.