Pediococcus acidilactici GR-5 alleviates hyperuricemia by degrading purine nucleosides and improving gut microbiota metabolism

Abstract

Reducing intestinal absorption by degrading purine nucleosides has been shown to alleviate hyperuricemia (HUA). The probiotic Pediococcus acidilactici GR-5, derived from the traditional food “Jiangshui”, achieved efficient degradation of purine nucleosides through its unique purine nucleoside phosphorylase-DeoD. In the purine nucleosides-induced HUA mouse model, GR-5 treatment reduced serum uric acid (UA) by about 52.17%. GR-5 may improves tissue inflammatory damage by inhibiting the NLRP3 inflammatory pathway, and restored the intestinal barrier by increasing tight junction proteins Occludin and ZO-1 expression, which outperforming the allopurinol. The mechanism of UA reduction may also involves inhibiting UA synthase activity and regulating UA transporters level. GR-5 colonization in intestine increased the abundance of Lactobacillus, improved the metabolism of purine, tryptophan and bile acid by gut microbiota, and increased the level of SCFAs. Overall, GR-5 may be a potential preventive agent for improving HUA and is expected to provide a healthy option for preventing diet-induced HUA.

Introduction

Hyperuricemia (HUA) refers to a chronic metabolic disease caused by purine metabolism disorder¹. HUA causes gout by promoting the formation and deposition of monosodium urate crystals in joint tissues². According to the Gout Report White Paper (2021), the prevalence of HUA in China is 13.3%, and the overall prevalence of gout is 1.1%, and it is gradually showing a trend of younger age³. Currently, the treatments for HUA include drugs, dietary interventions, and biological therapies⁴. Among therapeutic drugs, allopurinol and febuxostat mainly reduce the conversion of purine to uric acid (UA) by reducing the activity of xanthine oxidase (XOD) and adenosine deaminase (ADA) in the liver⁵. Drugs such as probenecid and benzbromarone reduce UA reabsorption by regulating UA transporters in the kidney and intestine⁶. However, drugs may cause hypersensitivity reactions, cardiovascular side effects, hepatotoxicity, gastrointestinal and skin discomfort in some patients⁷. Dietary intervention is to reduce the absorption of purine by limiting the intake of high-purine foods, but it is difficult to comply with dietary restrictions for a long time⁸. Unlike other UA-lowering therapies, biological therapy mainly lowers serum UA by degrading UA or purine in the intestine and reducing the intestinal absorption of these substances⁹.

As an economical and effective treatment, probiotic intervention can treat or prevent a variety of metabolic diseases by degrading adverse intestinal metabolites¹⁰. Because of its safety and sustained effect on the body, it has been widely accepted by the public. Purine metabolites are precursors of UA synthesis in the body. In the treatment of HUA with probiotics, probiotics can be used to degrade purine and UA in the intestine, reducing their absorption by the body and thus lowering serum UA levels¹¹. The degradation rates of guanine, xanthine and adenine by Lactobacillus plantarum X7023 within 16 h were 21.4%, 93.9% and 11.5%, respectively. However, after high-dose L. plantarum X7023 treatment of HUA mice, the serum UA level was only reduced by 19.0%¹². L. plantarum X7022 has a complete purine metabolic pathway and can degrade 82.1% of xanthine. After treating HUA mice with this strain for four weeks, serum UA levels significantly decreased by 35.5%¹³. Wu et al.¹⁴ screened a probiotic Limosilactobacillus fermentum GR-3 that can degrade UA from the traditional fermented food “Jiangshui” in Northwest China, which can reduce the serum UA of mice by 31.3% after feeding for 14 days. The functional fermented yogurt produced by using GR-3 has the function of regulating inflammation and lowering blood UA levels¹⁵. However, the ability of currently reported probiotics to lower serum UA by degrading purine and UA is low.

Purine nucleosides are important precursors for UA synthesis, and their solubility in water is significantly higher than that of other purine compounds and UA. At the same time, the absorption of inosine and guanosine by intestinal epithelial cells is significantly higher than that of hypoxanthine and guanine, and their contribution to serum UA in mice is higher¹⁶. Probiotics can degrade inosine, guanosine and adenosine into hypoxanthine, guanine and adenine through purine nucleoside phosphorylase (PNP, EC: 2.4.2.1)¹⁷. Using probiotics to degrade purine nucleosides in the intestine may reduce the absorption of purine nucleosides, thereby reversing the increase in serum UA caused by diet¹⁸. Lacticaseibacillus paracasei MJM60396 isolated from fermented foods can degrade adenosine, guanosine, and inosine, thereby preventing the body from excessive absorption of purine nucleosides from food¹⁹. Lactobacillus brevis DM9218 improved fructose-induced HUA by degrading inosine and regulating intestinal microecological imbalance¹⁷. Zhao et al.²⁰ isolated a strain of Lacticaseibacillus rhamnosus Fmb14 from traditional yogurt in Northwest China. Although the degradation rate of inosine was only 36.3% within 24 h, the serum UA level of model mice was reduced by 36.8% after oral administration of the Fmb14 strain for 12 weeks²¹. Based on the above status of probiotics in the treatment of HUA, reducing intestinal absorption of purine nucleosides by degrading purine nucleosides through probiotics may be an effective way to reverse the diet-induced increase in serum UA¹⁶.

The main form of purine in meat, seafood and beer is purine nucleosides, which are more easily absorbed and metabolized by the intestines than other purine substances^18,22. Current studies have preliminarily confirmed the effectiveness of purine nucleosides-degrading probiotics in alleviating HUA, but the degradation efficiency of purine nucleosides is low and the mechanism of treating HUA is still unclear¹¹. It was reported that Jiangshui has the efficacy in treating HUA and there may be probiotics that degrade purine nucleosides¹⁴. In this study, a probiotic strain of Pediococcus acidilactici GR-5 that efficiently degrades purine nucleosides was isolated from Jiangshui. The molecular mechanism of GR-5 degrading purine nucleosides was analyzed by genome, transcriptome sequencing and gene cloning. Through the HUA model mouse treatment experiment, the ability of probiotics to reduce serum UA and tissue inflammation indicators in model mice was explored. By analyzing the expression of related oxidases and UA transporters during probiotic treatment, the molecular mechanism of GR-5 in reducing serum UA was revealed. Finally, the structure, function and metabolite characteristics of gut microbiota after GR-5 treatment were analyzed. This study provides a potential probiotic option for the treatment or prevention of diet-induced HUA.

Results

Characteristics of purine nucleoside-degrading probiotic GR-5

The probiotic Pediococcus acidilactici GR-5, isolated from the Jiangshui of traditional fermented food in Northwest China, was able to almost completely degrade single or even mixed inosine, guanosine and adenosine (100 mg/L each) within 12 h (Figure S1). LC-MS results showed that GR-5 degraded inosine to hypoxanthine, guanosine to guanine and xanthine, and adenosine to adenine and hypoxanthine (Figure S2). In addition, GR-5 may also synthesize AMP, IMP, GMP and XMP through the purine salvage pathway. To elucidate the mechanism by which GR-5 degrades purine nucleosides, whole genome sequencing of GR-5 was performed. A total of 390,223 reads were obtained. The GR-5 genome size is 2,294,056 bp, the GC content is 42.21%, and contains a total of 2265 coding sequences (CDS), 15 rRNA, 58 tRNA, 37 sRNA, 3 plasmids and 3 trails of CRISPR-Cas sequences (Table S1). The Circos circle diagram of the GR-5 genome is shown in Fig. 1a. Functional annotation was performed using KEGG orthology (Fig. 1b). Among the predicted genes, 58.41% were successfully assigned to KEGG pathways. The analysis revealed that GR-5 contains 39 functional groups, 66.04% of which are involved in metabolism, including 46 genes involved in purine metabolism. A key gene deoD (K03784) encoding PNP (EC: 2.4.2.1) was identified on the chromosome of this strain, which can convert purine nucleosides into purine (Fig. 1c).

Fig. 1. Whole genome and transcriptome sequencing of Pediococcus acidilactici GR-5 under mixed stimulation of inosine, guanosine and adenosine (PN).

a Circular genome map of strain GR-5. The outermost circle is the genome size indicator; the second and third circles are the CDS on the positive and negative strands, and different colors indicate the functional classification of different COGs in the CDS; the fourth circle is rRNA and tRNA; the fifth circle is the GC content. b KEGG ortholog (KO) annotation and functional category summary of GR-5 predicted genes. c Genes related to purine nucleoside metabolism extracted from the GR-5 purine metabolism pathway (Pathway ID: ko00230). d Volcano plot of differentially transcribed genes in GR-5 under purine nucleoside stimulation (n = 3). e KEGG enrichment analysis.

RNA sequencing and DESeq2 differentially expressed gene (DEG) analysis were performed after GR-5 stimulation with purine nucleosides. Under purine nucleoside stimulation, 23 genes were upregulated and 102 genes were downregulated in GR-5 (log₂FC ≥ 1, p-adjust < 0.05) (Fig. 1d). KEGG enrichment analysis showed that DEGs were involved ABC transporters, oxidative phosphorylation, purine metabolism, and fatty acid biosynthesis pathways (Fig. 1e). Table 1 shows the expression of genes in the intercepted part of the GR-5 purine metabolism pathway (Pathway ID: ko00230, Fig. 1c). Under purine nucleoside stimulation, the expression of xanthine phosphoribosyltransferase (xpt, K03816), hypoxanthine phosphoribosyltransferase (hprT, K00760), and adenine phosphoribosyltransferase (apt, K00759) genes in the GR-5 purine salvage pathway was significantly upregulated (p < 0.01). This is consistent with the increased synthesis of GMP and AMP detected by LC-MS (Figure S2e). In addition, the expression of PNP (deoD, K03784), adenine deaminase (ade, K01486), and guanine deaminase (guaD, K01487) genes were all significantly upregulated (p < 0.05). This is consistent with the results of the study on the products obtained by GR-5 degradation of purine nucleosides (Figure S2a–d). The gene deoD encoding PNP (EC: 2.4.2.1) was upregulated by 3.15 times, indicating that it may play an important role in the degradation of purine nucleosides.

Table 1.

Analysis of differential transcription of genes related to purine nucleoside metabolism in GR-5

EC numbers	Gene name	Gene description	Transcripts Per Million		PN/CK	p-value	Regulate
			CK	PN
6.3.5.2	guaA	Glutamine-hydrolyzing GMP synthase	39.82 ± 9.49	106.87 ± 12.11	2.68	0.0016	up
1.1.1.205	guaB	IMP dehydrogenase	59.51 ± 11.43	117.27 ± 14.43	1.97	0.0056	up
2.4.2.7	apt	Adenine phosphoribosyltransferase	24.54 ± 5.08	55.55 ± 7.55	2.26	0.0041	up
2.4.2.1	deoD	Purine-nucleoside phosphorylase	2.91 ± 0.27	9.16 ± 0.88	3.15	0.0003	up
2.5.4.2	ade	Amidohydrolase family protein	1.06 ± 0.17	2.22 ± 0.12	2.09	0.0165	up
3.5.4.3	guaD	Amidohydrolase family protein	0.88 ± 0.33	2.51 ± 0.07	2.86	0.0011	up
2.4.2.22	xpt	Xanthine phosphoribosyltransferase	18.95 ± 4.33	45.09 ± 5.54	2.38	0.003	up
2.4.2.8	hprT	Hypoxanthine phosphoribosyltransferase	17.43 ± 4.78	33.75 ± 3.21	1.94	0.008	up

CK no purine nucleoside addition, PN purine nucleoside stimulation.

Characterization of the PNP-deoD gene

PNP is the key enzyme for purine nucleoside degradation. Through BLAST alignment in NCBI, the identity of the deoD gene in GR-5 with other Pediococcus acidilactici was higher than 98.16%. Except for Pasteurella dagmatis strain NCTC11617, which had an identity of 87.50%, no similar sequences were found in other strains (Sequences are provided in the supplementary material). This indicates that the deoD gene sequence found in this study may be unique to Pediococcus acidilactici. The amino acid sequence corresponding to the deoD gene in GR-5 also showed more than 99.15% identity with the PNP sequence reported in Pediococcus acidilactici, and a 68.80% to 79.91% similarity with other Lactobacilli (Fig. 2a). The tertiary structure modeling showed that the tertiary structure of the DeoD protein in GR-5 shares a sequence identity of 61.21% with the reported Bacillus cereus DeoD enzyme. The three-dimensional structure consists of two subunits forming a dimer, and three dimers are then constructed into a symmetrical homo-hexamer (Fig. 2b). To verify the purine nucleosides-degrading capability of DeoD in GR-5, the deoD gene from GR-5 was cloned into the pET28a vector, and heterologous expression and purification were carried out in Escherichia coli Rosetta (DE3) cells. The deoD gene size is 705 bp, and double enzyme digestion and sequencing results indicated successful cloning of deoD into DE3 cells (Figure S3a). The molecular weight of the purified DeoD protein was 25.58 kDa, and the elution effect was best at 350 mM imidazole (Figure S3b). Using the eluted protein to degrade purine nucleosides, DeoD exhibited a degradation rate of 61.37 ± 5.26% for inosine within 3 h (Fig. 2c), 69.28 ± 5.04% for guanosine (Fig. 2e), and 56.65 ± 6.07% for adenosine (Fig. 2g). However, the degradation ability of the purified protein was lower than that of GR-5 and IPTG-induced DE3 strain containing pET28a-deoD. Figure 2d–h show the HPLC chromatograms after DeoD degrades purine nucleosides.

Fig. 2. Structure prediction, heterologous expression and characterization of purine nucleoside degradation ability of GR-5 PNP-DeoD.

a Amino acid sequence alignment of DeoD in GR-5 and DeoD in other lactobacilli. b Using the crystal structure of PNP-DeoD from Bacillus cereus as a template, the tertiary structure modeling of the DeoD protein domain in GR-5 was conducted on the SwissModel server at ExPaSy (https://swissmodel.expasy.org/). Degradation rates and chromatographic peaks of (c, d) inosine, (e, f) guanosine and (g, h) adenosine by purified DeoD protein within 3 h (n = 5).

GR-5 reduced serum UA and inflammatory factor levels in HUA mice induced by purine nucleosides

In order to study the preventive and therapeutic effects of Pediococcus acidilactici GR-5 on HUA mice, a 3-week animal experiment was designed (Fig. 3a). Allopurinol, a drug for the treatment of HUA, is often used in comparison with probiotic therapy due to its side effects and was used as a drug control in this study^16,23. After 3 weeks of treatment, the weight of mice in the hyperuricemia group (HUA) was lower than that in other groups (Fig. 3b). GR-5 treatment helped restore the diet and drinking water of mice (Figure S4a, b). The organ and serum indicators of mice were detected. GR-5 restored the liver and kidney weights and colorectal length of HUA mice (Figure S4c). Oral gavage of purine nucleosides and PO stimulated an increase in the serum UA level of mice. Compared with the HUA group, GR-5 treatment reduced the serum UA of mice from 298.38 ± 65.02 µmol/L to 142.71 ± 25.28 µmol/L, a significant decrease of 52.17% (p < 0.0001) (Fig. 3c). The serum CRE and BUN levels both showed an upward trend in the HUA group, and GR-5 treatment restored the serum CRE and BUN to near healthy levels (Fig. 3d, e). HUA increased the levels of LPS and ALT in the serum of mice and reduced the level of T-AOC (Fig. 3f–h). The probiotic GR-5 treatment improved this trend and was superior to the allopurinol treatment group (ALLO). In addition, GR-5 treatment also reduced the increase in the levels of inflammatory factors IL-1β and IL-6 in the serum caused by HUA, and TNF-α even returned to around normal levels (Fig. 3i–k).

Fig. 3. Oral supplementation of Pediococcus acidilactici GR-5 reduced serum HUA and inflammatory markers in mice.

a Graphical overview of the experimental design of mice. HUA was induced by oral gavage of potassium oxonate and purine nucleosides. b Body weight changes of mice during the experiment, n = 10 per group. c–k Changes in serum UA, creatinine, blood urea nitrogen, LPS, T-AOC, ALT and inflammatory factors IL-1β, IL-6 and TNF-α levels in mice after GR-5 treatment (n = 10).

GR-5 intervention ameliorated tissue inflammation in HUA mice

HUA may induce renal inflammatory response and affect the intestinal barrier²⁴. Renal pathological sections showed that the mice in the HUA group had glomerular atrophy, tubular dilation, epithelial cell shedding, and inflammatory cell infiltration (Fig. 4a). GR-5 treatment was superior to allopurinol in relieving the above-mentioned renal symptoms, and also reduced the rupture of the liver central vein wall and lymphocyte infiltration, small intestinal villus structure disorder, and submucosal tissue thickening caused by HUA (Figure S5). Compared with the HUA group, GR-5 treatment significantly increased the activity of SOD and CAT in the kidneys of mice, while reducing the level of MDA (p < 0.01), and significantly reduced renal NF-κB by 24.39% (p < 0.05) (Fig. 4b). HUA-induced renal injury is associated with reciprocal activation of NF-κB and NLRP3 inflammatory pathways²⁵. WB results showed that compared with the NC group, the expression of NLRP3, ASC and Caspase-1 proteins in the kidney tissues of mice in the HUA group was significantly increased (p < 0.001) (Fig. 4c, d). GR-5 treatment significantly reduced the overexpression of NLRP3 inflammatory pathway proteins caused by HUA (p < 0.001), and was superior to the ALLO group. In addition, compared with NC, the expression of tight junction proteins Ocln and ZO-1 in the colon of the HUA group was significantly reduced (p < 0.01), which was significantly restored after GR-5 treatment compared with the HUA group (p < 0.01), indicating that GR-5 treatment ameliorated the integrity of the intestinal barrier of mice (Fig. 4e).

Fig. 4. GR-5 alleviated renal inflammation and restored intestinal barrier function in HUA mice by regulating oxidative stress and NLRP3 inflammatory pathway.

a Representative H&E staining of mouse kidneys after treatment, scale bar = 200 μm; (b) SOD, CAT, MDA (n  = 10), and NF-κB levels (n = 5). c, d Representative Western blots of NLRP3 inflammatory pathway-related proteins and bar graphs showing relative target protein signals normalized to β-actin (n = 3), ^#p < 0.05 compared to the NC group; *p < 0.05 compared to the HUA group. e Representative immunofluorescence images of colon tight junction proteins Ocln and ZO-1 (red) and bar graphs of red area relative to total area (red + blue) (n = 3). Cell nuclei were stained with DAPI (blue), scale bar = 100 μm.

GR-5 regulates UA transporter expression and UA synthase activity in HUA mice

The expression of UA transporters in the kidney and colon and the activity of UA synthase in the liver of HUA mice were analyzed to evaluate the effect of GR-5 treatment on UA excretion and production. WB results showed that compared with NC, the expression levels of ABCG2 and OAT1, the transporters responsible for UA secretion, in the kidneys of HUA group mice were significantly downregulated (p < 0.001), while the expression levels of GLUT9 and URAT1, the transporters responsible for UA reabsorption, were significantly upregulated (p < 0.05) (Fig. 5a, b). GR-5 treatment ameliorated the expression levels of the above four UA transporters in HUA mice and was superior to the allopurinol treatment group. Similar to the kidney, GR-5 upregulated the expression levels of ABCG2, OAT1, and NPT1 in the colon, and reduced the expression levels of GLUT9 and URAT1 (Fig. 5c, d). In addition, the UA level in the urine of mice in the GR-5 treatment group reached 167.90 ± 21.83 µmol/L, which was significantly higher than that in other groups (p < 0.01) (Fig. 5e). The activities of XOD and ADA in the liver were measured (Fig. 5f, g). The activities of XOD and ADA in the liver of mice in the HUA group were significantly higher than those in the NC group (p < 0.0001). Compared with the HUA group, GR-5 or allopurinol treatment significantly inhibited the activities of XOD and ADA (p < 0.001). GR-5 reduced the activities of XOD and ADA in the liver by approximately 36.43% and 32.26%, respectively.

Fig. 5. GR-5 reduces UA production and reabsorption by regulating UA transporter levels in the kidney and colon of mice and reducing hepatic uricase activity.

a, b Representative Western blots of renal UA transporters ABCG2, OAT1, GLUT9, and UTAT1 and bar graphs showing relative target protein signals normalized to β-actin (n = 3), ^#p < 0.05 compared to the NC group; *p < 0.05 compared to the HUA group. c, d Colonic UA transporter levels were measured by ELISA (n = 5). e UA content in urine after 21 days of treatment (n = 3). f, g Liver uricase activities of XOD and ADA were measured by ELISA (n = 10).

GR-5 improves the gut microbiota structure of HUA mice

Numerous studies have shown that the gut microbiota plays an important role in the overall health of the host²⁶. Unlike drugs, probiotics can alleviate diseases by regulating the gut microbiota. Full-length 16S rRNA sequencing evaluated the effect of GR-5 treatment on the gut microbiota of mice. The Venn diagram showed that there were 407 OUTs between the fecal microbiota of the four groups of mice (Fig. 6a). Compared with the HUA group, the GR-5 intervention group and the NC group shared more OUTs. Unweighted UniFrac principal coordinate analysis (PCoA) showed that HUA changed the structure of the gut microbiota of mice, and GR-5 or allopurinol treatment regulated this shift in the gut microbiota (Fig. 6b). The relative abundance of the gut microbiota of each group of mice was compared at the genus and species level. Compared with the NC group, the relative abundance of Eisenbergiella, Seramator, and Alistipes decreased in the HUA group, while the relative abundance of Prevotella and Bacteroides increased (Figure S6a). GR-5 intervention reversed this trend and significantly increased the abundance of the probiotic Lactobacillus to 23.62% (p < 0.05). At the species level, GR-5 intervention increased the abundance of Lactobacillus johnsonii, Lactobacillus intestinalis, and Seramator thermalis, and reduced the increase in Prevotella loescheii, Prevotella shahii, and Prevotellamassilia timonensis caused by HUA, and the regulatory effect on this trend was better than that of allopurinol treatment (Fig. 6c). Correlation analysis between serum UA levels in mice and the abundance of Lactobacillus intestinalis and Prevotella loescheii in the intestine showed that GR-5 may affect the UA metabolism by regulating the gut microbiota (Fig. 6d). The data of the allopurinol treatment group (blue) reduced the p-value in the correlation analysis, which also confirmed its limited role in regulating the gut microbiota (Figure S6b). Probiotic colonization in the intestine is essential for sustained function²⁷. GR-5 was detected in the cecal feces of mice in the GR-5 treatment group by PCR and agarose gel electrophoresis (Fig. 6e). qPCR analysis showed that GR-5 was detected in both the cecal feces and colonic mucosa (Fig. 6f, g) of mice in the GR-5 group, indicating that GR-5 has colonized in the mouse intestine.

Fig. 6. GR-5 colonizes the intestine and regulates the composition of the gut microbiota in HUA mice.

Fecal samples were collected from each group on day 21 of treatment (n = 3). a Venn diagram reveals the differences in OTUs of the gut microbiota between the groups. b Unweighted UniFrac principal coordinate analysis (PCoA) based on the OTU level. c Histograms of relative abundance of species at the species level. d Scatter plot of Spearman rho correlation analysis between gut microorganisms and serum UA levels, blue is the data of the ALLO group. e Representative agarose gel electrophoresis after PCR of the GR-5 deoD gene in fecal DNA (CK group was GR-5 DNA). f, g qPCR detection of the abundance of GR-5 relative to the total microorganisms and CFU in excreted feces and colonic mucosa-attached feces (n = 5).

GR-5 improves gut microbiota metabolism and SCFAs levels

The effects of GR-5 treatment on gut microbial metabolites in HUA mice were analyzed by non-targeted metabolomics and GC-MS-SCFA quantification. The partial least squares discriminant analysis (PLS-DA) score plot showed that GR-5 was obviously separated from the HUA group, but GR-5 was closer to the NC group, and the ALLO and HUA groups were clustered together (Fig. 7a). The orthogonal partial least squares discriminant analysis (OPLS-DA) score plot further showed that there were significant differences in metabolic characteristics between the GR-5 and ALLO groups (Fig. 7b). The differential metabolites were screened based on VIP > 1 and p < 0.05. The volcano plot showed that 53 metabolites were downregulated in the GR-5 group compared with the NC group, which were lower than those in the ALLO and HUA groups (Figure S7). There were only 22 common differentially expressed metabolites in the three differential metabolic sets (Fig. 7c). Cluster analysis of these 22 common differential metabolites showed that the trends of metabolite expression changes between the GR-5 and NC groups were closer, and ALLO and HUA were close (Fig. 7d). Compared with the GR-5 and NC groups, inosine was upregulated and xanthine was downregulated in the ALLO and HUA groups. Hypoxanthine was only downregulated in the HUA group. MetaboAnalyst was used to perform metabolic pathway enrichment analysis on the 22 identified metabolites. The main metabolic pathways affected by GR-5 treatment included Nucleotide metabolism, Bile acid biosynthesis, Purine metabolism, Arachidonic acid metabolism, Tryptophan metabolism, and ABC transporters (Fig. 7e). In addition, SCFAs analysis found that GR-5 treatment significantly restored the decreased levels of acetic acid, propionic acid, butyric acid, and valeric acid in mouse feces caused by HUA (p < 0.05) (Fig. 7f). Spearman correlation analysis showed that hypoxanthine, xanthine, and SCFAs in the gut microbiota metabolites were positively correlated with Lactobacillus johnsonii and Lactobacillus intestinalis, and negatively correlated with Prevotella loescheii and Duncaniella_freteri (Fig. 7g, p < 0.05). This suggests that GR-5 improves gut metabolism and relieves HUA by regulating gut microbiota.

Fig. 7. Intestinal colonization of GR-5 modulated gut microbiota metabolism and helped improve the severity of HUA.

Untargeted metabolomics and GC-MS-SCFA analysis of gut microbiota metabolites (n = 3). a The partial least squares discriminant analysis (PLS-DA) score plot of metabolites in each group and (b) The orthogonal partial least squares discriminant analysis (OPLS-DA) score plot of GR-5 and ALLO treatment groups. c Venn diagram showing a total of 22 differentially expressed metabolites in the three differential metabolite groups. d Heat map of these 22 differentially expressed metabolites and (e) metabolic pathway enrichment analysis of them using MetaboAnalyst. f Acetic, propionic, butyric, and valeric acid levels in feces. g Spearman correlation analysis between gut metabolites and gut microbiota.

Discussion

Increasing the degradation of intestinal purine nucleosides can reduce the intestinal absorption of purines^28,29. Fifty-five lactic acid bacteria were isolated from Chinese sauerkraut, with an average guanosine degradation rate of 10.20% and an inosine degradation rate of 11.30%³⁰. Zhao et al.³¹ also isolated Lactobacillus fermentum NCU003012 from fermented vegetables, which showed degradation rates of 72.21% for guanosine and 67.59% for inosine. The degradation efficiency of these reported strains for purine nucleosides was lower than that of the Pediococcus acidilactici GR-5 screened in this study, suggesting that GR-5 may have a stronger effect on lowering serum UA. Under the stimulation of purine nucleosides, the expression of GR-5 purine salvage pathway genes xpt, hprT and apt was significantly upregulated, and the synthesis of GMP and AMP increased, indicating that GR-5 contain purine salvage pathway³². The synthesis of purine nucleotides may be beneficial to the growth of GR-5 and beneficial strains in the intestine, helping to reduce the purine content in the intestine³³. The PNP-DeoD catalyzes the phosphate cleavage of the nucleoside-glycosidic bond, producing the corresponding free purine, and its significant upregulation in this study may play a key role in the degradation of purine nucleosides³⁴.

The deoD gene identified in the purine metabolic pathway of the GR-5 genome is one of the key PNP genes in the purine nucleosides degradation or purine salvage pathway, its gene and amino acid sequence are also unique to Pediococcus acidilactici³⁵. The study by Pugmire and Ealick³⁶ showed that PNP divided into homotrimers targeting 6-oxopurine and homohexamers accepting both 6-oxopurine and 6-aminopurine. In previous studies on purine nucleoside biosynthesis, deoD is typically knocked out to block nucleoside degradation, significantly increasing the accumulation of purine nucleosides, suggesting that DeoD plays a crucial role in nucleoside metabolism³⁷. However, the purine nucleoside degradation rate of the purified DeoD in this study was only about 56% within 3 h, and the in vitro purine nucleoside degradation efficiency was relatively low¹⁷, which may be related to the stability of the enzyme activity after DeoD was separated from the cells³⁸. GR-5 and DE3 strain containing deoD maintain efficient purine nucleosides degradation within the short reaction time of 3 h, which indicates that directly using the GR-5 strain for application may have advantages compared with the purified DeoD enzyme. Using DeoD in GR-5 to efficiently degrade purine nucleosides in the gastrointestinal tract and reduce the absorption of purine nucleoside may be effective in controlling serum UA in the body¹⁶.

Probiotics have been shown to be an effective means of treating or preventing HUA. In this study, Pediococcus acidilactici GR-5 reduced the serum UA level of mice by 52.17%, which may be related to the higher degradation efficiency of DeoD in GR-5 for purine nucleosides¹⁶. According to the search, there are currently no reports on the use of Pediococcus acidilactici to treat or prevent HUA. The increase in serum T-AOC levels indicates that GR-5 treatment has increased the total antioxidant level of mice³⁹. Serum LPS is filtered by the glomeruli and can be completely reabsorbed by the renal tubules⁴⁰. The increase in serum LPS in mice in the HUA group indicates that the kidneys of mice may be damaged, and allopurinol treatment did not significantly alleviate this trend⁴¹. Purine is oxidized to UA and releases superoxide anions, which induce oxidative stress⁴². Oxidative stress is the main cause of renal and intestinal damage in HUA patients⁴³. GR-5 treatment effectively reversed the decrease in SOD and CAT levels in the kidneys of mice caused by HUA, and also reduced the level of lipid oxidation product MDA. This suggests that GR-5 treatment may alleviate oxidative stress and its related complications in HUA⁴⁴. Oxidative stress also promotes the release of NF-κB, which promotes the release of inflammatory factor IL-1β by activating the NLRP3 inflammasome signaling pathway, exacerbating renal tubular epithelial cell and tubular damage⁴⁵. Compared with HUA, GR-5 intervention reduced the expression of NF-κB, inflammasome-related proteins NLRP3, ASC, and Caspase-1 protein in mouse renal tissue, indicating that GR-5 can alleviate HUA-induced renal damage by inhibiting NLRP3-related inflammatory signaling pathways. This is similar to the mechanism of Lactobacillus acidophilus F02 and Levilactobacillus brevis PDD-5 that have been reported to alleviate renal inflammation^46,47. Interestingly, the present study also found that allopurinol had a lower inhibition of NLRP3 inflammasome signaling and alleviation of renal injury than GR-5, which is consistent with the findings of Ro et al.¹² who believed that this may be due to the increased burden on the kidneys caused by long-term use of allopurinol⁴⁸. The improvement of renal inflammation and colonic permeability by GR-5 treatment is essential for maintaining renal and colonic UA excretion function²¹.

The balance of UA secretion and reabsorption contributes to UA homeostasis, but most HUA patients are accompanied by UA secretion disorders⁴⁹. The drug benzbromarone reduces UA reabsorption by inhibiting GLUT9 and URAT1, but has significant side effects⁵⁰. The currently reported Lactobacillus rhamnosus Fmb14 and Lactobacillus plantarum X7023 have been shown to promote UA excretion by regulating the expression of UA transporters, the highest UA level in the urine of mice in the GR-5 group also verifies the promotion of UA excretion by GR-5^12,21. The purine metabolism disorder caused by a high-purine diet increases the activity of UA synthesis-related enzymes in the liver and promotes the occurrence of HUA⁵¹. Allopurinol reduces the production of UA and excessive ROS by reducing the activity of liver XOD⁵². GR-5 has an inhibitory effect on XOD activity, and its inhibitory effect on ADA activity is more significant than allopurinol (p < 0.01). The reduction of XOD activity may also be the reason why GR-5 reduces oxidative stress damage in the body⁴². These results indicate that GR-5 may reduce the serum UA level in HUA mice by reducing the synthesis of UA and promoting UA excretion.

In the gut microbiota of gout patients, the number of Prevotella and Bacteroides is higher than that of normal individuals⁵³. These bacteria may be involved in the increase and maintenance of UA levels in patients with gout or HUA. Prevotella is also associated with inflammatory diseases such as rheumatoid arthritis and colitis⁵⁴. The changing trend of Prevotella in the gut microbiota of HUA mice induced by purine by Lian et al.⁵⁵ is also consistent with the results of this study. GR-5 treatment significantly reduced the relative abundance of Prevotella in mice, which may play a positive role in lowering serum UA levels and alleviating inflammation in the body. Purine shock causes gut microbiota imbalance, affects its regulation of purine metabolism and inflammation⁵⁶. Reducing the purine content in the intestine is the key to restoring the function of gut microbiota⁵⁷. GR-5 can not only relieve purine pressure by degrading purine nucleosides itself. At the same time, under GR-5 intervention, strains of the genus Lactobacillus with increased abundance in the gut microbiota can alleviate HUA through hydrolase-mediated degradation of purine nucleosides⁵⁸. These results indicate that GR-5 may alleviate HUA symptoms by regulating purine metabolism of gut microbiota. Lactobacillus johnsonii and Seramator thermalis can degrade prebiotics to produce SCFAs^59,60. The increase in the abundance of Lactobacillus johnsonii and Seramator thermalis may promote the recovery of intestinal SCFAs levels, enhance intestinal energy homeostasis and reduce inflammation.

The gut microbiota affects the occurrence and treatment effect of diseases through metabolites⁶¹. Compared with allopurinol, GR-5 treatment restored the metabolism of gut microbiota in HUA mice to a certain extent. Purine nucleosides are more easily absorbed by the intestine during purine metabolism, causing an increase in serum UA^16,22. GR-5 restores gut microbiota purine metabolism by reducing inosine levels and promoting the excretion of xanthine and hypoxanthine with feces. Tryptophan, bile acid and SCFA metabolism are the three most studied gut microbiota metabolisms⁶². Microbiota tryptophan metabolism produces active substances such as indole and tryptamine to regulate host inflammation, immune response and neurological function⁶³. The accumulation of L-tryptophan in mouse feces may be due to the impaired tryptophan metabolism function of gut microbiota caused by HUA, and GR-5 intervention may promote the absorption and metabolism of L-tryptophan by the microbiota⁶⁴. Excessive primary and secondary bile acids in feces caused by abnormal bile acid metabolism are associated with cirrhosis, inflammatory bowel disease, and cancer⁶⁵. The feces of mice in the HUA and ALLO groups showed increased cholic acid and allocholic acid content, and the bile acid biosynthesis and secretion pathways were enriched. GR-5 treatment reversed the dysregulation of bile acid metabolism, which may play a role in improving liver damage and intestinal inflammation⁶⁶. SCFAs produced by microbiota metabolism can promote the regeneration and repair of intestinal epithelial tissue cells⁶⁷. Butyrate improves inflammation and promotes UA excretion by upregulating the expression of GPR43 and ABCG2⁶⁸. Acetate, as a ligand of XOD, can inhibit XOD activity and reduce UA production⁶⁹. GR-5 treatment may also play a positive role in regulating the production and excretion of UA and improving inflammation by restoring intestinal SCFAs levels.

In summary, this study explored the mechanism of Pediococcus acidilactici GR-5 in treating or preventing HUA from the aspects of purine nucleoside degradation, UA synthesis and secretion, inflammation regulation, and improvement of gut microbiota and metabolism (Fig. 8). Unlike allopurinol treatment, GR-5 not only reduces serum UA levels by regulating the expression levels of UA synthase and transporter proteins^5,6, but also degrades purine nucleosides to reduce intestinal absorption to reduce UA levels. It may also reduce tissue inflammatory damage caused by HUA by regulating the NLRP3 inflammatory pathway, improving gut microbiota and metabolism, and avoiding the side effects of drug treatment on the liver and kidneys⁷. Although the biosafety of Pediococcus acidilactici GR-5 has not been studied, GR-5 is derived from traditional fermented foods, and Pediococcus acidilactici is also a probiotic that can be used in food⁷⁰. Given that Pediococcus acidilactici GR-5 can effectively reduce serum UA in mice and alleviate HUA symptoms, we are optimistic that GR-5 will provide a healthy option for the prevention of HUA caused by diet. However, this study only explored the effect of strain GR-5 on preventing HUA induced by purine nucleosides, and its preventive and even therapeutic effects on HUA caused by other purine-induced, genetic or intrinsic factors remain to be further study. In addition, it is essential to further elucidate the mechanism by which GR-5 regulates serum UA levels by affecting the gut microbiota and metabolism, and conduct human trials to determine the clinical efficacy of strain GR-5.

Fig. 8. The mechanism underlying the alleviating of purine nucleoside-induced HUA in mice by GR-5.

GR-5 reduces serum UA levels by degrading purine nucleosides, reducing UA synthesis and promoting secretion. It also reduces tissue inflammatory damage and repairs the intestinal barrier by inhibiting the expression of NLRP3 inflammasome-related proteins, improving gut microbiota and metabolism. This figure was created using Biorender.com, with key elements from https://BioRender.com, 2D chemical structure elements were created using InDrawforWeb (http://indrawforweb.integle.com/).

Methods

Materials and chemicals

The strain used for the test was a probiotic strain of Pediococcus acidilactici GR-5 that was isolated from Jiangshui sample in the laboratory earlier and has the ability to degrade purine nucleosides. GR-5 can completely degrade inosine (100 mg/L), guanosine (50 mg/L) and adenosine (100 mg/L) within 12 h (Figure S1). Inosine, guanosine, and adenosine (purity ≥99%) were purchased from Sigma-Aldrich (St. Louis, MO). Enzymes for heterologous gene expression were purchased from Takarabiomed Biotechnology Co., Ltd. (Beijing, China). Formic acid and methanol (HPLC grade) were purchased from J&K Scientific Ltd (Beijing, China). Probiotic strains were grown in de Man, Rogosa Sharpe (MRS) medium (Solarbio, Beijing, China). Biochemical kits were purchased from Nanjing Bioengineering Institute Co., Ltd. (Nanjing, China), ELISA kits were purchased from Shanghai Kexing Trading Co., Ltd. (Shanghai, China), and antibodies used for western blot (WB) and immunofluorescence staining (IF) were purchased from ProteinTech Group, Inc., (Chicago, USA).

Genome and transcriptome sequencing

After GR-5 was cultured anaerobically in MRS medium at 37 °C for 12 h, GR-5 genomic DNA was extracted using the TIANamp bacterial genomic DNA extraction kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. Genome sequencing of this strain was performed using PacBio RS II combined with Illumina HiSeq 2500 (Majorbio, Shanghai, China). Genomic data analysis was performed using Majorbio Cloud (https://cloud.majorbio.com)^71,72. The coding sequences in the genome were functionally annotated using KEGG (http://www.genome.jp/kegg). For transcriptome sequencing, GR-5 cells were washed three times with PBS and inoculated into M9 medium containing 1 g/L glucose. The medium was mixed with 100 mg/L of inosine, guanosine, and adenosine as the sole nitrogen source. The CK group used NH₄Cl as the sole nitrogen source. After 6 h of anaerobic culture at 37 °C, the culture medium was centrifuged at 12,000 rpm, and the supernatant was used for high-performance liquid chromatography (HPLC) and Liquid Chromatograph-Mass Spectrometer (LC-MS) detection of purine nucleoside degradation. The collected GR-5 cells were washed three times and total RNA was extracted using the RNeasy Mini Kit (Qiagen, Germany). After the total RNA was qualified by NanoDrop 2000 spectrophotometer (Thermo, Wilmington, DE) and agarose gel electrophoresis, the library was constructed using the TruSeqTM Stranded Total RNA Library Prep Kit (Illumina, San Diego, CA). Sequencing (NovaSeqXPlus platform) and bioinformatics analysis were performed by Majorbio (Shanghai, China)⁷³. The expression levels of genes and transcripts were quantitatively analyzed using the expression quantification software RSEM, and the quantitative index was TPM.

HPLC analysis of purine content

The concentrations of nucleosides and purines were measured using HPLC³⁰. The samples were centrifuged at 10,000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm membrane before injecting 10 μL of the filtered sample into a C18 column (150 × 4.6 mm, 5 μm; Agilent Technologies, Santa Clara, USA). The mobile phase A consisted of 10 mM NaH₂PO₄, and mobile phase B was 100% methanol (pH 4.75) (A:B = 9:1, isocratic elution), with a flow rate of 1.0 mL/min. The column temperature was set at 25 °C, and detection was performed at 254 nm using a VWD detector.

Analysis of purine nucleoside degradation products by LC-MS

The degradation products of purine nucleosides by GR-5 were analyzed using an LTQ Orbitrap EDT mass spectrometer (MS; Thermo Fisher Scientific, Bremen, Germany)⁷⁴. 5 µl of the filtered sample were injected into a reverse-phase C18 column (100 × 2.1 mm, 3.0 μm, Hypersil GOLD-C18, Thermo Fisher Scientific). The mobile phase consisted of 0.1% formic acid (A) and 100% methanol (B), with a flow rate of 0.3 mL/min. The LC conditions were as follows: 95% A decreased to 50% A within 10 min, held for 2 min, then increased to 95% A within 1 min and held for 2 min. MS analysis was performed in negative ion mode, scanning the m/z range from 50 to 600.

Heterologous expression of purine degradation gene in GR-5

After functional annotation of the coding sequences in the genome through KEGG (http://www.genome.jp/kegg), it was found that the purine metabolism pathway of GR-5 contains the k03784 (deoD) gene, which can metabolize purine nucleosides into free purine, and transcriptome sequencing also found that the expression of this gene was significantly upregulated. Using the crystal structure of PNP-DeoD from Bacillus cereus as a template, the tertiary structure modeling of the DeoD protein domain in GR-5 was conducted on the SwissModel server at ExPaSy (https://swissmodel.expasy.org/)⁷⁵. Using primers: deoD-F: 5’-CGCGGATCCATGAGTACACACATTGCGGCACA-3’ and deoD-R: 5’-CCCAAGCTTTTAGCTAATTGCCGTTTCTAACGCAACT-3’, the deoD gene (705 bp, gene sequence provided in supplementary material) was cloned from GR-5 genomic DNA and assembled into plasmid pET28a through BamH I and Hind III restriction sites. After verification in E. coli DH5α, it was introduced into E. coli Rosetta (DE3) cells. DeoD protein expression was induced by isopropyl β-D-thiogalactopyranoside (IPTG) at a final concentration of 100 mM. The expressed His-tagged DeoD protein was eluted using a Ni-NTA-Sepharose column and an imidazole gradient according to the manufacturer’s instructions. The degradation ability of the purified DeoD protein, the Rosetta strain containing the pET28a empty vector (R-P28a), and the Rosetta strain containing the deoD gene after IPTG induction (R-P28a-deoD) in M9 medium with inosine, guanosine, or adenosine as the sole nitrogen source was evaluated under static culture at 37 °C for 3 h.

Animal experiment protocol

The experiment was carried out in strict accordance with the Lanzhou University (Gansu, China) Laboratory Animal Management Standards and approved by the Lanzhou University Ethics Committee (approval number: EAF2023022). Four-week-old male Kunming mice (18–22 g) were purchased from the Lanzhou University Experimental Animal Center. The mice were kept in an environment with a temperature of 25 °C, a humidity of 50%, and 12 h of light per day (06:00-18:00). They were fed with standard feed and had free access to water. After 7 days of acclimatization, 40 mice were randomly divided into 4 groups, 10 mice in each group and 5 mice in each cage: normal control group (NC), HUA group (HUA), GR-5 treatment group (GR-5) and allopurinol treatment group (ALLO) (Fig. 3a). As previously described¹⁶, NC group was orally gavaged with 200 μL 5 g/L sodium carboxymethylcellulose (CMC-Na) daily, while the mice in the other groups were gavaged with potassium oxonate (PO, 200 mg/kg) + mixed purine nucleosides (inosine: guanosine: adenosine = 1:1:1, 300 mg/kg) suspended in 5 g/L CMC-Na solution daily to establish the HUA model. The selection of a mixture of inosine, guanosine and adenosine to establish the HUA model is to better simulate the complexity of purines in the diet and the fact that purine nucleosides in purine metabolism are more easily absorbed by the intestine^18,76. Treatment was carried out at the same time as modeling. The GR-5 group was orally gavaged with 200 μL ( ~ 1 × 10⁹ CFU) of GR-5 skimmed milk powder suspension daily²⁷. The ALLO group was orally gavaged with 200 μL (5 mg/kg) of allopurinol skimmed milk powder suspension daily¹⁶. The other groups were orally gavaged with 200 μL of skimmed milk powder daily.

The treatment lasted for 3 weeks. After the treatment, the mice were moved to a clean empty cage to collect feces and urine samples. After fasting for 12 h, mice were deeply anesthetized by 3% isoflurane inhalation for 2–5 min to reduce invasive pain. Blood samples were collected from the orbital sinus, and serum was obtained by centrifugation at 3000 × g for 10 min at 4 °C and stored at −80 °C. Mice were then euthanized by cervical dislocation, and tissue samples were collected, weighed, and quickly frozen in liquid nitrogen until analysis. Some tissues were fixed with 4% paraformaldehyde for hematoxylin and eosin (H&E) staining and IF analysis.

Biomarker analysis

Interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), nuclear factor-κB (NF-κB), ATP-binding cassette superfamily G member 2 (ABCG2), organic anion transporters 1 (OAT1), glucose transporter 9 (GLUT9), urate reabsorption transporter 1 (URAT1), and sodium-dependent phosphate transporter 1 (NPT1) were measured in serum or tissue homogenate samples using ELISA kits (Shanghai Kexing Trading Co., Ltd., China) according to the manufacturer’s protocol. UA, creatinine (CRE), blood urea nitrogen (BUN), and alanine aminotransferase (ALT) in serum and urine were measured using an automatic biochemical analyzer (Shenzhen Rayto Biotechnology Co., Ltd., China) and matching kits. Lipase (LPS), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), xanthine oxidase (XOD), and adenosine deaminase (ADA) in serum or tissue homogenate were measured using biochemical kits from Nanjing Bioengineering Institute Co., Ltd. (Nanjing, China). At least five samples were selected for independent measurement in each group. WB was performed according to the method described by Zhao et al.⁷⁷, and the antibodies for nucleotide-binding oligomerization domain-like receptor family containing pyrin domain 3 (NLRP3), apoptosis-associated spotted proteins (ASC), cysteinyl aspartate specific proteinase 1 (Caspase-1), ABCG2, OAT1, GLUT9, and URAT1 used in WB were purchased from ProteinTech Group, Inc., (Chicago, USA). The images were taken using a chemiluminescence imaging system (Viber fx6, France), and the target proteins were compared with the β-actin band for quantitative analysis using Image J.

Histopathological examination

Kidney, liver, and small intestine samples fixed with 4% paraformaldehyde were sent to Sevier Biotechnology Co., Ltd. (Hubei, China) for HE staining and histopathological examination using a microscope (Olympus IX71, Japan). For IF staining, colon paraffin sections were washed with PBS to remove the fixative and then immersed in 3% BSA blocking solution for 1 h at room temperature. The samples were mixed with primary antibody zonula occludens-1 (ZO-1) and occluding (Ocln) dilution and incubated overnight at 4 °C²¹. After washing with PBS, the secondary antibody goat anti-rabbit IgG was added and incubated at room temperature for 1 h. After washing with PBS again, DAPI was used to stain the cell nucleus. After washing, the samples were blocked with anti-fluorescence quenching blocking agent. The samples were observed using a laser scanning confocal microscope (Leica Stellaris 5, Germany). At least 3 samples were selected for each group, and semi-quantitative analysis was performed using Image J software.

16 S rRNA gene sequencing and GR-5 colonization analysis

Colonic mucosal contents and feces were collected, and the sample microbial DNA was extracted using the Omega Stool DNA Kit (D4015-02, Omega, USA) according to the instructions. After being analyzed by Nanodrop 2000 (Thermo Fisher, USA) and 1% agarose gel electrophoresis, the DNA was sent to Majorbio BPT Co., Ltd (Shanghai, China) for full-length 16S rRNA sequencing using the Pacbio Sequel platform. The sequencing data were analyzed on the Majorbio Cloud (https://cloud.majorbio.com)⁷⁸. To detect the colonization of GR-5, PCR primer sequences were designed based on the deoD gene (K03784) identified on the GR-5 chromosome: deoD-F and deoD-R, and 1% agarose gel electrophoresis was used to detect the presence of GR-5 in the PCR products of fecal DNA. Quantitative real-time PCR (qPCR) primers were designed based on the deoD gene: F: 5’-ATGAGTACACACATTGCGGCAC-3’; R: 5’- GGAGATTGATGGAATGCCCATGC-3’, and the partial fragment of GR-5 deoD gene (210 bp) copy number was measured relative to the total bacterial 16S rRNA copy number in the samples to determine the abundance of GR-5 colonization in the colon after GR-5 treatment²⁷. The universal primers 338 F and 518 R were used to measure the total bacterial 16S rRNA copy number.

Untargeted fecal metabolomics analysis

0.2 g of colonic contents were mixed with methanol/water (4:1, v/v) containing 0.02 mg/mL L-2-chlorophenylalanine, ground in a frozen tissue grinder for 5 min, and then extracted by cryo-ultrasound for 30 min⁷⁹. The samples were centrifuged at 4 °C and 13,000 g for 10 min. The supernatant was filtered through a 0.22 μm filter membrane, and metabolites in the supernatant were detected by ultra-high performance liquid chromatography-tandem fourier transform mass spectrometry (UHPLC-Q Exactive, Thermo Fisher Scientific, USA). All samples of equal volume were mixed to prepare quality control samples (QC). As previously described in ref. ⁸⁰, 2 μL of the sample was injected into an HSS T3 column (100 × 2.1 mm, 1.8 μm; Waters, USA). The mobile phase A was 0.1% formic acid in water, and the mobile phase B was acetonitrile (containing 0.1% formic acid) at a flow rate of 0.4 mL/min. Gradient elution was as follows: 0 min, 5% B; 2 min, 25% B; 9 min, 100% B; 13 min, 100% B; 16 min, 5% B. MS analysis was performed in positive and negative ion scanning mode with a scan range of m/z 70-1050. LC-MS raw data were processed by Progenesis QI (Waters Corporation, Milford, USA). Metabolite mass spectral information was matched with HMDB (http://www.hmdb.ca/) and Metlin (https://metlin.scripps.edu/), and data analysis was performed by the Majorbio Cloud Platform (https://cloud.majorbio.com)⁸¹. partial least squares discriminant analysis (PLS-DA) and orthogonal least squares discriminant analysis (OPLS-DA) were performed using the R package ropls (Version 1.6.2). Differential metabolites were determined by variable weight value (VIP) and student’s t test p-value. Metabolic pathway annotation of differential metabolites was performed using the KEGG database (https://www.kegg.jp/kegg/pathway.html), and KEGG pathway enrichment analysis was performed using MetaboAnalyst 5.0 (http://www.metaboanalyst.ca)⁸².

Gas Chromatography Mass Spectrometry (GC-MS) analysis of short-chain fatty acids (SCFAs) in feces

Samples were homogenated for 1 min with 500 μL of water and 100 mg of glass beads, and then centrifuged at 4 °C for 10 min at 12,000 rpm. 200 μL supernatant was extracted with 100 μL of 15% phosphoric acid and 20 μL of 375 μg/mL 4-methylvaleric acid solution as IS and 280 μL ether⁸³. Subsequently, the samples were centrifuged at 4 °C for 10 min at 12,000 rpm, and the supernatant was transferred into the vial prior to GC-MS analysis. The GC analysis was performed on trace 1310 gas chromatograph (Thermo Fisher Scientific, USA). The GC was fitted with a capillary column Agilent HP-INNOWAX (30 m × 0.25 mm ID × 0.25 μm) and helium was used as the carrier gas at 1 mL/min. Injection was made in split mode at 10:1 with an injection volume of 1 μL and an injector temperature of 250 °C. The temperature of the ion source and MS transfer line were 300 °C and 250 °C, respectively. The column temperature was programmed to increase from an initial temperature of 90 °C, followed by an increase to 120 °C at 10 °C /min, and to 150 °C at 5 °C /min, and finally to 250 °C at 25 °C /min which was maintained for 2 min. Mass spectrometric detection of metabolites was performed on ISQ LT (Thermo Fisher Scientific, USA) with electron impact ionization mode. Single ion monitoring (SIM) mode was used with the electron energy of 70 eV.

Statistical analysis

All measurements were performed at least three times independently, and the data are presented as mean ± SD (standard deviation). One-way analysis of variance (ANOVA) was performed using GraphPad Prism software (version 9.5.1) to analyze the significant differences between groups. p-value < 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Author contributions

Jing Ji: Writing-review & editing, Writing-original draft, Validation, Methodology, Formal analysis. Zi-Yi An: Writing-Review & Editing, Data curation, Conceptualization. Aman Khan: Conceptualization, Visualization. Liang Peng: Validation, Software. Sourabh Kulshreshtha: Writing-Review & Editing, Validation. El-Sayed Salama: Writing-review & editing. Hui Yun: Investigation, Supervision. Pu Liu: Investigation. Wei-Lin Jin: Formal analysis, Conceptualization. Xiangkai Li: Conceptualization, Project administration.

Data availability

The strain Pediococcus acidilactici GR-5 was deposited in the China General Microbiological Culture Collection Center (Beijing, China) with the deposit number CGMCC NO. 25729. The GR-5 sequencing data obtained in this study have been registered in NCBI GenBank (https://www.ncbi.nlm.nih.gov/), the 16S rRNA accession number is OP829805.1, and the GR-5 chromosome genome accession number is CP177348.1.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41538-025-00556-y.