Akk11 was supplied by Wecare Probiotics (Suzhou) Co., Ltd. and was isolated from infant feces. Akk11 was cultured anaerobically at 37 °C for approximately 48 h in a mucin-based basal medium, as described previously (Derrien et al., 2004). The cells were collected via centrifugation at 8,000 × g and 4 °C and then washed twice with sterile, anoxic phosphate-buffered saline (PBS) before being resuspended in the same solution. Furthermore, a pasteurized bacterial solution was created by applying heat treatment at 70 °C for 30 min in a digital water bath, a method typically used to eradicate microorganisms while preserving the bacteria’s potential immunomodulatory effects (Plovier et al., 2016).

A total of 40 male C57BL/6 mice, aged between 6 and 7 weeks and sourced from the Experimental Animal Center at Huazhong Agricultural University, were obtained. These specific pathogen-free mice were kept in a controlled environment with stable temperature and humidity, with a 12-h light/dark cycle. They were housed in groups of five per cage with unrestricted access to food and water. Prior to the experiment, the mice were maintained on a standard diet for 1 week and then randomly assigned to four groups using stratified randomization based on their body weight. This procedure ensured that the mean starting weight was identical across groups so that subsequent changes in body mass could be attributed solely to diet and/or Akk11 treatment rather than to pre-existing inter-group disparities. The standard diet was categorized into a control group (ND) and a high-fat diet consisting of 60% fat and 20% carbohydrates (D12492, kcal/100 g). The high-fat diet cohort was divided into three categories: the model group (HFD), a group receiving 2 × 10⁹ AFU/day of live Akk11 bacteria (HFD + Akk), and a group administered 2 × 10⁹ AFU/day of pasteurized Akk11 bacteria (HFD + PAkk). Both the model and control groups were treated with the same volume of sterile PBS via gavage.

The study spanned a duration of 5 weeks, during which the weight and weekly food consumption were recorded, alongside the collection of fresh feces from the mice. All 40 male mice remained healthy throughout the intervention; no unexpected deaths or removals occurred. Upon completion, all mice were euthanized via cervical dislocation, and samples of blood, white adipose tissue, the liver, the ileum, and the cecum were obtained. Lee’s index is defined as body weight (g) divided by the square of body length (cm²). The Institutional Animal Care and Use Committee of Huazhong Agricultural University approved all experimental procedures (approval No. HZAUMO-2024-0111).

Triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were quantified using the triglyceride assay kit (A110-1-1), low-density lipoprotein cholesterol assay kit (A113-1-1), and high-density lipoprotein cholesterol assay kit (A112-1-1) from Nanjing Jiancheng Bioengineering Institute. Both tests were performed in accordance with the instructions provided by the manufacturers.

Following a 24-h fixation in 4% paraformaldehyde, samples of colon and white adipose tissue were embedded in paraffin and cut into standard thickness sections. These sections underwent a series of dewaxing and hydration processes. To visualize cellular structures, the sections were stained with hematoxylin and eosin (H&E) (Cardiff et al., 2014). Pathological alterations were assessed using a light microscope (80i, Hitachi, Japan) at 100 × magnification (ND, n = 6; HFD, n = 9). Additionally, colon cells were stained with alizarin blue-periodic acid–Schiff (AB-PAS), and immunohistochemistry was used to identify the presence of tight junction proteins ZO-1, Muc-2, occludin, and claudin-1 in colon tissue at 200 × magnification (n = 5). The dimensions of white adipocytes, the relative proportion of goblet cells for AB-PAS staining, and the areas positive for immunohistochemistry were evaluated using Image Pro Plus 6.0 software for image analysis.

A total of 100 mg/mL mixed standard stock solutions of 5 SCFAs (acetic acid, propionic acid, butyric acid, isobutyric acid, and valeric acid) and 100 mg/mL caproic acid stock solution were prepared in water and ether, respectively. Five SCFAs and a caproic acid working solution series were both prepared by appropriate dilutions of a standard stock solution. A total of 375 μg/mL of internal standard (IS) solution containing 4-methylvaleric acid was similarly prepared with ether. A ten-point calibration curve was established by adding 220 μL of the working solutions, which contained 200 μL of the six-acid working solution series, 20 μL of the caproic acid working solution series, 100 μL of 15% phosphoric acid, 20 μL of 375 μg/mL IS solution, and 260 μL of ether, with a calibration range from 0.02 to 500 μg/mL (0.02, 0.1, 0.5, 2, 10, 25, 50, 100, 250, and 500 μg/mL). Stock solutions were stored at −20 °C before use, and working solutions were prepared before use.

The clear liquid obtained from the homogenized cecal contents was separated by centrifugation at 12,000 g for 10 min to eliminate any particulate matter, and this supernatant was utilized for analyzing short-chain fatty acids (SCFAs). The analysis was conducted using a Thermo Trace 1,310 gas chromatography system (Thermo Fisher Scientific, United States) fitted with an Agilent HP-INNOVAX capillary column (30 m × 0.25 mm inner diameter × 0.25 μm film thickness). The chromatographic settings included a split injection with a volume of 1 μL and a split ratio of 10:1. The injection port temperature was maintained at 250 °C, while the ion source temperature was set at 300 °C. The transmission line temperature was also maintained at 250 °C. The temperature program commenced at 90 °C, increased to 120 °C at a rate of 10 °C/min, then increased to 150 °C at a rate of 5 °C/min, and finally reached 250 °C at a rate of 25 °C/min, where it was held for 2 min. Helium served as the carrier gas with a flow rate of 1.0 mL/min. Detection was carried out using a Thermo ISQ LT mass spectrometer (Thermo Fisher Scientific, United States) with an electron impact ionization (EI) source and selected ion monitoring (SIM) mode, operating at an electron energy of 70 eV.

Cecal samples were promptly frozen in sterile cryovials and kept at −80 °C until the DNA extraction process. Genomic DNA was isolated from cecal contents with a kit supplied by Tiangen Biotech (Beijing) Co., Ltd., Beijing, China. DNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, United States). Following the quantification of the extracted DNA, a polymerase chain reaction (PCR) was performed targeting the V4 region of the bacterial 16S rRNA gene with the following primers: forward 5′- GTGCCAGCMGCCGCGGTAA −3′ and reverse 5′- GGACTACHVGGGTWTCTAAT −3′. The resulting PCR products were mixed and purified, followed by procedures including end repair, A-tailing, addition of sequencing linkers, and further purification to finalize the library preparation. The prepared library was quantified using the Qubit and Q-PCR methods. After confirming the library’s quality, sequencing was carried out on the NovaSeq 6,000 platform with a PE250 configuration. Paired-end reads were first merged using FLASH (v1.2.11) and subsequently quality-assessed and filtered with fastp (v0.23.1). Sequences were grouped into amplicon sequence variants (ASV) based on 99% similarity and annotated with the Silva 138.1 database. Alpha diversity metrics of the microbial community were computed using QIIME 2, while beta diversity was analyzed through weighted UniFrac distances and visualized using principal coordinate analysis (PCoA) and heatmaps. Statistical comparisons of microbial community structures between groups were performed using permutational multivariate analysis of variance (PERMANOVA) (999 permutations) and analysis of similarities (ANOSIM). Taxon-specific biomarkers were determined using linear discriminant analysis effect size (LEfSe). Additionally, PICRUSt2 was used to estimate microbial functional abundance.

Data are presented as mean values with standard error of the mean (SEM). Comparisons among mouse groups were conducted using a one-way ANOVA, accompanied by Tukey’s post-hoc analysis or the unpaired t-test. Statistical analyses were performed using GraphPad Prism version 8.00. A p-value of less than 0.05 was deemed statistically significant, indicated by the following symbols: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

All four experimental groups (ND, HFD, HFD + Akk, and HFD + PAkk) were included in every statistical model. However, because the normal diet (ND) group differed from the two Akk-supplemented groups in both diet background and treatment, we restricted the post-hoc pairwise comparisons to the three high-fat-fed cohorts (HFD vs. HFD + live Akk vs. HFD + pasteurized Akk) and separately compared ND to HFD.

To study the effects of the Akk11 strain on high-fat diet mice, we conducted a 5-week experiment using both live and pasteurized Akk11 for intervention (Figure 1A). It was observed that the addition of Akk11 did not significantly impact weight gain in mice fed a high-fat diet (Figure 1B). Akk11 supplementation did not alter daily food intake compared to the HFD group (Supplementary Table 1). However, statistical analysis revealed that mice supplemented with Akk11 exhibited significantly greater body length than both the high-fat diet and the normal diet groups (Figure 1C). Furthermore, Lee’s index, which provides an objective measure of obesity, showed that all Akk11 supplementations effectively reduced obesity levels, with pasteurized Akk11 demonstrating a stronger effect than its live counterpart (Figure 1D). While a high-fat diet led to elevated triglyceride (TG) levels, neither pasteurized nor live Akk11 supplementation resulted in a significant decrease in TG levels (Figure 1E). Additionally, when compared to the HFD group, both live and pasteurized Akk11 treatments significantly lowered low-density lipoprotein cholesterol (LDL-C) levels (Figure 1F). The high-density lipoprotein cholesterol (HDL-C) levels in the HFD + PAkk group were significantly higher than those in the HFD group (Figure 1G).

White adipose tissue is essential for maintaining energy balance by storing surplus energy as triglycerides and releasing it when necessary. When white fat cells malfunction, it can lead to metabolic issues such as obesity and diabetes. In our study, we assessed the size of white adipocytes in mice and conducted statistical analyses to determine the effect of Akk11 on adipose tissue. The findings indicated that mice fed a high-fat diet exhibited a notably larger area of white adipocytes than those on a normal diet (Figures 2A,B). However, treatment with Akk11 significantly decreased the white adipocyte area in the high-fat diet group (Figures 2A–E). Following administration of live bacteria, the white adipose tissue area decreased from 4,659 to 2,483 μm², while in the PAkk group, it decreased from 4,659 to 3,559 μm² compared to the high-fat diet group. These findings suggested that both live and pasteurized Akk11 treatments effectively diminish the area of white adipose tissue, thus helping to mitigate obesity.

To assess the impact of live and pasteurized Akk11 treatments on intestinal health, we first measured the number of goblet cells in these groups. AB-PAS staining of the colon revealed that a high-fat diet led to a reduction in the number of goblet cells in the colonic mucosa, whereas live Akk11 showed significant improvement, with pasteurized Akk11 demonstrating even greater effectiveness (Figure 3A).

A high-fat diet usually leads to the disruption of the intestinal barrier. Immunohistochemical analysis revealed that such a diet markedly reduced the levels of tight junction proteins Muc-2 and occludin but did not significantly affect the expression of claudin-1 (Figures 3C–E). After supplementing with pasteurized Akk11, the expression levels of Muc-2 and occludin returned to normal in the colon (Figures 3C,D), and that of ZO-1 also showed a similar trend, although there was no statistical difference (Figure 3B). These findings suggested that pasteurized Akk11 plays a significant role in enhancing intestinal barrier function and helps preserve the integrity of the intestinal mucosa to a certain degree.

The composition of the microbial community in the cecal contents of mice was notably influenced by dietary changes and treatments with Akk11. The evaluation of the alpha diversity index revealed that a high-fat diet led to a marked decrease in gut microbiota diversity. Although pasteurized Akk11 significantly enhanced the Chao1 index, indicating an increase in microbial richness, this value remained lower than that of the normal diet group (Figure 4A). The structure of the fecal microbiome was examined using PCoA and Jaccard distance analysis. Notably, pasteurized Akk11 resulted in alterations in the beta diversity of the intestinal microbiota under a high-fat diet. Additionally, Bray-Curtis distance analysis of beta diversity showed that the administration of pasteurized Akk11 induced changes in gut microbial beta diversity (ANOSIM, R = 0.248, p < 0.05), while no significant differences were observed with live Akk11 (Figure 4B).

Notably, analyses of gut microbial taxonomy revealed a significant increase in the relative abundance of potentially beneficial bacterial groups, such as Akkermansia and Lachnospiraceae_NK4A136_group, following treatment with pasteurized Akk11 (Figures 4C,D). To explore the unique bacterial community structures across the different treatment groups, linear discriminant analysis effect size (LEfSe) using amplicon sequence variant (ASV) was used to identify the taxa with the greatest difference. In particular, the pasteurized Akk11-treated group showed an increase in the levels of key bacteria, including Akkermansia, Lachnospiraceae, and Alistipes finegoldii, compared to the HFD group. These findings suggest that Akkermansia, Lachnospiraceae, and Alistipes_finegoldii serve as significant biomarkers associated with the supplementation of pasteurized Akk11 (Figure 4E). Overall, these data indicate that pasteurized Akk11 is associated with an increased proportion of potentially health-promoting taxa.

Short-chain fatty acids (SCFAs) are important metabolites that influence gut microbiota. Our findings demonstrated that acetic acid levels were higher in the HFD + Akk group than in the HFD + PAkk group (Figure 5A). The propionic acid, isobutyric acid, and valeric acid levels were increased in the HFD + Akk group when compared to the HFD group (Figures 5B,D,E). However, butyric acid levels increased with the treatment of pasteurized Akk11, and the HFD + PAkk group exhibited significantly elevated butyric acid levels compared to the HFD + Akk group (Figure 5C). Both live and pasteurized Akk11 supplementation led to significant increases in isobutyric acid and valeric acid levels, although the live Akk11 group demonstrated more pronounced effects than the pasteurized variant (Figures 5D,E). These findings suggest that live Akk11 may generate specific SCFAs during its growth in the gut, while pasteurized Akk11 appears to enhance the intestinal environment by influencing bacteria that produce these fatty acids.

A correlation heatmap analysis using Spearman’s correlation was performed to examine the correlations among physiological parameters, intestinal SCFAs, and microbial genera in mice fed a high-fat diet. The genera Muribaculum, UCG-005, Siraeum_group, Parasutterella, and Prevotella ceae UCG-001 exhibited negative correlations with physiological parameters associated with decreased white adipocyte size, lower serum triglyceride levels, and preservation of the intestinal barrier to mitigate endotoxin effects (Figure 6A). Additionally, Akkermansia displayed positive correlations between body length and the integrity of the intestinal barrier (Figure 6A). The presence of Erysipelatoclostridium was positively associated with the concentrations of acetic acid, propionic acid, and isovaleric acid. Intestinimonas showed a positive relationship between propionic acids and butyric acids, while Bilophila was positively associated with butyric acid levels (Figure 6A). Functional predictions of gut microbiota through PICRUSt2 indicated that the high-fat diet group had a generally elevated abundance of functional genes associated with transport compared to those on a normal diet. Notably, pasteurized Akk11 treatment led to an even more pronounced increase in these functional genes (Figure 6B).