Fecal samples were collected from a group of 85 healthy cats aged 2–4 years, fully immuned to deworming, disease-free and in good health. The weight of the cats ranged from 2.5 to 4 kg. Samples were collected after the cat had defecated, then transferred to a 50 ml sterile centrifuge tube and subjected to a rapid freezing protocol using liquid nitrogen, subsequently stored at −80°C. After thawing the frozen fecal samples at room temperature for 15 min, weigh 1 g of the samples and put them in 9 ml of MRS liquid culture medium (1% peptone, 0.5% beef powder, 2% glucose, 0.4% yeast barm) (Solarbio, Beijing, China) at 37°C for 24 h. Then, pick out the liquid culture medium with sterile inoculation ring and coated it on MRS solid culture medium (1% agar, w/v) containing 2% CaCO₃, and let it stand at 37°C for 24 h. After that, the single colony with obvious dissolution circle greater than 1 mm was selected to be enriched and cultured in MRS liquid medium, and then stood at 37°C for 24 h. All growth environments were micro-aerobically and we provided micro-aerobic environments through standing cultures. After that, single colonies were selected again and cultured in MRS liquid. The purification was repeated twice. After that, 80% glycerol was used to preserve bacteria and frozen at −80°C.

Bacteria was inoculated at 1% inoculum into MRS liquid culture medium at 37°C for 24 h, 1% inoculum from which was inoculated into MRS liquid culture medium with cholesterol (0.1 g/L), and after 24 h, 0.2 ml of bacteria liquid was taken into 10 ml centrifuge tube. A total of 4.8 ml absolute ethanol was then added into the centrifuge tube, still for 15 min, and then centrifuged at 6,659 × g for 10 min. Aspirate 2 ml of supernatant into a test tube, then 2 ml of ferric alum chromogenic agent solution was added. After cooling to room temperature, the OD of liquid was measured at 560 nm with an enzyme-labeled instrument within 15–90 min. Using sterile cholesterol standard solutions with cholesterol concentrations of 0.02, 0.04, 0.06, 0.08, and 0.10 mg/ml, respectively, the standard curve was drawn with OD 560 nm value and standard concentration. The cholesterol concentration in the experimental group was recorded as Cb, and the cholesterol concentration in the control group was recorded as Ca.

The lactic acid bacteria strains in the logarithmic growth period were diluted with physiological saline to a bacterial solution with a concentration of 10⁸ CFU/ml, and the bacteriostatic effect of the lactic acid bacteria strains on pathogenic bacteria was detected by agar diffusion oxford cup. E. coli ATCC 14028, S. aureus ATCC 43300, Salmonella CVCC 3377 were selected as pathogenic bacteria. All pathogens were grown in NA medium (1% peptone, 0.3% beef powder, 0.5% NaCl) (Solarbio, Beijing, China) and incubated at 37°C for 24 h. All pathogens were grown in NA medium (1% agar, w/v). Then, the bacterial solution of each lactic acid bacteria strains (10⁸ CFU/ml) were used to fill a 6 mm Oxford cup previously punched on LB agar plates. All plates were incubated at 37°C for 24 h, and the radius of the antibacterial circle around the wells were measured. According to the size of the inhibition zone, it can be divided into three types: no inhibition (r = 0 mm), inhibition (r = 0–10 mm), and obvious inhibition (r > 10 mm), and lactic acid bacteria with inhibition zone radius >15 mm were screened, and sterile MRS liquid culture medium was used as blank control.

The genomic DNA of lactic acid bacteria was extracted by bacterial genome extraction kit (Solarbio, Beijing, China), and the target fragment was amplified by 16S rDNA universal primers 27F and 1492R. The purified and recovered PCR products were sent to Shenggong Bioengineering Co., Ltd. for sequencing. The sequencing results were compared with BLAST homology on NCBI website. The nucleic acid sequence of the lactic acid bacteria model strain with the highest homology was selected for homology analysis, and constructed phylogenetic tree by MEGA software. Combined with biological characteristics and alignment of the 16S rRNA gene sequence (SEQ ID NO: 1), the strain was determined.

Single colony of each isolate was selected and mixed it evenly in sterile PBS solution to make a solution with Maxwell’s ratio turbidity of 2. A total of 400 μl of liquid was taken into API liquid culture medium (Meria French), mixed evenly, and then 200 μl of liquid was inoculated onto glucose fermentation test strips, such as glycerol and erythritol, and incubate at 37°C for culture, observed and recorded changes in test strips.

Activated bacterial liquid was inoculated into MRS liquid culture medium with 1% inoculation amount, and cultured at 37°C for 48 h. The OD 600 nm value and pH value of bacterial liquid were determined every 4 h, and the growth curve and acid production curve of the strain were drawn.

MRS liquid medium was adjusted to pH 2, 3, and 4 by HCL (98%), respectively, and each strain was inoculated in the medium according to the inoculation amount of 0.01%. After 2 and 4 h of culture, 100 μl above bacterial liquid was inoculated at solid medium and cultured at 37°C for 24 h, and then the number of live bacteria was counted to determine the acid resistance of each strains (Yang E. et al., 2018). Similarly, the above culture medium were inoculated with bile salt content of 0%, 0.15%, 0.3%, and 0.6%, and the number of living bacteria was counted after 24 h at 37°C to determine the bile salt tolerance of lactic acid bacteria, and the strains with tolerance to bile salt concentration greater than 0.3% were screened out.

Using disk diffusion test (K-B method), depending on the size of the bacteriostatic ring, it was determined whether different isolates were sensitive to common antibacterial drugs such as amoxicillin clavulanate potassium, ceftiofur, cefquinome, and doxycycline. Under aseptic conditions, single colonies were picked from sterilized inoculation rings and inoculated in MRS medium for 24 h at 37°C. Each plate was coated with 100 μl of bacterial liquid, which was dripped on the surface of MRS solid culture medium. After uniform coating, the drug-containing paper was removed by sterile tweezers and stuck to the surface of the culture medium. After standing for a period of time, it was cultured in a constant temperature box at 37°C for 24 h (Kang et al., 2020). The bacteriostatic results were observed and the radius of bacteriostatic circle of each tablet group was measured. At the same time, according to the Experimental Specification for Antimicrobial Drug Sensitivity, the drug sensitivity effect was judged, and the results were divided into three types: sensitive (r > 15 mm), intermediate (r = 15–10 mm), and drug resistance (r < 10 mm), and antibiotic sensitivity characteristics of various lactic acid bacteria were detected.

The adhesion potential of lactic acid bacteria was investigated using epithelial cell lines. Cells were grown in an environment supplemented with 10% fetal bovine serum at 37°C and 5% carbon dioxide, and monolayer cells were prepared in a six-hole tissue culture plate. Epithelial cell lines were inoculated at a concentration of 5 × 10⁸ and incubated in a 5% carbon dioxide incubator at 37°C. Cells grown in an environment of 5.0 × 10⁸ lactic acid bacteria were added and washed twice with PBS. The number of lactic acid bacteria adhered to 20 cells in the field of random oil mirror was observed by microscope, and the adhesion capacity of lactic acid bacteria was calculated by number, which was divided into non-adhesion (less than one bacteria adhered to 20 cells), adhesion (1–8 bacteria adhered to 20 cells) and strong adhesion (more than 8 bacteria adhered to 20 cells) (Shukla and Goyal, 2014).

All animal experiments were approved by the Laboratory Animal Ethical Committee and its Inspection of the Institute of Feed Research of CAAS (IFR-CAAS-20230301). Twenty healthy 4 week old male BAL b/c mice raised at 22 ± 1°C and 55 ± 10% humidity on a 12 h light/dark cycle on the pilot base of the Chinese Academy of Agricultural Sciences. After 1 week of adaptation, they were randomly divided into five groups, with four mice in each group. They were L-27-2 group (supplemented with Lactobacillus plantarum L-27-2 in HFD), L-14-1 group (supplemented with Pediococcus sp. L-14-1 in HFD), F203 group (supplemented with Enterococcus faecium F203 in HFD), M group (HFD model group), and CON group (normal diet group). The experiment was conducted for 28 days, with four mice per cage. All mice were fed with an unrestricted amount of food and water daily, and no additional antibiotics or probiotics were given during the experiment. Group L-27-2, group L-14-1, group F203, and group M were fed a HFD (60% fat, 20% protein, and 20% carbohydrate), while group CON was fed a normal diet (ND) (5% fat, 18% protein, and 77% carbohydrate). Mice were given the corresponding lactic acid bacteria freeze-dried powder at a dose of 10⁹ CFU/kg daily, diluted with sterile PBS, gastric administration every morning and M group and CON group were given the same amount of sterile PBS for 28 days. Total intake and weight were recorded every 14 days. The mice were euthanized by neck removal. Blood samples were collected from the eyeball and stored at −20°C. Colonic contents were rapidly frozen in liquid nitrogen, and then stored to −80°C. In addition, fecal samples and other issues were stored at −20°C. Fat samples were soaked in tissue fixative and stored at 4°C to observe the effects of lactic acid bacteria on body weight, abdominal fat, liver, serum immunoglobulin, lipid metabolism-related proteins, and fecal microorganisms in mice induced by the HFD.

Feed intake and weight of each mice were measured and recorded on day 0, 14, and 28 after the start of the experiment. On the 28th day, the mice were dissected, liver and abdominal fat washed with sterile saline, and filter paper was used to absorb water, record appearance, weigh, and calculate liver and obesity indices.

Mice orbital blood was collected in a centrifuge tube, and the whole blood was kept at room temperature for 30 min, so as not to shake violently to avoid hemolysis. After the whole blood was naturally solidified, serum was collected. Inflammatory cytokines 6 (IL-6) and indexes related to lipid metabolism [total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low density lipoprotein (LDL-C)] were detected by ELISA. The serum was put on ice for later use. Competitive ELISA kit (Jiangsu Yancheng Jiangsu Meimian Industrial Co., Ltd., Yancheng, China) was used for detection. First, the standard was diluted to draw the standard curve. The test sample and standard competitive antigen (HRP-labeled) were added, gently shaken and stirred, and stood at room temperature in the dark for 60 min. Then the plate was washed five times and pat dry. TMB was added to stop color development, and the absorbance value (OD 450 nm) of each well was measured. If the detection result was valid within 15 min, draw a standard curve with OD 450 nm value and standard concentration. Then the concentrations of IL-6, TC, TG, HDL-C, and LDL-C in the sample were determined by comparing the external diameter of the sample with the standard curve. Because of the use of competitive ELISA detection kits, the OD 450 nm value was negatively correlated with sample concentration.

Fat samples were fixed in tissue fixative, sliced into 4 μm and baked at 65 rve. Xylene solution A and xylene solution B were added sequentially for 1 min, slicing for dewaxing, 100% ethanol was added sequentially for 10 min, 95% ethanol for 1 min, 80% ethanol for 1 min and 70% ethanol for 1 min, and xylene was removed by eluting with water. Then the hematoxylin dye solution was added for 3 min and rinse with water. One percent hydrochloric acid alcohol was added for 30 s and washed with water. One percent ammonia water was added for 30 s and rinsed with water. Eosin dye solution was added for 2 min and rinsed with water. A total of 70% ethanol, 80% ethanol, 95% ethanol, and 100% ethanol were added each for 1 min in turn. Blow dry, pass through xylene solution and blow dry again. The film was read in the mirror.

Liver tissue (0.1 g) of mice were collected, extracted in RIPA lysis buffer containing protease/phosphatase inhibitor by tissue homogenizer, and homogenized at 4,722 × g for 30 s, 4 times for 2 min. The homogenate samples were centrifuged at 10,625 × g at 4°C for 3 min, then the supernatant was separated and stored at 4°C. Lipid metabolism related indexes (TC and TG) were detected by ELISA. The ELISA kit assay (Jiangsu Meimian Industrial Co., Ltd., Yancheng, Jiangsu) was used to test. First, the standard was diluted and used to draw the standard curve. The test sample and standard competitive antigen (HRP-labeled) were added, gently shaken and stirred, and stood at room temperature in the dark for 60 min. Then the plate was washed five times and pat dry. TMB was added to stop color development, and the absorbance value (OD 450 nm) of each well was measured. If the detection result was valid within 15 min, draw a standard curve with OD 450 nm value and standard concentration. Then the concentrations of TC, TG in the sample were determined by comparing the outer diameter of the sample with the standard curve.

Microbial genomic DNA was extracted from 20 samples of colon contents by a E.Z.N.A. Mag-Bind Soil DNA Kit (Omega, M5635-02, USA) and Quibit dsDNA HS (Thermo, USA) was used to test the concentration of DNA samples. The extracted DNA samples were stored in a freezer at −80°C and used for the PCR amplication.

The sequence of forward primer was CCTACGGGNGGCWGCAG, and the reverse primer was GACTACHVGGGTATCTAATCC. The PCR reaction conditions were as follows: 94 ermo, USA, 5 cycles at 94°C for 30 s, 45°C for 20 s, 65°C for 30 s; 20 cycles at 94°C for 20 s, 55°C for 20 s, 72°C for 30 s; 72°C for 5 min. PCR products were purified and quantified bt QIAquick gel extraction kit (QIAGEN, Hilden, Germany) and Quant-iT PicoGreen dsDNA assay kit (Life Technologies, Carlsbad, CA, USA), respectively. The extracted DNA samples were sequenced for 16S rDNA in Sangon Biotech (Shanghai) Co., Ltd.

Gut microbiota evaluation was made using QIIME and the R package 3.5.1, whereas Alpha diversity indices and bacterial abundance data of different groups were compared by performing the Kruskal–Wallis test followed by pairwise Mann–Whitney U comparison. Then, resulting P-values were corrected with the Bonferroni method. The alpha-diversity analysis was conducted to investigate the microbial diversity using Shannon diagram, and beta-diversity analysis was conducted to investigate the structural variation of microbial communities across samples using UniFrac distance metrics and visualized via principal coordinate analysis (PCoA). Besides, difference comparison was used to identify features with significantly different abundances between groups using STAMP (version 2.1.3) and LefSe (version 1.1.0). Correlation coefficients and P-values between communities/OTUs were calculated using SparCC (version 1.1.0). Except the 16S rRNA data has been described in detail, the data obtained in this study are presented as the mean ± SEM. Besides, all statistical analyses were carried out by applying the IBM SPSS Statistics software 19.0 for Windows, while differences between two groups were assessed using the unpaired two-tailed Student’s t-test. In addition, more than two groups were evaluated by a one-way analysis of variance (ANOVA) followed by Newman–Keuls post-hoc tests. Here, it should be noted that a P-values of P < 0.05 was considered statistically significant. The following P-values were used: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

A total of 221 lactic acid bacteria strains were isolated from 85 cats for calcium dissolving rings, and 16 strains with calcium dissolving rings larger than 1 mm were selected (Supplementary Table 1). The colony morphology was observed by Gram staining and microscopic examination (Supplementary Table 2). After that, the ability of 16 strains to degrade cholesterol was measured, and 8 strains with more than 15% of the ability to degrade cholesterol were screened (Figure 1). The strains that inhibited Salmonella, S. aureus, and E. coli were screened, and nine strains with antibacterial circle radius greater than 15 mm were screened (Table 1). A total of six strains with both cholesterol-degrading and antibacterial abilities were selected, and three strains (L-27-2, L-14-1, and F203) with better ranking were selected to test other growth performance.

Neighbor-Joining method was used to draw phylogenetic trees (Figure 2). Evolutionary distance was calculated by the p-distance method, and the unit was the number of base differences at each locus. This analysis involved 34 nucleotide sequences including a noncoding sequence with codon at base 1, base 2, and base 3. Removed all ambiguous positions from each sequence pair, and there were 998 locations in the final dataset. According to these 998 loci, the difference in the base number of each strain was shown at the nodes of the tree. Based on the baseline sequence and phylogenetic tree results, the fecal strains L-27-2, L-14-1, and F203 were identified as L. plantarum, Pediococcus sp., and E. faecium. The login number in Genbank are: OR919791, OR919790, and OR919789.

The physiological and biochemical characteristics of three isolated strains were tested. It was found that L-27-2, L-14-1, and F203 had the ability to ferment common monosaccharides and polysaccharides, such as glucose and sucrose, of which F203 could ferment most types of carbohydrates (23 types), and at the same time, the color reaction results of all carbohydrates were integrated. The strains L-27-2, L-14-1, and F203 were identified as L. plantarum, Pediococcus sp., and E. faecium, respectively. The fermentation properties of L-27-2 were different from the bacteria found by others, which could ferment arabinose but had no obvious effect on ribose (Cui et al., 2021). Compared to Lactococcus lactis strains detected by Passerini et al. (2013), the fermentation properties of L-14-1 showed an additional ability to utilize lactose and xylose. There are few studies on carbohydrate fermentation by E. faecium (Halstead et al., 2015; Kim et al., 2020). Comparing F203 with Kim MA, we found that E. faecium has the ability to ferment sucrose, and it also has the ability to ferment lactose and maltose (Table 2).

The growth performance of L-27-2, L-14-1, and F203 was investigated. It was found that L-27-2, L-14-1, and F203 all reached a stable stage after 18 h of growth, among which L-27-2 and L-14-1 could reach higher biomass, with an OD 600 nm of about 1.8, while F203 had less biomass and lower OD 600 nm (Figure 3A). L-27-2, L-14-1, and F203 can all produce acid with a pH value of about 3.5(Figure 3B), and this value remains unchanged after the strains entered the growth stable period, showing a lower pH compared to the former research (pH = 4.1), which may be related to the higher ability of these three strains to produce SCFA (LeBlanc et al., 2004; Sun et al., 2023), and the time to enter the growth stable period was obviously earlier than other lactic acid bacteria. These growth characteristics can make L-27-2, L-14-1, and F203 better stored and clinically applied (Wang et al., 2022).

Due to the acidic environment and high bile salt environment in the gastrointestinal tract, a good probiotic preparation needs to have good acid and bile salt resistance (Holzer, 2007; Mulaw et al., 2019; Naqqash et al., 2022). The acid resistance and bile salt resistance of L-27-2, L-14-1, and F203 were determined. It was found that L-27-2, L-14-1, and F203 maintained high vitality after 4 h in pH 2 environment, and survival rates reached 93.71%, 96.80%, and 70.17%. L-27-2 was well adapted to pH 3, and the survival rate of L-14-1 and F203 at pH 3 was 96.96% and 86.21%, respectively. The pH 4 environment was conducive to the growth of L-27-2 and L-14-1, and the survival rate of F203 was 91.56% at pH 4 (Table 3), which showed the same trend as Reuben et al.’s (2020) experimental results. In addition, survival rates for L-27-2, L-14-1, and F203 were 69.76%, 68.27%, and 60.23% at 0.6% bile salt concentration, respectively, and survival rates increased with the decrease of bile salt concentration. The results showed that L-27-2, L-14-1, and F203 could tolerate acid environment and high bile salt (0.6%) environment well (Table 4). Because the gastrointestinal pH was more than 2 and the salt concentration in the intestinal lining was less than 0.3%, it could tolerate the acidic environment with pH 2 and the bile salt environment of 0.6%, which proved that L-27-2, L-14-1, and F203 could exist well in the intestinal tract and would not lead to the reduction of biomass due to the special environment in the intestinal tract (Mukherjee et al., 2013; Winston and Theriot, 2020).

The antibiotic sensitivity of three strains was tested, and it was found that all of these strains showed good sensitivity to common clinical antibiotics (Table 5), indicating that these three strains could not easily cause drug resistance (Levy and Marshall, 2004; Nowakiewicz et al., 2021).

The adhesion capacity of L-27-2, L-14-1, and F203 to epithelial cell lines was shown in the Figure 4. At least 20 L-27-2 cells adhere to a single epithelial cell, and at least 10 L-14-1 and F203 cells adhere to a single epithelial cell (Figure 4). These three strains of lactic acid bacteria showed good adhesion to epithelial cells, indicating that lactic acid bacteria can adhere to intestinal epithelial cells well, which also made them more capable of competing with pathogenic bacteria (Uraipan and Hongpattarakere, 2015; Sirichokchatchawan et al., 2018; Yang J. et al., 2018), becoming the dominant bacteria in the intestinal tract and improving the intestinal environment (Salonen et al., 2009; Liu et al., 2018, 2021).

Studies have shown that the HFD could lead to obesity in mice (Kim et al., 2023). By feeding mice with the HFD, it was observed that the intake did not decrease significantly for 28 days after feeding with HFD. The results showed that the weight and obesity index increased significantly (P < 0.05) and the liver index increased (P > 0.05). Supplementation with L-27-2, L-14-1, and F203 could slow weight gain, reduce fat accumulation, and reduce liver index (P > 0.05) and fat index (P < 0.05) in mice fed with HFD (Figures 5A–D). However, the effect of L-27-2 in lowering the obesity index and the tendency of the three strains to lower liver weight was not significant, which may be related to the short duration of the trial. According to former research, lactic acid bacteria have obvious effects on reducing obesity and weight (Crovesy et al., 2017; Khare and Gaur, 2020; Zhong et al., 2022). This conclusion was consistent with our results which confirm the potential of L-27-2, L-14-1, and F203 to reduce body weight gain in animals.

To further confirm the effects of L-27-2, L-14-1, and F203 on blood lipids, the blood lipid content (TG, TC, LDL-C, and HDL-C) of mice was biochemically analyzed. The results showed that the increase in TC, TG, and LDL-C was closely related to hyperlipidemia, obesity, and other diseases (Retterstøl et al., 2018; Webb et al., 2022; Duan and Li, 2023). However, all three strains can reduce TG in serum, however, TC and LDL-C showed only a decreasing tendency, and their differences with M group were not significant (P > 0.05) (Figures 6A, C). The serum TG content of L-27-2 decreased significantly (P < 0.05), while that of L-14-1 and F203 decreased (P > 0.05) (Figure 6B). In addition, feeding F203 could also increase the concentration of HDL-C in the blood (P < 0.01) (Figure 6D), and the increase of HDL-C could promote the reverse transport of cholesterol and reduce lipid accumulation. These results indicated that lactic acid bacteria L-27-2, L-14-1, and F203 could reduce the increase in blood lipid in mice induced by the HFD, thereby reducing the occurrence of nutrition-related metabolic diseases such as hyperlipidemia and diabetes.

Research has shown that HFD mice may have higher levels of inflammation than ND mice, including an increase in pro-inflammatory cytokines and related inflammatory proteins (Terzo et al., 2020; Kuerman et al., 2021; Chan et al., 2022). The occurrence of these inflammations may also be related to the fragile body state of mice, which made them more susceptible to colonization by pathogenic bacteria (Huang et al., 2023). To demonstrate the effects of L-27-2, L-14-1, and F203 on obesity-induced inflammation in vivo, after feeding HFD mice with lactic acid bacteria for 28 days, serum IL-6 levels in mice were detected by ELISA detection kit. Then, it was found that the serum IL-6 content of mice was significantly increased by HFD (P < 0.01), but the blood IL-6 of mice fed with L-27-2 and L-14-1 was significantly decreased (P < 0.01), and the IL-6 of mice fed with F203 was decreased, but not significant (P > 0.05) (Figure 7A). These results showed that L-27-2, L-14-1, and F203 could help alleviate increased weight gain by HFD and inflammation caused by obesity.

It has been found that the HFD can increase the content of total cholesterol TC and TG in the liver, and eventually lead to liver lipid deposition and even nonalcoholic liver disease (Recena Aydos et al., 2019; Ding et al., 2022). Therefore, in order to investigate the effects of L-27-2, L-14-1, and F203 on lipid accumulation in the liver of HFD mice induced by the HFD, the contents of TG and TC in the liver of mice were determined by ELISA. It was found that HFD could increase the content of TC and TG in the liver of mice, while feeding L-27-2 could decrease the content of TC (P < 0.05) (Figure 7B), and feeding L-27-2, F203 could decrease the content of TG in the liver of HFD mice (P < 0.05) (Figure 7C).

Studies have shown that accelerating the excretion of TC and TG from the body is helpful in reducing lipid deposition in vivo, which is manifested by the increase of TC and TG in feces (Kong et al., 2019). Therefore, in order to explore the ability of L-27-2, L-14-1, and F203 to metabolize lipids in obese mice induced by the HFD, the contents of TG and TC in the feces of mice were determined by ELISA. It was found that feeding L-27-2, L-14-1, and F203 could increase TC content in feces of mice fed with HFD (Figure 7E), but the TG metabolism in feces did not increase significantly (P > 0.05) (Figure 7D). Therefore, L-27-2, L-14-1, and F203 may be helpful for TC metabolism under the HFD.

Lack of NPC1l1 has an effect on cholesterol metabolism, and inhibition and lack of NPC1L1 has been shown to protect mice from obesity induced by the HFD (Jia et al., 2021; Meng et al., 2022). Therefore, in order to investigate the effects of L-27-2, L-14-1, and F203 on the expression of NPC1L1 protein in the intestine, the expression of NPC1L1 in the jejunum of mice was measured by ELISA. The results showed that L-27-2, L-14-1, and F203 could reduce the expression of NPC1L1 protein induced by HFD (P > 0.05) (Figure 7F), and inhibition of this protein might be the specific pathway of L-27-2, L-14-1, and F203 acting on cholesterol metabolism.

Studies have shown that in obese mice fed the HFD, fat accumulates significantly in the abdominal cavity and is accompanied by inflammation (van der Heijden et al., 2015; Airaksinen et al., 2018). Therefore, in order to explore the effects of L-27-2, L-14-1, and F203 on fatty tissue inflammation in obese mice induced by the HFD, white fat in the abdomen of mice was taken for pathological section, and it was found that fatty inflammation in HFD mice was alleviated to varying degrees after feeding L-27-2, L-14-1, and F203, of which L-27-2 had the best effect and the average diameter of adipocytes was smaller and fewer inflammatory cells were observed (Figure 7G). This showed that L-27-2, L-14-1, and F203 have certain inhibitory effects on fatty inflammation produced by the HFD.

In this study, it was found that the HFD could reduce the number of Bacteroides and increase the abundance of Firmicutes in the gut of mice, but feeding L-27-2, L-14-1, and F203 could slow this change, thus reducing the number of Firmicutes and increasing the number of Bacteroidetes in the gut of HFD mice (Figure 8A). Using PCoA diagram analysis, it was found that feeding L-27-2, L-14-1, and F203 could change the intestinal microbial diversity of mice, among which L-27-2 and F203 were significantly different from HFD mice, while L-14-1 was slightly different, and the intestinal microbial diversity of mice fed L-27-2, L-14-1, and F203 increased significantly (Figure 8B). At a subordinate level, the HFD might increase the number of Erysipelatoclostridium in the gut of mice. Feeding L-27-2, L-14-1, and F203 might reduce the accumulation of Erysipelatoclostridium and Lachnoclostridium in mice fed the HFD. However, Erysipelatoclostridium enrichment in the body was associated with the occurrence of many diseases (Milosavljevic et al., 2021), and Lachnoclostridium has been shown to be associated with rectal adenoma and cancer (Liang et al., 2020). In addition, Faecalibaculum, as a kind of short-chain fatty acid-producing bacteria, has been shown to have the effect of preventing tumor growth (Zagato et al., 2020). This study found that Faecalibaculum was clearly enriched in mice fed L-27-2 and F203, indicating the positive effect of lactic acid bacteria on the intestinal flora of mice (Figure 8C). These results indicated that L-27-2, L-14-1, and F203 have effects on intestinal microorganisms in mice, which was related to the increasing SCFA, lipid metabolism, and the decreasing of pathogenic bacteria colonization in mice. All results were uploaded to NCBI with SRA number as PRJNA1050058.