Lactobacillus plantarum culture solution was added to an Oxford cup and the growth inhibition diameter was measured after 24 h of culture with MRS liquid culture medium as control. Absorption method was partially modified to test antibacterial activity of L. plantarum. L. plantarum supernate an MRS medium were mixed into 100 μL of S. mutans culture solution, respectively. When the inhibition ratio is less than two after 18 to 24 h of culture, the sample demonstrated good antibacterial activity. The bacteriostatic value was obtained by Equation 1:

where A represented the bacteriostatic value, C₁ the control group’s average colony forming units (CFU), and T₁ the experimental group’s average CFU.

Supernatant of Lactobacillus was to handle S. mutans. A final concentration of 12.5 to 100% (v/v) was employed, with MRS liquid medium serving as the control. Every 4 hours, 100 microliters of various concentrations of culture fluid were collected and put in a 96-well plate to monitor the growth conditions. The ELISA Reader was used to measure the OD value at 600 nm. An ELISA reader was used to measure optical density at 600 nm.

To evaluate the biofilm formation and removal, crystal violet staining was employed, utilizing various concentrations of the supernatant ranging from 0 to 100% (v/v), with MRS liquid medium serving as the control. Following a 24 h culture period, it was washed with PBS for 3 times and a 15 min methanol fixation. 0.1% crystal violet was utilized for staining, followed by the dissolution of 33% glacial acetic acid. The optical densities were again measured at 600 nm to assess the effectiveness of the different concentrations on biofilm dynamics.

By adding 50% (v/v) L. plantarum supernatant with the same quantity at various time intervals (0 h, 6 h, 12 h, and 24 h), which was used to observe the production and removal of biofilm. MRS medium was used as the control (12 h and 24 h). Staining and determination steps are the same as above, and the biofilm reduction rate is obtained by Equation 2:

Once a biofilm had formed after 24 h of culture, a dyeing working solution was made by diluting PI and N₀ fluorescent dye with NaCl solution (Shanghai Beibo Biotechnology Co., Ltd.). After gently rinsing the side wall three times with NaCl and draining, add 200 μL of the dyeing working solution and let it sit in the dark for fifteen minutes. After washing the bacteria once with NaCl solution, resuspend them by adding the proper amount of NaCl solution. The red signal is received through the red channel, and the green signal is received through the green channel. Look through a fluorescent microscope and snap pictures.

Specific modifications to the previously established methods were applied. Adjust the concentration of L. plantarum solution to OD₆₀₀ = 0.6 ± 0.05, and the concentration of S. mutans solution to OD₆₀₀ = 0.5 ± 0.05, and the bacteria separately and in a mixed manner. The absorbance of the upper liquid were measured at various time intervals. The self-polymerization and co-polymerization ability of strains at different times were judged, respectively. The Auto-aggregation ability was obtained by Equation 3:

A₀ refers to the absorbance measured at 0 h, while A_t represents absorbance values taken at 2, 4, 6, and 24 h.

The Co-aggregation ability was obtained by Equation 4:

Where A_x is the absorbance of 0 h L. plantarum, A_y is the absorbance of 0 h S. mutans, A_mix is the absorbance of the mixture at the 2, 4, 6, and 24 h time points.

Lactobacillus plantarum was inoculated into MRS medium with pH of 3.0 and 0.3% (w/v) bile salt (Beijing Solarbio Science & Technology Co., Ltd.), respectively. The MRS culture medium served as the control. Following incubation for 3 and 24 h, the results were analyzed by evaluating the optical density at a wavelength of 600 nm. The survival rate was obtained by Equation 5:

Lactobacillus plantarum suspension was inoculated into artificial gastric juice (Shanghai yuanye Bio-Technology Co., Ltd.) and artificial intestine juice, respectively. The survival rate was obtained by Equation 5.

Lactobacillus plantarum and S. mutans were inoculated in MRS and BHI broth containing 10% glucose. After being diluted to the same concentration (OD₆₀₀ = 1). The glucose content after 24 h was measured by the glucose kit method (Nanjing Jiancheng Bioengineering Company, China), and the solution without bacterium was used as control. The content rate of glucose was obtained from Equation 6:

A 0.5% (v/v) inoculum of both L. plantarum and S. mutans cultures were introduced into Triglyceride-MRS and Triglyceride-BHI media, incubated at 37°C for 24 h. The triglyceride content was measured utilizing the triglyceride kit method, both with and without bacteria for comparative analysisthe triglyceride content ratio is calculated according to Equation 6.

All procedures were conducted at the Animal Experimental Center of Jinzhou Medical University, with the animal studies receiving prior examination and approval from the Animal Ethics Committee of the same institution. The approval agreement number of animal program is 240,139. Male Sprague Dawley rats of 3-week-old were purchased and housed under controlled temperature (20°C ± 5°C) and Temperature (50% ± 10%) conditions. Rats were randomly allocated into three groups, each containing three rats. The control group (Control) was given conventional feed and sterile water, while the caries and obesity group (CA_OB) and caries and obesity treated with L. plantarum ATCC14917 group (LP) were given a high-fat and high-sugar feed and 5% sucrose water. The 56-day experiment included a 0–3 day antibiotic interference period during which ampicillin (0.5 μg/mL) inhibited the oral flora of the rats. The colonization time of S. mutans is 4–8 days. Before each use, a bacterial solution (10⁸ CFU/mL) was made using BHI culture medium. Each rat was anesthetized by inhalation. Using sterile cotton swabs, dip the S. mutans suspension until it is saturated. Then, in turn, daub in the respective group’s teeth, tongue, and oral mucosa, remaining at each location for 15 s. Using a blunt syringe, rinse the rat’s mouth, making sure the solution reaches every area of the mouth and plays its full part. The experimental solution was 1 mL/tube. Rats handled once daily in the morning and evening. The LP group was given 2 × 10⁸ CFU/mL L. plantarum bacterial solution orally from day 14 to day 56. Until the completion of the trial, L. plantarum was administered four times per week. To ensure microbial colonization, refrain from eating or drinking for 30 min prior to and following the inoculation. The same experimenter always performed the procedure. Every week, the rats’ weight was recorded. The animals were put down at the conclusion of the experiment, and samples were gathered.

Mandibular specimens were scanned by Micro-CT [PINGSENG Healthcare (Kunshan) Inc.]. The enamel was separated from the mandible with a fixed threshold and the volume of enamel was calculated to evaluate the dental caries. The demineralization rate of enamel was obtained from Equation 7:

Where V₀ was the volume of enamel in control group, V_t was the volume of enamel in LP group, and V_m was the volume of enamel in CA_OB group.

Serum was extracted by centrifuging blood samples at 5000 × g for 10 min after the animals were put under anesthesia. The serum was stored at-80°C until further analysis.

Utilizing the assay kits, levels of Total triglyceride (TG), Total cholesterol (T-CHO), High-density lipoprotein-cholesterol (HDL), Low-density lipoprotein-cholesterol (LDL), and Blood glucose were evaluated (Nanjing Jiancheng Bioengineering Company, China).

Hematoxylin and eosin (H&E) staining was performed on the rat liver and epididymis adipose tissues. Rat liver and epididymis fatty tissue was cleaned with regular saline, fixed for 24 h in a 10% paraformaldehyde solution, and the excess fixative was rinsed off with tap water. Following ethanol gradient dehydration, the tissue was embedded in paraffin and cut using a microtome into slices that were 4–5 microns thick. Following hematoxylin–eosin staining, followed by photography to document the findings.

Fresh feces from each rat were collected on the 57th day under aseptic conditions on the ultra-clean workstation, put in a 5/mL freezing tube, kept in the refrigerated at-80°C, and then delivered to the firm for testing. The FastPure Stool DNA Isolation Kit (MJYH, shanghai, China) was used to extract the total DNA. The hypervariable region V3–V4 of the bacterial 16S rRNA gene were amplified with primer pairs 338F (5’-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5’-GGACTACHVGGGTWTCTAAT-3′). Identification, purification and quantification of PCR products were carried out. The sequencing was based on the platform of Shanghai Maggie Biomedical Technology Co., Ltd.

The statistical program GraphPad Prism 8.0.2 was utilized for the mapping and analysis. To analyze the differences among the groups, a one-way analysis of variance was utilized, with a significance level set at p < 0.05 to indicate noteworthy differences. Data were expressed as mean ± standard deviation, and each experimental condition was replicated three times to ensure reliability and consistency in the results obtained.

Lactobacillus plantarum had a substantial inhibitory effect on S. mutans growth, and it measured 18.85 ± 1.05 mm in diameter (Figure 1A). Lactobacillus exhibited a 2.24 antibiosis activity to S. mutans. The aforementioned findings demonstrated that Lactobacillus had a potent antibacterial impact (Figures 1B,C).

The bacteriostatic effects of the five groups of L. plantarum supernate started to take effect at 4 h, with the corresponding bacteriostatic rates being 60.90, 72.30, 79.18, 79.83, and 80.62%. The bacteriostatic efficacy of L. plantarum supernatant at concentrations of 12.5 and 25% declined with time, from 8 to 24 h (p < 0.05). Nonetheless, L. plantarum supernate still plays a good bacteriostatic role at concentrations of 50 to 100% (v/v), and the bacteriostatic rate was higher than 79% (p > 0.05) (Figure 2).

Studies have proved the influence of Lactobacillus on the biofilm of S. mutans within 24 h. L. plantarum supernate (0, 12.5, 25, 50, 75, 100%) exhibited varying degrees of inhibitory effects on S. mutans biofilm. The corresponding inhibitory rates were 15.91, 66, 80.79, and 83.99% (p > 0.05). The biofilm formation process is characterized by four crucial time points: the initial bacterial adhesion occurring at 0 h, initial bacterial colonization at 6 h, early biofilm development at 12 h, and the maturation of the biofilm at 24 h (Figure 3).

In addition, with the intervention of L. plantarum, fluorescent staining was also used to measure the biofilm activity of S. mutans. After 24 h of culture, it was found that the colonies in the control group covered the whole field of vision, and there was no gap in the middle. The membrane was a dense network structure, mainly with green fluorescence and scattered with red fluorescence (Figure 4A). Live bacteria decreased gradually, the block structure decreased, the biofilm became sparse gradually, the red part increased, and the activity of the biofilm was obviously inhibited (Figure 4B).

With time, L. plantarum ATCC 14917’s capacity for self-aggregation grew. The auto-aggregation rates of the strain were observed over various time intervals: 2 h, 4 h, 6 h, and 24 h, yielding rates of 22.62, 31.56, 39.32, and 70.91%, respectively. At 2 hours, the co-aggregation ability was minimal; by 4 hours, it had clearly increased. The strain under test started to agglutinate with S. mutans, according to the data, and after 24 h, the rate of cross-agglutination had reached 48.32% (Figure 5).

The properties of probiotics were evaluated using the average intestinal bile salt content, which was 0.3% (w/v). Under conditions of low pH, the survival rate of L. plantarum decreased significantly, falling from 73.20 to 15.18%. For the duration ranging from three to twenty-four hours in a medium containing 0.3% bile salts, the survival rate remained consistently near 80% (Table 1).

The number of viable bacteria decreased by 0.68 and 0.3 after 3 hours, respectively. Lactic acid bacteria have a strong ability to adapt to the tough environment of the gastrointestinal tract, as evidenced by the fact that even while their development was limited, a constant survival rate could still be ensured (Table 2).

Bacteria require glucose as their primary growth substrate, and different strains exhibit diverse abilities to metabolize it. The amount of glucose content was found to be 32.54 and 236.13% in the culture medium, and the rate of lowering blood sugar and the increasing blood sugar are 67.46 and 136.13%, respectively (Figure 6A). On the other hand, the triglyceride content in the Triglyceride-MRS and Triglyceride-BHI media was 63.84 and 254.50%, respectively, and the lipid-lowering and fat-increasing rates were 36.16 and 154.50% (Figure 6B). The data above demonstrated the strain’s strong probiotic qualities.

Rat molar enamel volumes were computed for every experimental group. The model demonstrating the establishment of caries is further validated by the considerable amount of demineralized enamel found in the CA_OB group when compared to the Control group. The volume of demineralized enamel in LP group is similar to that in Control group. In addition, the demineralization rate of enamel of the LP group is similar to that of the Control group. The above results indicate that L. plantarum has an obvious inhibitory effect on dental caries in animal models (Figure 7).

After an eight-week feeding period, a clear change in weight was noted among the rats in the LP group, with the CA_OB group displaying a much greater weight than their Control counterparts. Rats within the LP group showed significantly lower weights compared to those in the CA_OB group (Figure 8A). Compared with the Control group, the serum TG, T-CHO, LDL-C and Blood glucose levels in the CA_OB group were significantly increased, while the serum HDL-C level was significantly decreased, indicating that the blood lipid metabolism in CA_OB group was abnormal. However, the LP group significantly inhibited the lipid parameters in rat serum (Figures 8B–F).

The results indicated no significant pathological changes in the liver tissue of rats from the Control group. In the CA_OB group, liver cells were obviously swollen, filled with fat vacuoles of different sizes, and inflammatory cells were obviously infiltrated. Rats from the LP group had far fewer fat vacuoles in their liver tissue, although there were still some localized inflammatory reactions (Figure 9A). The CA_OB group’s fat cells were noticeably larger and there were less cells in the same field of vision when compared to the group on a regular diet. Rat epididymal adipocyte hypertrophy may be inhibited following treatment with L. plantarum, suggesting that probiotic supplementation has a beneficial impact on lipogenesis (Figure 9B).

To assess the diversity of bacterial communities, the dilution curve was primarily constructed using the Alpha diversity index from samples taken at various sequencing depths. The Shannon index is an important metric that tends to smooth out the dilution curve, serving as a reflection of the diversity present within microbial communities. This index indicates that the data volume gathered from each group is sufficient to meet the sequencing requirements (Figure 10A). Results from the Principal Coordinate Analysis highlighted that the contributions of Principal Component 1 and Principal Component 2 exceeded 60%, allowing for a comprehensive representation of the community composition. The LP group was similar to the Control group at the OTU level, and the CA_OB group and the LP group could be distinguished more clearly (Figure 10B). The PCoA data revealed that the LP group and the Control group nearly completely overlapped at the genus level, further supporting the idea that lactic acid bacteria might govern the gut flora (Figure 10C). To evaluate whether the differences between groups were statistically significant, we employed supervised partial least squares analysis. The findings from this analysis clearly illustrated a notable separation between the groups at both the OTU level and the genus level (Figures 10D,E). The ANOSIM analysis further showed the significant differences between groups (Figure 10F).

Intestinal microbiota may shield the host against pathogens that cause intestinal disorders in addition to enhancing nutrition absorption and preserving immunity. On the genus of level, the LP group increased relative abundance of Lactobacillus, Limosilactobacillus, unclassified_f__Muribaculaceae, Blautia, Faecalibaculum. The LP group led to the decrease of the abundance of pathogenic bacteria, including unclassified_o__Clostridia_UCG-014, unclassified_f__Oscillospiraceae, Turicibacter, unclassified_f__Lachnospiraceae, Clostridium_sensu_stricto_1 (Figure 11A).

On the family of level, the three groups of bacteria were mainly the following: Lactobacillaceae, Lachnospiraceae, Muribaculaceae, Oscillospiraceae, unclassified_o__Clostridia_UCG-014, Peptostreptococcaceae, Erysipelotrichaceae (Figure 11B).

This analysis illuminated the communities or species that exhibited significant variation across all taxonomic levels, including phylum, class, order, family, and genus. It was evident that the relative importance of microbial biomarkers escalated in tandem with increasing Linear Discriminant Analysis (LDA) scores. Compared with the Control group, in the CA_OB group, g__unclassified_f__[Eubacterium]_coprostanoligenes_group, f__Selenomonadaceae, g__unclassified_f__Oscillospiraceae, g__Quinella, g__unclassified_f__Desulfovibrionaceae. Contrasting to the CA_OB group, The profusion of g__Phascolarctobacterium, f__Acidaminococcaceae, g__Elusimicrobium and c__Elusimicrobia within the LP group was markedly elevated (Figures 12A,B). The degree of microbial ecological imbalance was measured using the microbial dysbiosis index. The degree of microbial imbalance increases with increasing value. In comparison to the Control group, the CA_OB group had a considerably higher microbial dysbiosis index. The microbial dysbiosis index clearly decreased following L. plantarum intervention, suggesting that L. plantarum may control the intestinal flora of CA_OB rats (Figure 12C).

Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2), Kyoto Encyclopedia of Genes and Genomes (KEGG), Evolutionary Genealogy of Genes: Non-supervised Orthologous Groups (EggNOG).

Bacterial colony function is largely associated with the metabolism pathway, according to the 16S rRNA sequencing data and KEGG functional predictions (Figure 13A). We could further demonstrate that bacteria are related to global and overview maps, carbohydrate metabolism, and amino acid metabolism by combining 16S rRNA sequencing data with KEGG data information of Pathway Level 2 (Figure 13B). Based on the 16S rRNA data and the PICRUSt2 channel display of EggNOG and KEGG databases, we predicted the biological functions of bacteria. 16S rRNA sequencing data combined with KEGG data information of Pathway Level 3, we could further confirm that bacteria are related to purine metabolism, peptidoglycan biosynthesis, oxidative phosphorylation, amino acid metabolism, streptomycin biosynthesis, nitrogen metabolism, taurine and hypotaurine metabolism (Figure 13C). In an effort to delve deeper into the functional roles of bacteria, we utilized the EggNOG database. Predictions indicated that the primary functions of the bacteria were closely associated with metabolic processes. These processes predominantly involved translation, the structure and biogenesis of ribosomes, as well as the transport and metabolism of amino acids and carbohydrates (Figure 13D).

At the genus level, Limosilactobacillus was negatively connected with associated with obesity variables (TC, TG, LDL, Blood Glucose), while Clostridium_sensu_stricto_1, Turicibacter, unclassified_f__Oscillospiraceae, Colidextribacter, Quinella, Desulfovibrio were positively linked to some relating to obesity factors (Figure 14A). At the family level, Lactobacillaceae, unclassified_p__Firmicutes had a negative correlation with linked to obesity indexes, while Streptococcaceae, Desulfovibrionaceae, Clostridiaceae, Oscillospiraceae, Selenomonadaceae, UCG-010 were found to have a positive correlation with obesity measures (Figure 14B).