Bacterial strains and mice

S. mutans strains BNCC 700,610 and ATCC 25,175 were purchased from the BeNa Culture Collection Centre (Beijing, China). S. mutans CCTCC AB 99,010 was purchased from the China Center for Type Culture Collection (Wuhan, China). The 3 strains were cultured in the brain-heart infusion (BHI) broth both separately for 24 h with a bacterial load of 108 CFU/mL in aerobic conditions at 37℃ and then mixed in a volume ratio of 1:1:1. All references to S. mutans fluids in this article refer to a mixture of the 3 strains.

L. plantarum VHProbi V38, Ligilactobacillus salivarius VHProbi A17, L. rhamnosus VHProbi M14 and L. paracasei VHProbi OF10 were kept in China Center for Type Culture Collection (NO: M2022173, M2022172, M2022171 and M2022170, Wuhan, China). The 4 strains were individually incubated for 24 h in Man, Rogosa, and Sharpe (MRS) broth with a bacterial load of 108 CFU/mL. They were then mixed in equal volumes to prepare the probiotic mixture for the test. All references to Lactobacillus liquid in this article refer to a mixture of the 4 strains.

Twenty 3-week-old weanling specific pathogen-free (SPF) male Wistar rats were provided by the Qinglong Mountain Animal Breeding Centre (Jiangsu, China) under license number SCXK(Su)2017-0001. The rats were fed a special pellet diet (Diet 2000, Jiangsu Synergy Biological Co., Ltd.) and housed in an SPF-class animal house after the caries model was constructed. Housing conditions included a 12 h/12 h light/dark cycle, free feeding and watering, a temperature of 20–26 °C, and relative humidity between 40% and 70%.

Bacteriostatic activity testing

The antibacterial activities of Lactobacillus strains against S. mutans were quantified using a modified agar-diffusion method and co-culture in vitro [25,26,27,28].

A volume of 100 mL melted BHI agar medium, maintained at ~ 45℃, was inoculated with 300 µL mixed bacterial suspension of S. mutans (OD600 0.5–0.6). The medium (7 mL) containing S. mutans was quickly poured into solidified agar plates. After medium solidification, Oxford cups were placed on the surfaces of the cultures, and 100 µL of the supernatant of lactic acid bacteria (LAB) isolates was dripped into the Oxford cup. All experiments were performed in triplicate. The growth inhibitory activity of the supernatant was calculated by measuring the diameter of the inhibition zone.

The BHI broth (10 mL) was initially mixed with 0.3% (v/v) of the lactic acid bacteria culture (108 CFU/mL) and S. mutans culture (108 CFU/mL) as the inoculants, and cultured at 37℃ for 48 h. The control group was inoculated with S. mutans in BHI broth only. Samples were taken to determine the viable bacterial counts (CFU/mL) for S. mutans using Mitis Salivarius Bacitracin agar. All tests were conducted in triplicate. The inhibition rate (%) was calculated as follows:

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(1)

Biofilm elimination testing

Biofilm removal assay in polystyrene 24-well (flat bottom) plates was performed using the method by Zhang et al. [23] and Khan et al. [29]. A total of 600 µL of the S. mutans cultures in BHI broth was transferred to a 24-well plate with chamber slides and incubated for 24 h to form a biofilm at 37℃ under aerobic conditions. The plate was washed twice with phosphate buffer solution (PBS) (pH 7.0, 0.05 M potassium phosphate). 600 µL of cell suspension (or the supernatant) of lactic acid bacteria was inoculated into the wells. The cell suspension of lactic acid bacteria was harvested by centrifugation at 5,000 × g for 15 min, washed 3 times with PBS, and resuspended in PBS at a concentration of approximately 108 CFU/mL. MRS broth alone was used as the control. After inoculation, all plates were incubated at 37℃ for 24 h to measure the ability of the samples to remove biofilms. All tests were conducted in triplicate. The culture medium was then decanted, and the plates were gently washed 3 times with 600 µL sterilized PBS to remove planktonic and loosely bound cells.

The biofilm removal rate was determined by counting the number of bacteria on the chamber slides. The chamber slides were removed and placed in sterile homogeneous bags. The slides were then subjected to a 10-minute ultrasonic wash to diffuse the organisms into the buffer solution. After a series of dilutions, 100 µL of the samples were cultured into Mitis Salivarius Bacitracin (MSB) agar plates containing 400 U/L bacitracin (G-clone, 65000u/g, China) to determine the concentration of S. mutans. The following formula was used to calculate the removal rate of biofilm:

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(2)

Animal experimentsRat caries model and general procedures

Twenty rats (3 weeks old) were allowed to adapt to the diet and environment for 3 days. They were then divided randomly into 4 groups (n = 5/group), comprising a normal group (N), caries control group (C), probiotic group (P), and antagonist group (A). The rats in each group were then given antibiotic water at a concentration of 200 mg/L of penicillin and 1500 mg/L of streptomycin for 3 consecutive days. On day 7, the rats were taken off antibiotic water and fasted from food and water for 2 h. Saliva was collected from each group using sterile cotton swabs. The swabs were suspended in sterile saline, and the bacterial count was detected using MSB agar. The rats were fed with sterilized chow and water. The caries model was started when the MSB plates were negative. The rats in the C and P groups were subjected to 7 consecutive days of dental caries modeling. In brief, 1 mL of S. mutans culture (108 CFU/mL) was dipped into a sterile cotton stick. The sterile cotton swab was then applied to the rat’s oral cavity for 15 s per quadrant, as described by Beiraghi et al. [30]. On day 7, oral swabs were spread on MSB agar plates supplemented with 400 µg/L bacitracin (G-clone, 65000u/g, China) to confirm S. mutans colonization in the oral cavity. After successful molding, the C group was treated with high-sugar feed sterilized by Co60 irradiation and observed for 5 weeks. The P group was continuously treated with 1 mL probiotic mixture (108 CFU/mL) for 1 week and observed for 4 consecutive weeks. The N group consumed a regular diet and water throughout the 6-week experimental period. The A group was treated with 2 mL mixed fermentation solution (the probiotic mixture: S. mutans culture = 1:1) for one week and observed for 5 consecutive weeks. During the experiment, all groups except the N group were fed 5% glucose water and a high-sugar diet sterilized by Co60 irradiation for six weeks. The ingredients of the high-sugar diet (Diet 2000 caries modelling feed) were: whole meal flour 6%, sucrose 56%, skimmed unsweetened milk powder 28%, yeast 4%, alfalfa meal 3%, liver powder 1% and salt 2%. The regular diet was rat maintenance feed (HFK 1025, Beijing, China). The experimental protocol is shown in Table 1.

This study was carried out at the laboratory of BIOGENE Biotech. Co. Ltd. (Nanjing, China), which has been approved by Laboratory Animal Ethics Committee Nanjing BIOGENE Biotech. Co. Ltd. All methods were carried out in accordance with relevant guidelines and regulations. All methods were in accordance with ARRIVE guidelines.

Table 1 The experimental protocol in different groupsObservations and microbial analysis

We observed the mortality, mental activity, and hair shine of mammary rats and recorded the weight of rats every week.

The whole experiment lasted 6 weeks, during which 4 samples were collected on days 7, 14, 28, and 42. The N group was tested in the first week only. The samples were collected by rubbing a sterile cotton swab in clockwise circles on the molar occlusal surface of the mouth of each group. The swab was immediately placed in a centrifuge tube containing 1 mL of saline for 2 min. After a series of dilutions, bacterial counts were performed on the MSB plates with the appropriate dilution.

Caries scoring and X-ray test

At the end of the 6-week trial, the rats were placed in the euthanasia chamber (Fengshi FSZZ-2 A, Suzhou, China), then the lid was fastened. A CO2 gas line was connected and CO2 was injected into the chamber at a rate of 10–30% of euthanasia box volume per min. After the rats have collapsed and lost the ability to move, the gas flow rate can be increased to a maximum of 0.5 Kpa. The CO2 was not switched off until the rats were immobile, not breathing and its pupils were dilated. Then the rats were observed for two minutes to determine death. The skull was removed, the soft tissues on the teeth and jaws were peeled off with a scalpel, and the jaws were cleaned with ultrasound for 20 min. Then the skull was placed in an autoclave at 121 °C for 15 min. The attached soft tissue was again peeled off with a scalpel, and the jaw was cleaned and dried at room temperature. The teeth on the left side of the rat were used to assess caries scores. The upper and lower jaws were soaked in 10% paraformaldehyde for 24 h and then washed and dried. All specimens were immersed in a 0.4% ammonium purpurate staining solution for 12 h, rinsed, and semi-sectioned along the occlusal surfaces of maxillary and mandibular molars using a diamond cutter (thickness: 0.1 mm). Caries on the rat molars were observed and evaluated under a stereomicroscope according to the caries diagnosis and scoring method reported by Keyes [31]. The method of assigning values involves judging by eye the linear extent of lesions in one plane and recording the depth of penetration under 4 headings, including enamel only (E), slight dentinal (Ds), moderate dentinal (Dm), and extensive dentinal (Dx). The scoring rules are shown in Table 2 [31].

The teeth on the right side of the rat were used for x-ray detection. Rat mandibles were scanned using a small animal X-ray machine, and the pictures were analyzed after scanning.

Table 2 Number of linear units assigned to each molarStatistical analysis

SPSS Statistics 22.0 (SPSS, Inc., Chicago, IL, USA) was used for analysis. All data are presented as the mean ± SD. Where applicable, ANOVA or a 2-tailed Student’s t-test was used to analyze the differences between treatments. A p-value < 0.05 was considered statistically significant. A p-value < 0.01 was considered highly statistically significant.