Lacticaseibacillus paracasei strain, fermentation product, live bacterial preparation, preparation method therefor, and application thereof
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- COREE CO LTD
- Filing Date
- 2025-11-25
- Publication Date
- 2026-06-04
Smart Images
Figure CN2025137304_04062026_PF_FP_ABST
Abstract
Description
Lactobacillus paracasei strains, fermentation products, live bacterial preparations, their preparation methods and applications
[0001] Cross-reference to related applications
[0002] This application claims priority to Chinese Patent Application No. 202411730108.6 and Chinese Patent Application No. 202411730140.4, filed in China on November 28, 2024, the entire contents of which are incorporated herein by reference. Technical Field
[0003] This invention relates to the field of microbial technology, specifically to a strain of Lactobacillus paracasei, fermentation products, live bacterial preparations, their preparation methods, and applications. Background Technology
[0004] Dental caries is a progressive destructive disease of the hard tissues of the teeth caused by the combined effects of multiple factors, including microorganisms, the host, the environment, and time. Commonly known as cavities or tooth decay, dental caries is the most common chronic bacterial infection of the oral cavity. It can lead to pulpitis and periapical periodontitis, and can aggravate or induce systemic diseases. Oral microorganisms adhere to the tooth surface to form dental plaque biofilm. Dental plaque biofilm is a complex of extracellular polysaccharides, proteins, and minerals secreted by microorganisms that adhere to the solid surface of tooth enamel or the tooth root.
[0005] Studies have found that probiotics can prevent tooth decay by inhibiting the growth of cariogenic bacteria and biofilm formation. This includes: ① probiotics competing with cariogenic bacteria for nutrients or attachment sites; ② co-aggregating with pathogens and exerting antibacterial effects; ③ probiotics producing organic acids, hydrogen peroxide, bacteriocins, etc., which interfere with the metabolism of cariogenic bacteria. Other mechanisms for preventing tooth decay include immune regulation, local pH regulation, and increased saliva flow.
[0006] CN202210615263.8 discloses a strain of Lactobacillus salivarius CCTCC NO: M2022172 that can prevent or treat dental caries and periodontal disease, and its application. This Lactobacillus salivarius has a significant inhibitory effect on Streptococcus mutans and Actinomyces viscerata that cause dental caries, as well as on Porphyromonas gingivalis, Clostridium nucleatum and Aggregates hemiactiformis that cause periodontal disease. It can be used as an oral probiotic to effectively prevent and improve oral diseases.
[0007] Research on the inhibitory effect and mechanism of *Lactobacillus paracasei* on *Streptococcus mutans* is insufficient, resulting in poor caries prevention efficacy. Therefore, there is a need for a *Lactobacillus paracasei* strain that can significantly inhibit *Streptococcus mutans* and achieve superior caries prevention. Summary of the Invention
[0008] The technical problem to be solved by the present invention is to provide a strain of Lactobacillus paracasei that has excellent effects in preventing or treating dental caries.
[0009] This *Lactobacillus paracasei* is tolerant of the oral environment, possesses good self-aggregation ability, and co-aggregates with *Streptococcus mutans*. It can inhibit the proliferation of *Streptococcus mutans* and common pathogenic bacteria, effectively reducing the formation of dental plaque biofilm caused by *Streptococcus mutans*, lowering the content of mature dental plaque biofilm, and is not prone to forming biofilm itself. It also reduces the level of water-insoluble extracellular polysaccharides produced by *Streptococcus mutans*, and significantly reduces the expression levels of *Streptococcus mutans* cariogenic genes gtfD, ftf, and LuxS. Furthermore, it significantly inhibits the adhesion of *Streptococcus mutans* to hydroxyapatite tooth models. This bacterium can be used to formulate functional products for the prevention and treatment of dental caries.
[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0011] On one hand, the present invention provides a strain of Lacticaseibacillus paracasei, the preservation number of which is CGMCC No. 27974.
[0012] In one embodiment, the *Lactobacillus paracasei* strain contains the 16S rRNA gene represented by SEQ ID NO:1.
[0013] In one embodiment, the *Lactobacillus paracasei* strain of the present invention is tolerant of the oral environment and does not readily form biofilms on the tooth surface.
[0014] On the other hand, the present invention also provides a live bacteria preparation comprising the *Lactobacillus paracasei* strain and excipients as described above.
[0015] In one embodiment, the live bacteria preparation contains 4.0 × 10⁻⁶ bacteria. 11 Live bacteria at CFU / g or higher.
[0016] In another embodiment, the excipient is selected from one or more of starch, sucrose, trehalose and glycerol.
[0017] On the other hand, the present invention also provides a food or health product comprising the live bacteria preparation described above.
[0018] In another aspect, the present invention also provides the use of the Lactobacillus paracasei strain or live bacterial preparation described above in the preparation of articles for the prevention or treatment of dental caries.
[0019] In one embodiment, the strain or the live bacterial preparation is used to inhibit the growth of Streptococcus mutans.
[0020] In one embodiment, the bacterial strain or the live bacterial preparation is used to reduce the formation of dental plaque biofilm caused by Streptococcus mutans and to reduce the content of mature dental plaque biofilm.
[0021] In one embodiment, the strain or the live bacterial preparation is used to inhibit the level of water-insoluble extracellular polysaccharide production by Streptococcus mutans.
[0022] In one embodiment, the strain or the live bacterial preparation is used to reduce the expression levels of the cariogenic genes gtfB, gtfC, gtfD, ftf, and LuxS in Streptococcus mutans.
[0023] In one embodiment, the strain or the live bacterial preparation is used to reduce the adhesion rate of Streptococcus mutans on a hydroxyapatite tooth model.
[0024] In one embodiment, the article is food, health product, medicine, or oral care product.
[0025] In another embodiment, the oral care product is selected from one or more of mouthwash, beverages, mouth sprays, chewing gum, dental gels, toothpaste, tooth powder, dental floss, dental cleaning solutions, and dental cleaning foams.
[0026] In another aspect, the present invention also provides a fermentation product obtained by fermentation using the Lactobacillus paracasei strain described above.
[0027] In one embodiment, the fermentation product is fermented milk, and the content of active *Lactobacillus paracasei* strain in the fermented milk is 4.0 × 10⁻⁶. 8 CFU / mL or higher.
[0028] In one embodiment, the fermentation product is fermented oat milk, and the viable count of *Lactobacillus paracasei* strain in the fermented oat milk is 8.5 × 10⁻⁶. 8 CFU / mL or higher.
[0029] In another aspect, the present invention also provides a method for preparing the live bacterial preparation as described above, comprising the following steps:
[0030] The *Lactobacillus paracasei* strain described above was cultured in an optimized liquid medium.
[0031] Collect bacterial cells;
[0032] The bacteria were resuspended with a preservative, freeze-dried under vacuum, and then pulverized to obtain the active bacterial agent. Beneficial effects
[0033] Compared with the prior art, the *Lactobacillus paracasei* strain and its live bacterial preparation of the present invention have the following advantages:
[0034] Beneficial effects:
[0035] (1) The Lactobacillus paracasei strain of the present invention can tolerate the oral environment and has good self-aggregation ability; its co-aggregation ability with Streptococcus mutans can inhibit the proliferation of Streptococcus mutans and common pathogenic bacteria, effectively reduce the formation of dental plaque biofilm caused by Streptococcus mutans, reduce the content of mature dental plaque biofilm and is not easy to form biofilm itself, reduce the level of water-insoluble extracellular polysaccharides produced by Streptococcus mutans, significantly inhibit the adhesion ability of Streptococcus mutans on hydroxyapatite tooth model, and significantly reduce the expression levels of cariogenic genes gtfD, ftf and LuxS of Streptococcus mutans.
[0036] (2) The *Lactobacillus paracasei* strain of the present invention has beneficial in vitro probiotic functions. This strain exhibits excellent performance in inhibiting a variety of pathogens.
[0037] (3) The Lactobacillus paracasei strain of the present invention can be used to ferment and produce fermented products such as fermented milk and oat milk.
[0038] (4) The production process parameters of the active bacterial agent of the present invention are simple, easy to control, and have a short cycle, which ensures the high survival rate of Lactobacillus paracasei strains and the resulting product can be stored for a long time with stable product quality. Attached Figure Description
[0039] Figure 1 shows the copolymerization ability of the *Lactobacillus paracasei* HOM1212 strain of the present invention with *Streptococcus mutans* CICC10438. Note: ** indicates a highly significant difference compared to *Streptococcus mutans* CICC10438 (p<0.01). ## The results showed a highly significant difference compared to the commercial Lactobacillus paracasei S strain (p<0.01).
[0040] Figure 2 illustrates the inhibitory effect of the *Lactobacillus paracasei* HOM1212 strain of the present invention on the biofilm of *Streptococcus mutans* CICC10438, and specifically shows the biofilm content. Note: ** indicates a highly significant difference compared to *Streptococcus mutans* CICC10438 (p<0.01). ## The results showed a highly significant difference compared to the commercial Lactobacillus paracasei S strain (p<0.01).
[0041] Figure 3 illustrates the inhibitory effect of the *Lactobacillus paracasei* HOM1212 strain of the present invention on the caries gene of *Streptococcus mutans* CICC10438, and specifically shows the relative expression levels of the gene. Note: ** indicates a highly significant difference compared to *Streptococcus mutans* CICC10438 (p<0.01). ## Compared with commercial Lactobacillus rhamnosus GG, it showed a highly significant difference (p<0.01).
[0042] Figure 4 illustrates the inhibitory effect of the *Lactobacillus paracasei* HOM1212 strain of the present invention on the *Streptococcus mutans* CICC10438 tooth adhesion model, and specifically shows the fluorescence values. Note: ** indicates a highly significant difference compared to *Streptococcus mutans* CICC10438 (p<0.01). # The results showed a significant difference compared to commercial Lactobacillus rhamnosus GG (p<0.05).
[0043] Preservation Information:
[0044] The *Lacticaseibacillus paracasei* strain HOM1212 of this invention was deposited on July 24, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; accession number CGMCC No. 27974. This strain was identified at the Institute of Microbiology, Chinese Academy of Sciences.
[0045] The strain was sent to the Institute of Microbiology, Chinese Academy of Sciences for identification.
[0046] The identification conclusions are as follows: Under the conditions of this laboratory, based on a comprehensive analysis of experimental data including cell morphology, physiological and biochemical characteristics, 16S rRNA gene sequence, and dnaK gene sequence of the submitted bacterial strain, and referring to relevant research papers in *Bergey's Manual of Systematic Bacteriology* and the *International Journal of Systematic and Evolutionary Microbiology*, the identification result of the submitted bacterial strain (strain number: HOM1212) is: *Lactobacillus paracasei*. Synonym: *Lactobacillus paracasei*.
[0047] The cell morphology of this strain is rod-shaped; its physiological and biochemical characteristics are Gram-positive, catalase-negative (-), and oxidase-negative (-); the 16S rRNA gene sequence is shown in SEQ ID NO:1, and the dnaK gene sequence is shown in SEQ ID NO:2. Detailed Implementation
[0048] This invention discloses the bacterial strain and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.
[0049] It should be noted that, unless otherwise specified, the experimental methods used in this invention are all conventional methods.
[0050] Unless otherwise specified, all reagents and materials used in this invention are prepared using conventional methods or obtained commercially.
[0051] As identified above, *Lacticaseibacillus paracasei* and *Lactobacillus paracasei* are synonyms of the same species. Therefore, in this invention, *Lacticaseibacillus paracasei* and *Lactobacillus paracasei* can be used interchangeably.
[0052] Streptococcus mutans is the most common bacterium causing dental caries. The relationship between Streptococcus mutans and dental caries is mainly due to its ability to use sucrose as a substrate to produce water-insoluble extracellular glucans, fructans, and intracellular polysaccharides. Glucans mediate bacterial adhesion and promote plaque formation.
[0053] The *Lactaseibacillus paracasei* strain HOM1212 used in this invention was isolated from the feces of healthy infants in Beijing. Its physicochemical properties include: an optimal growth temperature of 37°C; antibacterial properties; good self-aggregation ability and good co-aggregation ability with *Streptococcus mutans*; inhibition of the proliferation of *Streptococcus mutans* and common pathogenic bacteria; tolerance to the oral environment; significant inhibition of *Streptococcus mutans* adhesion to hydroxyapatite tooth models; effective reduction of plaque biofilm formation caused by *Streptococcus mutans*; reduction of mature plaque biofilm content; low biofilm formation rate; reduced production of water-insoluble extracellular polysaccharides by *Streptococcus mutans*; and significant reduction of the expression levels of *Streptococcus mutans* cariogenic genes gtfD, ftf, and LuxS. It can be used in the preparation of functional products for the prevention or treatment of dental caries.
[0054] To further understand the present invention, the following detailed description of the *Lactobacillus paracasei* and its uses, in conjunction with embodiments, is provided. The scope of protection of the present invention is not limited by the following embodiments.
[0055] Example 1: Isolation and Identification of Lactobacillus paracasei strain HOM1212
[0056] (1) Preparation of culture medium
[0057] MRS liquid culture medium:
[0058] Mix 1 L of MRS medium (OXOID, CM1163) and double-distilled water thoroughly. Sterilize at 121°C for 20 min and set aside.
[0059] Modified MRS solid culture medium:
[0060] MRS medium (OXOID, CM1163), 0.05 g bromocresol green (Shanghai Sangon Biotech), 1 L double-distilled water, mixed thoroughly. Adjust pH to 5.5, sterilize at 121℃ for 20 min, and set aside.
[0061] (2) Isolation of laboratory strains
[0062] Weigh 1g of healthy infant feces and mix thoroughly with 9mL of 0.9% physiological saline. Perform serial dilutions using a 10-fold dilution method, selecting two suitable dilutions. Spread 100μL of each sample onto a modified MRS solid plate and incubate at 37℃ for 72h. Select single colonies with smooth surfaces, milky white or milky yellow coloration around the edges, streak, and purify. Observe colony morphology under a microscope. Select single colonies for pure culture in MRS liquid medium, preserve the culture with glycerol, and label it HOM1212.
[0063] (3) Isolation of commercial strains Lactobacillus paracasei S strain and Lactobacillus rhamnosus GG strain
[0064] Weigh 1g of bacterial powder or 1mL of product (containing either the commercial strain *Lactobacillus paracasei* S or *Lactobacillus rhamnosus* GG), mix thoroughly with 9mL of 0.9% physiological saline, and serially dilute the sample using a 10-fold dilution method. Select two appropriate dilutions, and spread 100μL onto modified MRS agar plates, incubating at 37℃ for 48h. Pick smooth, single colonies, streak, and purify. Observe the colony morphology under a microscope. Pick single colonies for pure culture in MRS liquid medium, preserve with glycerol, and label as *Lactobacillus paracasei* S and *Lactobacillus rhamnosus* GG strains.
[0065] (4) Identification of strains
[0066] The selected bacterial strain was inoculated into MRS liquid medium and cultured at 37°C for 24 h. Total bacterial DNA was extracted from the culture, and 16S rRNA gene amplification was performed. PCR amplification and agarose gel electrophoresis were conducted using universal primers 27F (5'-AGTTTGATCMTGGCTCAG-3', SEQ ID NO:3) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO:4). The gel was excised and the DNA was recovered, and sequencing was performed (by CGMCC). Then, BLAST was used to compare the results in the NCBI database, and strain HOM1212 was identified as *Lactaseibacillus casei*. The sequence of the 16S rRNA gene of strain HOM1212 is shown in SEQ ID NO:1.
[0067] The sequence of the dnaK gene of strain HOM1212 is shown in SEQ ID NO:2:
[0068] Example 2: Preparation process of active bacterial powder of Lactobacillus paracasei strain HOM1212
[0069] (1) Culture of bacterial strains
[0070] The *Lactobacillus paracasei* strain HOM1212 isolated in Example 1 was inoculated into MRS liquid medium at a 3% inoculum and cultured at 37°C for 24 hours, activating for two generations. When inoculated into MRS medium and cultured at 37°C, the viable count reached 1.8 × 10⁻⁶. 10 CFU / mL or higher.
[0071] (2) Freeze drying
[0072] The fermentation broth of *Lactobacillus paracasei* strain HOM1212 cultured in (1) above was centrifuged to collect bacterial sludge. The sludge was washed with 0.9% sterile saline, mixed with a preservative (100 g / L skim milk powder), and freeze-dried in a freeze dryer. The bacterial cake was then pulverized using a fine grinder to obtain the freeze-dried bacterial powder. The viable count of the freeze-dried bacterial powder was higher than 4.0 × 10⁻⁶. 11 CFU / g.
[0073] Example 3: Test on the ability of Lactobacillus paracasei HOM1212 strain to inhibit common pathogenic bacteria
[0074] (1) Activation of pathogenic bacteria
[0075] Five pathogenic bacteria were selected for this experiment: *Escherichia coli* (ATCC 8739), *Salmonella typhimurium* (ATCC 14028), *Staphylococcus aureus* (ATCC 6538), *Pseudomonas aeruginosa* (ATCC 9027), and *Listeria monocytogenes* (ATCC 19111). The pathogenic strains were purchased from ATCC (American Type Culture Collection).
[0076] The pathogenic strains were inoculated into nutrient agar medium at a rate of 1% of the total culture medium volume and cultured at 37°C with shaking at 200 rpm for 12 hours. The indicator strains were then prepared with fresh culture medium to an OD of 0.1 for later use.
[0077] (2) Activation of strains
[0078] In this experiment, the commercial strain *Lactobacillus rhamnosus* GG was selected as the control strain. The GG and HOM1212 strains isolated in Example 1 were inoculated into MRS liquid medium at an inoculum of 1% of the total culture medium volume, and cultured statically at 37°C for 24 hours. After two activations, the fermentation broth was obtained. The broth was centrifuged at 11000 rpm for 10 minutes, and the supernatant was used for an antibacterial test. The MRS liquid medium served as a negative control.
[0079] (3) Plate preparation
[0080] Pour the sterilized nutrient agar culture into a petri dish, add 100 μL of indicator bacterial solution to the culture medium, mix well, and let it stand to solidify.
[0081] (4) Antibacterial test
[0082] Using sterile forceps, gently place the Oxford cups onto the plate, maintaining a certain distance between the wells. Add 150 μL of fermentation supernatant to each well, incubate at 4°C for 12 h to diffuse, then incubate at 37°C for 18 h. Observe and measure the diameter of the inhibition zone. Table 1 shows the inhibitory effect of the strain on the pathogenic bacteria.
[0083] Table 1
[0084] Note: "–" indicates no antibacterial activity, <11mm; "+" indicates 11mm ≤ inhibition zone <16mm; "++" indicates 16mm ≤ inhibition zone <23mm; "+++" indicates ≥23mm.
[0085] Table 1 shows that *Lactobacillus paracasei* strain HOM1212 has inhibitory effects on all five pathogenic bacteria. The antibacterial ability of *Lactobacillus paracasei* strain HOM1212 is comparable to that of the commercial strain *Lactobacillus rhamnosus* GG.
[0086] Example 4: Tolerance of Lactobacillus paracasei HOM1212 strain to artificial saliva containing lysozyme
[0087] (1) Activation of the strain
[0088] The *Lactobacillus paracasei* strain HOM1212 isolated in Example 1 and the commercial strain *Lactobacillus paracasei* S were inoculated into MRS liquid medium at a 3% inoculum and cultured at 37°C for 24 h, followed by two activation cultures. After activation, the strains were centrifuged at 11,000 rpm for 10 min, the supernatant was removed, and the viable count was adjusted to 1.5 × 10⁻⁶ cells / mL with sterile phosphate-buffered saline (PBS). 9 CFU / mL.
[0089] Streptococcus mutans CICC10438 Streptococcus mutans CICC10438 was selected from children with dental caries and purchased from the China Industrial Microbial Culture Collection Center. CICC10438 was inoculated at a 3% inoculum into brain and heart infusion (BHI) liquid medium and anaerobically cultured at 37°C for 24 hours, followed by two activation cultures. After two activations, the strain was centrifuged at 11,000 rpm for 10 minutes, the supernatant was removed, and the viable count was adjusted to 1.5 × 10⁻⁶ cells / day using sterile PBS. 9 CFU / mL.
[0090] (2) Experiment on the strain's tolerance to artificial saliva containing lysozyme
[0091] The strain was transferred into sterile artificial saliva containing 1 mg / mL lysozyme and cultured at 37°C. The viable cell counts were measured at 0 h and 6 h to assess the strain's tolerance to artificial saliva containing lysozyme.
[0092] (3) Tolerance
[0093] Where T1 represents the number of viable bacteria of the strain at t=6 hours; T0 represents the number of viable bacteria of the strain at t=0 hours. Table 2 shows the tolerance of the strains to artificial saliva containing 1 mg / mL lysozyme.
[0094] Table 2
[0095] Note: ** indicates a significant difference compared to Streptococcus mutans CICC10438 (p<0.01). # # The results showed a significant difference compared to the commercial Lactobacillus paracasei S strain (p<0.01).
[0096] Table 2 shows that compared with *Streptococcus mutans* CICC10438 and the commercial strain *Lactobacillus paracasei* S, *Lactobacillus paracasei* HOM1212 strain exhibited significantly stronger tolerance in artificial saliva containing 1 mg / mL lysozyme (p<0.01). After 6 hours of incubation in artificial saliva containing 1 mg / mL lysozyme, the survival rate of *Lactobacillus paracasei* HOM1212 strain was 92.55% ± 3.61%. This study indicates that oral saliva contains abundant lysozyme, thus enabling *Lactobacillus paracasei* HOM1212 strain to maintain a high survival rate in the oral cavity, thereby exerting a beneficial effect.
[0097] Example 5: Self-aggregation ability of Lactobacillus paracasei strain HOM1212
[0098] (1) Activation of the strain
[0099] The *Lactobacillus paracasei* strains HOM1212 and S isolated in Example 1 were inoculated into MRS liquid medium at a 3% inoculum and cultured at 37°C for 24 h, followed by two activation cultures. After activation, the strains were centrifuged at 11,000 rpm for 10 min, the supernatant was removed, and the suspension was resuspended three times with sterile PBS buffer.
[0100] Streptococcus mutans CICC10438 was inoculated into BHI liquid medium at a 3% inoculum and cultured at 37°C for 24 h, followed by two activation cultures. After two activations, the strain was centrifuged at 11,000 rpm for 10 min, the supernatant was removed, and the strain was resuspended three times in sterile PBS buffer.
[0101] (2) Self-aggregation experiment
[0102] The initial absorbance of the strain was adjusted to A0 = 0.6 ± 0.02 using sterile PBS buffer and allowed to stand at room temperature. The absorbance (A0) of the suspension was measured at 1 h, 2 h, and 3 h. t (OD=600).
[0103] (3) Self-aggregation ability
[0104] Where A t The absorbance values are indicated at t = 1, 2, and 3 hours. Table 3 shows the self-aggregation ability of the strains.
[0105] Table 3
[0106] Note: ** indicates a highly significant difference compared to Streptococcus mutans CICC10438 (p<0.01). ## The results showed a highly significant difference compared to the commercial Lactobacillus paracasei S strain (p<0.01).
[0107] Table 3 shows that the self-aggregation ability of the strains gradually increased with time. The self-aggregation abilities of *Lactobacillus paracasei* strain HOM1212 and the commercial *Lactobacillus paracasei* S strain were both higher than those of *Streptococcus mutans* CICC10438 (p<0.01). Compared with the commercial *Lactobacillus paracasei* S strain, *Lactobacillus paracasei* strain HOM1212 had a significantly higher self-aggregation ability (p<0.01). This study indicates that a higher self-aggregation ability contributes to the reproduction and survival of the strains in the oral cavity.
[0108] Example 6: Co-aggregation ability of Lactobacillus paracasei strain HOM1212 and Streptococcus mutans CICC10438
[0109] (1) Activation of the strain
[0110] The *Lactobacillus paracasei* strains HOM1212 and S isolated in Example 1 were inoculated into MRS liquid medium at a 3% inoculum and cultured at 37°C for 24 h, followed by two activation cultures. After activation, the strains were centrifuged at 11,000 rpm for 10 min, the supernatant was removed, and the suspension was resuspended 2-3 times with sterile PBS buffer.
[0111] Streptococcus mutans CICC10438 was inoculated into BHI liquid medium at a 3% inoculum and cultured anaerobically at 37°C for 24 h, followed by two activation cultures. After two activations, the strain was centrifuged at 11,000 rpm for 10 min, the supernatant was removed, and the strain was resuspended three times in sterile PBS buffer.
[0112] (2) Coagulation experiment
[0113] Equal volumes of the probiotic *Lactobacillus paracasei* strain HOM1212 and *Lactobacillus paracasei* strain S were mixed with *Streptococcus mutans* strain CICC10438, respectively. In the control group, the probiotics were replaced with sterile PBS buffer. The absorbance of the supernatant was measured at 1 h, 2 h, and 3 h, respectively. The results are shown in Figure 1.
[0114] (3) Coagulation ability
[0115] Where A0 and B0 represent the initial absorbance of *Lactobacillus paracasei* strain HOM1211 and *Streptococcus mutans* strain CICC10438, and C... t This indicates the absorbance of the mixture measured at t = 1, 2, and 3 hours.
[0116] As shown in Figure 1, the co-aggregation ability of the probiotic *Lactobacillus paracasei* strain HOM1211 and strain S with *Streptococcus mutans* CICC10438 increased with time. Compared with the PBS control group, the co-aggregation ability of *Lactobacillus paracasei* strain HOM1211 with *Streptococcus mutans* CICC10438 showed a highly significant difference at 2 h and 3 h (p < 0.01). Compared with *Lactobacillus paracasei* strain S, the *Lactobacillus paracasei* strain HOM1212 showed a highly significant difference at 1 h (p < 0.01). This study indicates that the high co-aggregation ability of *Lactobacillus paracasei* strain HOM1211 with *Streptococcus mutans* can reduce the adhesion and colonization of free *Streptococcus mutans* in the oral cavity and reduce the formation of dental biofilms.
[0117] Example 7: Inhibitory effect of Lactobacillus paracasei HOM1212 strain on Streptococcus mutans CICC10438
[0118] (1) Activation of the strain
[0119] The *Lactobacillus paracasei* strains HOM1212 and S isolated in Example 1 were inoculated into MRS liquid medium at a 3% inoculum and cultured at 37°C for 24 h, followed by two activation cultures. After activation, the strains were centrifuged at 11,000 rpm for 10 min, the supernatant was removed, and the bacterial count was adjusted with sterile PBS.
[0120] Streptococcus mutans CICC10438 was inoculated into BHI liquid medium at a 3% inoculum and cultured at 37°C for 24 h, followed by two activation cultures. After two activations, the strain was centrifuged at 11,000 rpm for 10 min, the supernatant was removed, and the bacterial count was adjusted with sterile PBS.
[0121] (2) Co-culture experiment with Streptococcus mutans CICC10438
[0122] The viable counts of the probiotic strains *Lactobacillus paracasei* HOM1212, *Lactobacillus paracasei* S, and *Streptococcus mutans* CICC10438 were adjusted to 1.5 × 10⁻⁶. 8 CFU / mL, 125 μL of Lactobacillus paracasei strain HOM1212 and Lactobacillus paracasei strain S were respectively inoculated into BHI liquid medium along with Streptococcus mutans CICC10438 and cultured anaerobicly at 37°C for 24 h. In the control group, Lactobacillus paracasei strain HOM1212 was replaced with sterile PBS. After co-culture, colony counting was performed using BHI solid medium.
[0123] (3) Inhibition rate:
[0124] Table A shows the viable count of *Streptococcus mutans* CICC10438 after co-culturing the probiotic *Lactobacillus paracasei* strain HOM1212 and strain S with *Streptococcus mutans* CICC10438, respectively; Table B shows the viable count of *Streptococcus mutans* CICC10438 in the control group. Table 4 shows the inhibitory effect of the probiotics *Lactobacillus paracasei* strain HOM1212 and strain S on *Streptococcus mutans* CICC10438.
[0125] Table 4
[0126] Note: ** indicates a highly significant difference compared to the commercial Lactobacillus paracasei S strain (p<0.01).
[0127] Table 4 shows that both the probiotic strains *Lactobacillus paracasei* HOM1212 and *Lactobacillus paracasei* S can inhibit the growth of *Streptococcus mutans* CICC10438. Compared with the commercial *Lactobacillus paracasei* S strain, *Lactobacillus paracasei* HOM1212 strain has a stronger inhibitory effect on *Streptococcus mutans* CICC10438 (p<0.01).
[0128] Example 8: Biofilm formation ability of Lactobacillus paracasei strain HOM1212
[0129] (1) Activation of the strain
[0130] The *Lactobacillus paracasei* strain HOM1212 and *Lactobacillus casei* strain S isolated in Example 1 were inoculated into MRS liquid medium at an inoculum volume of 3% and cultured at 37°C for 24 h, with activation repeated twice. After activation, the strains were centrifuged at 11000 rpm for 10 min, the supernatant was removed, and the suspension was resuspended three times with sterile physiological saline. Sterile PBS was used as a blank control to adjust the OD value. 600 =0.5±0.02.
[0131] Streptococcus mutans CICC10438 was inoculated at a 3% inoculum into BHI liquid medium and anaerobically cultured at 37°C for 24 h, activating twice. After two activations, the strain was centrifuged at 11000 rpm for 10 min, the supernatant was removed, and the suspension was resuspended 2-3 times in sterile PBS. Sterile PBS was used as a blank control to adjust the OD value. 600 =0.5±0.02.
[0132] (2) Biofilm formation ability of strains
[0133] The probiotic strains of Lactobacillus paracasei HOM1212 and Lactobacillus paracasei S were inoculated into MRS + 2% sucrose medium, while Streptococcus mutans was inoculated into BHI + 2% sucrose medium. After mixing thoroughly, the mixtures were transferred to 96-well plates for culture and anaerobic culture at 37°C for 48 hours.
[0134] (3) Determination of biofilm content
[0135] Rinse the 96-well plate twice with sterile water, air dry, and stain with 200 μL of 0.1% crystal violet for 15 min. After staining, remove the crystal violet, rinse twice with sterile water, and incubate at 37°C for 30 min to dry. Then add 200 μL of 30% glacial acetic acid to dissolve and reflux for at least 30 min. Read the values using a microplate reader at a wavelength of 570 nm. Table 5 shows the biofilm formation ability of the strain.
[0136] Table 5
[0137] Note: ** indicates a highly significant difference compared to Streptococcus mutans CICC10438 (p<0.01).
[0138] As shown in Table 5, the biofilm formation ability of the two probiotic strains, *Lactobacillus paracasei* HOM1212 and *Lactobacillus paracasei* S, was significantly lower than that of *Streptococcus mutans* CICC10438. Compared with *Streptococcus mutans* CICC10438, the biofilm formation of the two probiotic strains *Lactobacillus paracasei* HOM1212 and *Lactobacillus paracasei* S showed a highly significant difference (p<0.01). Therefore, ingestion of *Lactobacillus paracasei* HOM1212 does not exacerbate biofilm formation or tooth decay.
[0139] Example 9: Inhibitory effect of Lactobacillus paracasei HOM1212 strain on biofilm formation by Streptococcus mutans CICC10438
[0140] (1) Activation of the strain
[0141] The *Lactobacillus paracasei* strains HOM1212 and S isolated in Example 1 were inoculated into MRS liquid medium at a 3% inoculum and cultured at 37°C for 24 h, activating twice. After activation, the supernatant was removed by centrifugation at 11,000 rpm for 10 min.
[0142] Streptococcus mutans CICC10438 was inoculated into BHI liquid medium at a 3% inoculum and anaerobically cultured at 37°C for 24 h, activating twice. After centrifugation at 11,000 rpm for 10 min, the supernatant was removed.
[0143] (2) Probiotics Lactobacillus paracasei strain HOM1212 and Lactobacillus paracasei strain S were co-cultured with Streptococcus mutans CICC10438, respectively.
[0144] The viable counts of the probiotic *Lactobacillus paracasei* strain HOM1212 and *Lactobacillus casei* strain S, along with *Streptococcus mutans* strain CICC10438, were adjusted to 1.5 × 10⁻⁶ using fresh MRS + 2% sucrose and BHI + 2% sucrose medium. 8 CFU / mL was added at a 1:1 ratio. In the control group, the probiotics were replaced with MRS + 2% sucrose medium. After mixing evenly, the mixture was added to a 96-well plate and anaerobic fermented at 37°C for 24h and 48h.
[0145] (3) Determination of biofilm content
[0146] After fermentation, the samples in the 96-well plate were rinsed twice with sterile water, air-dried, and stained with 200 μL of 0.1% crystal violet for 15 min. After staining, the crystal violet was removed, rinsed twice with sterile water, and placed in an incubator at 37℃ to air-dry for 30 min. Then, 200 μL of 30% glacial acetic acid was added to dissolve the samples for at least 30 min. The results were read using a microplate reader at a wavelength of 570 nm. The results are shown in Figure 2.
[0147] As shown in Figure 2, when the probiotic *Lactobacillus paracasei* strain HOM1212 and strain S were co-cultured with *Streptococcus mutans* CICC10438 for 24 h and 48 h, respectively, *Lactobacillus paracasei* strain HOM1212 significantly inhibited biofilm formation of *Streptococcus mutans* CICC10438 (p<0.01). However, strain S only significantly inhibited biofilm formation of *Streptococcus mutans* CICC10438 at 48 h (p<0.01). Compared with strain S, *Lactobacillus paracasei* strain HOM1212 showed a much stronger ability to inhibit biofilm formation by *Streptococcus mutans* CICC10438 at both 24 h and 48 h (p<0.01), indicating that strain HOM1212 has a stronger ability to inhibit biofilm formation by *Streptococcus mutans* CICC10438.
[0148] Example 10: Inhibitory effect of Lactobacillus paracasei HOM1212 strain on mature biofilm of Streptococcus mutans CICC10438
[0149] (1) Preparation of probiotic supernatant
[0150] Probiotic strains of *Lactobacillus paracasei* HOM1212 and *Lactobacillus paracasei* S were inoculated into fresh MRS liquid medium at a 3% inoculum and cultured anaerobically at 37°C for 24 h. After two generations of activation, the supernatant was obtained by centrifugation at 11,000 rpm for 10 min. The pH was adjusted to 6.5 with 1.0 M NaOH, and the bacterial cells were removed by filtration through a 0.22 μm filter membrane. The supernatant was then stored at 4°C for later use.
[0151] (2) Preparation of mature biofilm of Streptococcus mutans CICC10438
[0152] Streptococcus mutans CICC10438 was inoculated at a 3% inoculum into fresh BHI liquid medium and cultured anaerobically at 37°C for 24 h, activating for two generations. After centrifugation, Streptococcus mutans CICC10438 was resuspended twice in sterile PBS and then OD was calculated using fresh BHI medium containing 0.2% sucrose. 600 Adjust the concentration to 0.5, mix well, and add 100 μL to a 96-well plate. Incubate anaerobically at 37°C for 24 h.
[0153] (3) Effects of the supernatants of probiotics Lactobacillus paracasei strain HOM1212 and Lactobacillus paracasei strain S on the mature biofilm of Streptococcus mutans CICC10438
[0154] Remove the supernatant of Streptococcus mutans CICC10438 after the culture is completed, rinse once with sterile PBS, and then add 100 μL of the treated strain supernatant. In the control group, the probiotic supernatant was replaced with fresh MRS medium (pH=6.5), and the cultures were co-cultured at 37°C for 24 h.
[0155] (4) Determination of biofilm content
[0156] After fermentation, the samples in the 96-well plates were rinsed twice with sterile water, air-dried, and stained with 200 μL of 0.1% crystal violet for 15 min. After staining, the crystal violet was removed, rinsed twice with sterile water, and placed in an incubator at 37°C to air-dry for 30 min. Then, 200 μL of 30% glacial acetic acid was added to dissolve the samples for at least 30 min. The results were read using a microplate reader at a wavelength of 570 nm. Table 6 shows the inhibitory effects of the fermentation supernatants of *Lactobacillus paracasei* strain HOM1212 and strain S on the mature biofilm of *Streptococcus mutans* CICC10438.
[0157] Table 6
[0158] Note: ** indicates a highly significant difference compared to Streptococcus mutans CICC10438 (p<0.01).
[0159] As shown in Table 6, after adjusting the fermentation supernatant of the probiotic Lactobacillus paracasei strain HOM1212 and Lactobacillus paracasei strain S to pH 6.5, the fermentation supernatant still had a highly significant inhibitory effect on the mature biofilm of Streptococcus mutans CICC10438 (p<0.01), indicating that the non-acidic metabolites produced by Lactobacillus paracasei strain HOM1212 have the effect of inhibiting the formation of biofilm by Streptococcus mutans.
[0160] Example 11: Effect of Lactobacillus paracasei HOM1212 strain on the content of water-insoluble extracellular polysaccharides produced by Streptococcus mutans CICC10438
[0161] (1) Activation of the strain
[0162] The *Lactobacillus paracasei* strain HOM1212 and *Lactobacillus rhamnosus* strain GG isolated in Example 1 were inoculated into MRS liquid medium at a 3% inoculum and cultured at 37°C for 24 h, activating twice. After activation, the strains were centrifuged at 11,000 rpm for 10 min, the supernatant was removed, and the samples were washed twice with sterile PBS for later use.
[0163] Streptococcus mutans CICC10438 was inoculated into BHI liquid medium at a 3% inoculum and cultured anaerobically at 37°C for 24 h. After the strain was activated twice, the supernatant was removed by centrifugation at 11,000 rpm for 10 min, and the strain was washed twice with sterile PBS for later use.
[0164] (2) Probiotics Lactobacillus paracasei strain HOM1212 and Lactobacillus rhamnosus strain GG were co-cultured with Streptococcus mutans CICC10438, respectively.
[0165] The viable counts of the probiotic strains *Lactobacillus paracasei* HOM1212 and *Lactobacillus rhamnosus* GG were adjusted to 1.5 × 10⁻⁶ using fresh MRS and 2% sucrose. 8 CFU / mL, the viable count of Streptococcus mutans CICC10438 was adjusted to 1.5 × 10⁻⁶ CFU / mL using fresh BHI + 2% sucrose medium. 8 CFU / mL. Probiotics *Lactobacillus paracasei* HOM1212 and *Lactobacillus rhamnosus* GG were added to *Streptococcus mutans* CICC10438 at a 1:1 ratio. In the control group, the probiotic culture was replaced with fresh MRS + 2% sucrose. After mixing evenly, the mixture was added to 24-well plates and anaerobic fermented at 37°C for 48 hours.
[0166] (3) Determination of water-insoluble extracellular polysaccharide content
[0167] After culturing the samples in 24-well plates, the supernatant was removed, 2 mL of sterile water was added, and 1 mL was centrifuged. The samples were resuspended three times with sterile water to remove water-soluble extracellular polysaccharides. The water-insoluble extracellular polysaccharides were dissolved in 1.0 M NaOH and stirred at 37 °C for 2 h. The content was determined using the phenol-concentrated sulfuric acid method. Table 7 shows the effects of the probiotic strains *Lactobacillus paracasei* HOM1212 and *Lactobacillus rhamnosus* GG on the content of water-insoluble extracellular polysaccharides produced by *Streptococcus mutans* CICC10438.
[0168] Table 7
[0169] Note: ## indicates a highly significant difference compared to the commercial Lactobacillus rhamnosus GG strain (p<0.01).
[0170] Studies have shown that water-insoluble extracellular polysaccharides are key factors in the formation of tooth biofilms and dental caries by *Streptococcus mutans*. Table 7 shows that both *Lactobacillus paracasei* strain HOM1212 and *Lactobacillus rhamnosus* strain GG significantly reduced the formation of water-insoluble extracellular polysaccharides in *Streptococcus mutans* CICC10438, with inhibition rates of 90.83% ± 0.28% and 75.44% ± 0.29%, respectively. This indicates that both *Lactobacillus paracasei* strain HOM1212 and *Lactobacillus rhamnosus* strain GG can inhibit the formation of water-insoluble polysaccharides by *Streptococcus mutans*, thus preventing dental caries.
[0171] Example 12 Effect of Lactobacillus paracasei HOM1212 strain on the expression level of cariogenic gene in Streptococcus mutans CICC10438
[0172] (1) Activation of the strain
[0173] The *Lactobacillus paracasei* strain HOM1212 and *Lactobacillus rhamnosus* strain GG isolated in Example 1 were inoculated into MRS liquid medium at a 3% inoculum and cultured at 37°C for 24 h, activating for two generations. After activation, the strains were centrifuged at 11,000 rpm for 10 min, the supernatant was removed, and the samples were washed twice with sterile physiological saline.
[0174] Streptococcus mutans CICC10438 was inoculated into BHI liquid medium at a 3% inoculum and cultured anaerobically at 37°C for 24 h. After the strain was activated twice, the supernatant was removed by centrifugation at 11,000 rpm for 10 min and the strain was washed twice with sterile physiological saline.
[0175] (2) Probiotics Lactobacillus paracasei strain HOM1212 and Lactobacillus rhamnosus strain GG were co-cultured with Streptococcus mutans CICC10438, respectively.
[0176] The probiotic strains *Lactobacillus paracasei* HOM1212 and *Lactobacillus rhamnosus* GG were adjusted to the same viable count as *Streptococcus mutans* CICC10438 using fresh MRS + 2% sucrose and BHI + 2% sucrose media, respectively. 200 μL of *Lactobacillus paracasei* HOM1212 and *Lactobacillus rhamnosus* GG were added to the upper chamber of a Transwell plate, while 600 μL of *Streptococcus mutans* CICC10438 was added to the lower chamber. The control group had only MRS + 2% sucrose medium in its upper chamber. The plates were then anaerobically cultured at 37°C for 48 h.
[0177] (3) RT-qPCR technology
[0178] RNA was extracted from Streptococcus mutans CICC10438 and subjected to quality testing. If the purity, concentration, and integrity of the RNA met the requirements for subsequent experiments, it would proceed to the next step of reverse transcription. After reverse transcription of RNA into cDNA, the expression levels of the internal reference gene and the target gene were measured using real-time quantitative PCR. The results were then processed and analyzed. The cariogenic genes and primer designs are shown in Table 8. The housekeeping gene gyrA was selected as the internal reference gene. The results are shown in Figure 3.
[0179] Table 8. Internal reference genes and cariogenic genes of Streptococcus mutans and their primer design
[0180] Studies have shown that gtfD is associated with the formation of dextran in biofilms, while ftf is associated with the formation of fructan in biofilms. Inhibiting the expression of gtfD and ftf can reduce biofilm formation, thereby lowering the risk of dental caries. Furthermore, the LuxS gene is involved in the regulation of oxidative stress tolerance in Streptococcus mutans, and can reduce the adaptability of Streptococcus mutans to the environment.
[0181] As shown in Figure 3, compared with the control group of *Streptococcus mutans* CICC10438, the *Lactobacillus paracasei* HOM1212 strain significantly reduced the expression of the cariogenic genes gtfD, ftf, and LuxS of *Streptococcus mutans* CICC10438 (p<0.01), while the *Lactobacillus rhamnosus* GG strain significantly reduced the expression of ftf and LuxS (p<0.01), but did not reduce the expression of gtfD (p>0.05). Compared with the commercial strain *Lactobacillus rhamnosus* GG, the *Lactobacillus paracasei* HOM1212 strain showed a more significant effect in inhibiting the expression of gtfD / LuxS genes (p<0.01). This indicates that the *Lactobacillus paracasei* HOM1212 strain can effectively reduce the expression of the above three cariogenic genes of *Streptococcus mutans*, thereby affecting the limitation of cariogenicity of *Streptococcus mutans* in the oral cavity.
[0182] Example 13 Effect of Lactobacillus paracasei HOM1212 strain on the adhesion of Streptococcus mutans CICC10438 on a dental model
[0183] (1) Preparation of hydroxyapatite
[0184] Hydroxyapatite was suspended in sterile potassium chloride (KCl) buffer, washed 10 times with distilled water, and dried in a constant temperature oven at 60°C for 48 hours. 12 mg of the treated hydroxyapatite and 1 mL of sterile KCl buffer were added to a suitable tube and placed at 4°C for 16 hours for adhesion experiments.
[0185] (2) Activation of the strain
[0186] In Example 1, the probiotic strains *Lactobacillus paracasei* HOM1212 and *Lactobacillus rhamnosus* GG isolated were activated twice with *Streptococcus mutans* CICC10438. The activated strains were then washed three times with sterile KCl buffer. The viable counts of *Lactobacillus paracasei* HOM1212 and *Lactobacillus rhamnosus* GG were adjusted to 1.5 × 10⁻⁶ cells / year. 8 The CFU / mL count of Streptococcus mutans CICC10438 was adjusted to 7.5 × 10⁻⁶. 7 CFU / mL.
[0187] (3) Pre-incubation of probiotic Lactobacillus paracasei strain HOM1212 and Lactobacillus rhamnosus strain GG with artificial saliva containing bovine serum albumin (BSA).
[0188] In the experimental group, bacterial suspensions of the probiotic *Lactobacillus paracasei* strain HOM1212 and *Lactobacillus rhamnosus* strain GG were mixed with saliva at a 1:1 volume ratio and incubated anaerobically at 37°C for 1 h. After incubation, the samples were filtered through a 0.22 μm filter membrane. In the control group, the probiotics were replaced with sterile KCl buffer.
[0189] (4) Establishment of a dental model and adhesion of *Lactobacillus paracasei* strain HOM1212 and *Lactobacillus rhamnosus* strain GG to *Streptococcus mutans* CICC10438.
[0190] The pre-incubated saliva was mixed with hydroxyapatite at room temperature for 1 hour. After mixing, the saliva was removed, and the sample was washed three times with sterile KCl buffer. Streptococcus mutans CICC10438 was added, and the mixture was mixed at room temperature for 1 hour. After mixing, the sample was washed three times with sterile KCl buffer to remove unattached Streptococcus mutans CICC10438. The sample was sonicated for 15 minutes, and the attached bacterial cells were stained with Nile red for 1 hour. The cell-related fluorescence intensity was measured (excitation wavelength 530 nm, emission wavelength 570 nm). The results are shown in Figure 4.
[0191] The adhesion of *Streptococcus mutans* to teeth is a crucial step in its biofilm formation process. As shown in Figure 4, both *Lactobacillus paracasei* strain HOM1212 and *Lactobacillus rhamnosus* strain GG reduced the adhesion ability of *Streptococcus mutans* CICC10438 to the tooth model (p<0.01). This indicates that *Lactobacillus paracasei* strain HOM1212 can effectively inhibit the adhesion of *Streptococcus mutans* to teeth, thereby reducing biofilm and caries formation.
[0192] Example 14 Fermented Milk Experiment
[0193] Weigh 12g of skim milk powder, 2g of glucose, and 2g of yeast powder. Add double-distilled water to make up to 100mL, stir well, homogenize with a high-pressure homogenizer (60℃, 22MPa), sterilize at 95℃ for 10min, and cool to 37℃ for later use.
[0194] The *Lactobacillus paracasei* strain HOM1212 prepared in Example 1 was inoculated into MRS liquid medium and cultured at 37°C for 18 hours. This method was repeated twice to obtain a highly viable bacterial culture for later use.
[0195] The obtained bacterial suspension was eluted and resuspended with 0.9% physiological saline, and then subjected to a 1×10⁻⁶ solution. 7 Inoculate milk with CFU / mL, mix thoroughly, and incubate at 37℃ for 16 hours to obtain fermented dairy products. The fermented milk contains more than 4.0 × 10⁻⁶ active *Lactobacillus paracasei* strain HOM1212. 8 With a concentration of CFU / mL and a pH of up to 4.60, it has a rich, mellow, and sweet-and-sour taste.
[0196] Example 15 Fermented Oat Milk Experiment
[0197] Weigh 120g of enzymatically hydrolyzed oat flour and 20g of yeast powder, add double-distilled water to make up to 1000mL, homogenize at 200bar, sterilize at 90℃ for 10min, and cool to 37℃ for later use.
[0198] The seed culture of Lactobacillus paracasei HOM1212 prepared in Example 1 was inoculated into MRS liquid medium and cultured at 37°C for 18 hours. After two subcultures, a high concentration of bacterial culture was obtained and stored at low temperature for later use.
[0199] The bacterial suspension was eluted and resuspended with 0.9% physiological saline, and then subjected to a 1×10⁻⁶ solution. 7 Inoculate the oat milk with an inoculum concentration of CFU / mL, mix thoroughly, and incubate at a constant temperature of 37℃ for 6 hours to obtain fermented oat milk. The viable count of *Lactobacillus paracasei* strain HOM1212 in the fermented milk is higher than 8.5 × 10⁻⁶. 8 CFU / mL, pH can be reduced to 4.50, and it has a rich flavor.
[0200] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details shown and described herein.
Claims
1. A Lacticaseibacillus paracasei strain, characterized in that, The Paracaseiclostridium strain has a preservation number of CGMCC No. 27974.
2. The strain of Paracaseicolus paracasei according to claim 1, characterized in that, The Paracaseiclostridium strain comprises a 16S rRNA gene represented by SEQ ID NO:
1.
3. A live bacterial preparation, characterized in that, The live bacterial preparation comprises the Paracaseiclostridium strain according to claim 1 or 2 and an excipient.
4. The live bacterial preparation according to claim 3, characterized in that, The live bacteria preparation comprises 4.0 x 10 11 CFU / g or more of live bacteria.
5. The live bacterial preparation according to claim 3 or 4, characterized in that, The excipient is selected from one or more of starch, sucrose, trehalose and glycerol.
6. A food or health product comprising the live bacterial preparation according to any one of claims 3 to 5.
7. Use of the Paracaseiclostridium strain according to claim 1 or 2 or the live bacterial preparation according to any one of claims 3 to 5 in the preparation of an article for preventing or treating dental caries.
8. Use according to claim 7, characterized in that, The strain or the live bacterial preparation is used for inhibiting the growth of Streptococcus mutans.
9. Use according to claim 7, characterized in that, The strain or the live bacterial preparation is used for reducing the formation of dental plaque biofilm caused by Streptococcus mutans, and reducing the content of mature dental plaque biofilm.
10. Use according to claim 7, characterized in that, The strain or the live bacterial preparation is used for inhibiting the level of water-insoluble exopolysaccharide produced by Streptococcus mutans.
11. Use according to claim 7, characterized in that, The strain or the live bacterial preparation is used for reducing the expression level of cariogenic genes gtfB, gtfC, gtfD, ftf and LuxS of Streptococcus mutans.
12. Use according to claim 7, characterized in that, The strain or the live bacterial preparation is used for reducing adhesion rate of Streptococcus mutans on hydroxyapatite tooth model.
13. The use according to claim 7, characterized in that, The article is a food, a health product, a pharmaceutical product or an oral care product.
14. Use according to claim 13, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The oral care product is selected from one or more of mouthwash, beverage, mouth spray, chewing gum, dental gel, toothpaste, tooth powder, dental floss, dental cleaning liquid, dental cleaning foam.
15. A fermentation product, characterized in that, The fermentation product is obtained by fermentation using the Paracaseiclostridium strain according to claim 1 or 2.
16. The fermentation product of claim 15, wherein, The fermentation product is fermented bovine milk, the content of active Paracasei strain in the fermented bovine milk is 4.0 x 10 8 CFU / mL or more.
17. The fermentation product of claim 15, wherein, The fermentation product is a fermented oat milk, in which the number of viable bacteria of the Paracasei strain is above 8.5 x 10 8 CFU / mL.
18. A method for preparing the live bacterial preparation according to any one of claims 3 to 5, comprising the following steps: scaling up the Paracaseiclostridium strain according to claim 1 or 2 in an optimized liquid medium; collecting the bacterial cells; adding a protective agent for resuspension, vacuum freeze-drying, and grinding to obtain the live bacterial preparation.