CN121203922A

CN121203922A - Pediococcus pentosaceus with caries and periodontal disease preventing or treating effects, and preparation and application thereof

Info

Publication number: CN121203922A
Application number: CN202511783763.2A
Authority: CN
Inventors: 王永平; 杨树俊; 王永存; 王小珊; 孟祥烁; 格日乐其木格
Original assignee: Weikaihaisi Shandong Bioengineering Co ltd
Current assignee: Weikaihaisi Shandong Bioengineering Co ltd
Priority date: 2025-12-01
Filing date: 2025-12-01
Publication date: 2025-12-26

Abstract

This invention relates to the field of probiotics technology, specifically to a strain of *Pediococcus pentosaceus* with efficacy in preventing or treating dental caries and periodontal disease, its preparations, and applications. This *Pediococcus pentosaceus* was deposited on May 15, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 27356. The *Pediococcus pentosaceus* provided by this invention can significantly inhibit the growth and reproduction of oral pathogens such as *Streptococcus mutans*, *Porphyromonas gingivalis*, *Fusobacterium nucleatum*, *Actinomyces villosa*, and *Aggregobacter actinomycetes*, thus promoting oral flora balance and can be widely used for the prevention or treatment of dental caries, periodontitis, and other oral diseases.

Description

Pediococcus pentosaceus with caries and periodontal disease preventing or treating effects, and preparation and application thereof

Technical Field

The invention relates to the technical field of probiotics, in particular to Pediococcus pentosaceus with the efficacy of preventing or treating dental caries and periodontal disease, and a preparation and application thereof.

Background

Caries is a chronic progressive destruction of hard tissue of teeth that occurs under the combined action of a number of bacteria-based factors. The caries formation process is mainly that pathogenic bacteria in the oral cavity metabolize carbohydrates in food residues to generate acidic substances which act on hard tissues of teeth for a long time to cause demineralization of enamel and dentin, thereby forming caries cavities. If the intervention is not performed in time, the lesions can continuously develop, so that not only can the teeth pain be caused and the chewing function be influenced, but also complications such as pulpitis, periapical periodontitis and the like can be caused, and even the whole body health is influenced. Periodontal disease is a group of diseases characterized by inflammation and destruction of periodontal tissues, mainly comprising gingivitis and periodontitis, and is frequently clinically manifested by gingival red swelling and bleeding, periodontal pocket formation and alveolar bone absorption, and the later stage of periodontal disease can also cause loosening, displacement and even shedding of teeth, which seriously affects the functions and the beauty of the oral and jaw systems of patients. When the oral cavity is in microecological unbalance, the conditional pathogenic bacteria are converted into dominant pathogenic bacteria, periodontal diseases are caused by invasion of periodontal tissues, initiation of inflammatory reaction and other mechanisms, and a plurality of inconveniences are brought to the daily life of patients.

Pathogenic bacteria closely related to caries and periodontal disease occurrence mainly include Streptococcus mutans, fusobacterium nucleatum, porphyromonas gingivalis, actinomyces viscosus and Actinobacillus actinomycetemcomitans. Streptococcus mutans is a recognized major cariogenic bacteria which can produce a large amount of acidic metabolites, has strong dental surface adhesion capability, can form a biological film on the surface of teeth, continuously damages hard tissues of the teeth, and is a core pathogenic bacteria causing occurrence and development of dental caries. Porphyromonas gingivalis is one of key pathogenic bacteria of periodontitis, and the strain can generate various toxins and enzymes, destroy the structure and functions of periodontal tissue cells, inhibit immune defense reaction of a host, accelerate periodontal pocket formation and alveolar bone resorption, and cause serious threat to periodontal health. Fusobacterium nucleatum is an important periodontal disease related pathogenic bacteria which is often present in deep periodontal pockets, has pathogenic effects, and can promote the colonization and inflammatory diffusion of bacterial flora in periodontal tissues, and aggravate the destruction of periodontal tissues. The actinomycetes viscosus can participate in the formation of dental plaque biomembrane, and acidic substances generated by metabolism of the actinomycetes viscosus can damage hard tissues of teeth, can trigger inflammatory reaction of gingival tissues, and are common related pathogenic bacteria of dental caries and gingivitis. Actinobacillus conglomerates is a main pathogenic bacterium for invasive periodontitis, has strong invasive capability, can directly invade periodontal tissues, causes rapid progress of inflammation, causes severe alveolar bone resorption, and has relatively light onset age, thus having great harm to oral health of teenagers and young adults. These pathogens interact and cooperate to cause disease in the oral cavity, which in turn promotes the occurrence and development of dental caries and periodontal disease.

The traditional oral disease treatment method mainly comprises mechanical cleaning and drug treatment, wherein the mechanical cleaning such as tooth brushing, tooth washing and the like can reduce dental plaque and dental calculus in a short period of time, but bacteria at hidden parts such as periodontal pockets and the like are difficult to thoroughly clean, and the oral microecological balance cannot be fundamentally regulated, and the drug treatment such as antibiotics can temporarily inhibit pathogenic bacteria from growing, but the bacterial drug resistance is easy to increase after long-term use, and the normal flora structure in the oral cavity can be damaged, so that the oral microecological unbalance is further aggravated. Therefore, it is of great importance to find a safer, milder and effective way of intervention. Probiotics, which are a class of living microorganisms beneficial to host health, have been developed to exert probiotic effects by modulating intestinal microecological balance, and their use in the oral health field has received widespread attention in recent years. The probiotics can inhibit the growth and reproduction of oral pathogenic bacteria through the mechanisms of secreting antibacterial substances, competing with pathogenic bacteria for field planting, regulating host immune response and the like, improve the oral micro-ecological environment, and provide a new thought for preventing and treating dental caries and periodontal disease.

The Chinese patent application with publication number CN 118146972A discloses a probiotic composition for improving oral health, which comprises Lactobacillus helveticus K6 and various probiotics such as Lactobacillus acidophilus, lactobacillus crispatus, pediococcus pentosaceus, and the like, can effectively coagulate harmful oral bacteria, improve the micro-ecological environment of the oral cavity, prevent dental caries, slow gum swelling and pain, and reduce oral odor. According to the description, the pediococcus pentosaceus plays a role in inhibiting food-borne pathogenic bacteria, is beneficial to improving immunity, can reduce cholesterol of a human body and regulate metabolic problems of the human body, has no function localization related to inhibition of pathogenic bacteria related to dental caries and periodontal disease, cannot exert direct antagonism on core pathogenic bacteria of dental caries and periodontal disease, and has limitation in targeted prevention and treatment of oral diseases.

The Chinese patent application with publication number CN 120888446A discloses a probiotic composition for preventing and/or inhibiting oral pathogenic bacteria, which comprises a plurality of strains such as lactobacillus rhamnosus strain A21149, lactobacillus plantarum strain A21241, pediococcus pentosaceus strain A21358 and the like, and the metabolite of the probiotic composition can effectively inhibit a plurality of pathogenic bacteria such as streptococcus mutans, porphyromonas gingivalis, helicobacter pylori and the like, and has application prospects in the aspects of preventing and relieving dental caries, periodontitis, halitosis and other oral problems. Although the probiotics composition provided by the technical scheme comprises Pediococcus pentosaceus strain A21358, the specification clearly records that the core efficacy of the strain is that the growth and infection of helicobacter pylori can be effectively inhibited and the inflammatory reaction caused by the helicobacter pylori can be lightened through enhancing various mechanisms such as aggregation, reducing urease activity, regulating inflammatory factors and the like, and the probiotics composition does not play a main inhibition role on core pathogenic bacteria of dental caries and periodontal disease. More importantly, the composition needs to be matched with a plurality of strains according to specific mass parts to achieve better common oral pathogenic bacteria inhibition effect, the effect of a single strain is limited, the combination of the strains is complex, and the difficulty of product preparation and quality control is increased.

Chinese patent application publication No. CN 116445367A discloses pediococcus pentosaceus JYPR 9330 for improving oral health and its bacterial agent, and the function of pediococcus pentosaceus JYPR 9330 is limited to pathogenic streptococcus mutans for inhibiting dental caries. For key pathogenic bacteria related to periodontal disease such as porphyromonas gingivalis, fusobacterium nucleatum, actinobacillus conglomerates and the like, the strain does not show inhibition effect, can not meet the requirements of preventing and treating periodontal disease, has a narrow application range, and is difficult to comprehensively solve various oral health problems caused by oral microecological imbalance.

In addition, the three technical schemes have common defects. On the one hand, the potential hazard of the related strains is not systematically evaluated by the schemes, the oral probiotics directly act on the oral environment and directly contact with the hard tissues of the teeth and the oral mucosa, the characteristics of acid production capacity, corrosiveness to the teeth and the like are directly related to the use safety, if the acid production capacity of the strains is too strong, the hard tissues of the teeth can be additionally damaged, but the caries risk is increased, but the key safety index is not evaluated by the prior art. On the other hand, these approaches lack evaluation of dental inflammation levels, and do not clarify the specific effects of the strain in relieving oral inflammation, nor have they involved studies of the immunoregulatory function of gingival epithelial cells. The occurrence and development of oral diseases are closely related to the immune response of a host, the inflammatory response caused by pathogenic bacteria is an important mechanism for causing periodontal tissue damage, gingival epithelial cells are taken as important components of oral mucosa, the balance of the immunoregulation function is important for maintaining oral health, the prior art fails to pay attention to the regulation effect of strains on the immune response of the gingival epithelial cells, and effective intervention on the oral diseases cannot be realized from the aspect of inflammatory regulation, so that the efficacy of the oral care products in oral health maintenance is not comprehensive enough, and the requirements of people on the multiple functions and deep levels of oral care products are difficult to be met.

Therefore, a probiotic bacterial strain and related products which can inhibit caries and periodontal disease pathogenic bacteria in a targeted manner, have good biological safety and can regulate oral immune inflammatory reaction are developed, and the probiotic bacterial strain has important practical significance and wide application prospect.

Disclosure of Invention

Aiming at the technical problems that the existing oral safety is lacking, inflammatory immune response caused by oral pathogenic bacteria can be regulated, and meanwhile, streptococcus mutans, porphyromonas gingivalis, fusobacterium nucleatum, actinomyces viscosus, actinomyces companion and other oral pathogenic bacteria are inhibited, the invention provides the pediococcus pentosaceus with the effects of preventing or treating dental caries and periodontal disease, a preparation and application thereof.

The technical scheme of the invention is as follows:

In a first aspect, the present invention provides pediococcus pentosaceus (Pediococcus pentosaceus) HC3368 having the efficacy of preventing or treating dental caries and periodontal disease, which has been deposited in the China general microbiological culture Collection center with the accession number CGMCC No.27356 at the accession number of North Star West-way No.1, the accession number of which is the Kao-yang area North Star, of Beijing, at the year of 2023, and 15.

In a second aspect, the invention provides a probiotic preparation prepared from Pediococcus pentosaceus HC3368 as described above.

Further, the probiotic preparation comprises Pediococcus pentosaceus HC3368 and/or fermentation products of Pediococcus pentosaceus HC 3368.

Further, the preparation method of the probiotic preparation comprises the following steps:

Inoculating Pediococcus pentosaceus HC3368 into MRS liquid culture medium according to the inoculation amount of 1% by volume, culturing at 37 ℃ for 24 h, centrifuging fresh bacterial liquid at 8000 r/min for 10min, discarding supernatant to obtain bacterial cells, washing the bacterial cells, and re-suspending the bacterial cells into suspension to obtain the product.

Inoculating Pediococcus pentosaceus HC3368 into MRS liquid culture medium according to the inoculation amount of 1% by volume, culturing at 37 ℃ for 24h, centrifuging fresh bacterial liquid at 4 ℃ for 8000 r/min for 10 min, and obtaining fermentation supernatant.

Further, the probiotic preparation can be a health product, a functional food, a medicine or an oral care product. Preferably, the probiotic preparation is an oral care product, and the oral care product is limited to local action in an oral cavity, specifically can exert efficacy in contact areas in the oral cavity such as oral mucosa, tooth surfaces and the like through ways of smearing, rinsing, cleaning and the like, and common products include toothpaste, polishing powder, tooth powder, dental floss, tooth cleaning liquid, mouthwash, oral spray, tooth cleaning foam and the like.

In a third aspect, the present invention provides the use of pediococcus pentosaceus HC3368 as described above for the preparation of a probiotic preparation having dental caries and periodontal disease preventing or treating efficacy.

Further, the probiotic preparation prepared from Pediococcus pentosaceus HC3368 has an inhibitory effect on oral pathogenic bacteria including Streptococcus mutans, fusobacterium nucleatum, porphyromonas gingivalis, actinomyces viscosus and Actinobacillus actinomycetes.

Further, the probiotic preparation prepared from Pediococcus pentosaceus HC3368 can regulate inflammatory immune response caused by oral pathogenic bacteria, and reduce the levels of proinflammatory cytokines IL-1 beta, IL-6 and IL-8.

The invention has the beneficial effects that:

the Pediococcus pentosaceus HC3368 provided by the invention has strong tolerance to artificial intestinal gastric juice, and can germinate in the artificial intestinal gastric juice. The strain does not produce hemolysin, can not dissolve blood cells, and has good biological safety. The strain can remove DPPH and HRS free radicals, inhibit lipid peroxidation, has a certain antioxidant function activity, can degrade cholesterol, and has the probiotic property of reducing serum cholesterol.

The pediococcus pentosaceus HC3368 provided by the invention has weaker acid generating capacity and corrosiveness to teeth than oral pathogenic bacteria, and has no potential harm to teeth. Pediococcus pentosaceus HC3368 can produce certain extracellular polysaccharide, has good adhesion performance to teeth and gingival epithelial cells, and is beneficial to the colonization of the oral cavity by the strain. Pediococcus pentosaceus HC3368 can also inhibit oral pathogenic bacteria such as Streptococcus mutans, fusobacterium nucleatum, porphyromonas gingivalis, actinomyces viscosus and Actinobacillus actinomycetes by producing proteins such as bacteriocin, and both bacterial suspension and fermentation supernatant can exert the effect. The mechanism of inhibiting effect includes copolymerization effect with pathogenic bacteria in oral cavity, and can reduce the biofilm formed by pathogenic bacteria in oral cavity on tooth surface, so as to reduce the occurrence risk of caries, periodontal disease and other oral diseases. In addition, pediococcus pentosaceus HC3368 can regulate inflammatory immune response caused by oral pathogenic bacteria, and reduce the levels of proinflammatory cytokines IL-1 beta, IL-6 and IL-8.

The pediococcus pentosaceus HC3368 provided by the invention can protect oral health without being compounded with prebiotics and/or other probiotics, and has no side effect and excessive risk after long-term administration.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the description of the embodiments or the prior art will be briefly described below, and it will be obvious to those skilled in the art that other drawings can be obtained from these drawings without inventive effort.

FIG. 1 is a colony morphology of Pediococcus pentosaceus HC3368 of example 1.

FIG. 2 is a crystal violet staining micrograph of Pediococcus pentosaceus HC3368 of example 1.

FIG. 3 is an API 50 CHL carbon source metabolism identification map of Pediococcus pentosaceus HC3368 in example 2.

FIG. 4 is a RAPD fingerprint of Pediococcus pentosaceus HC3368 of example 2.

FIG. 5 is a rep-PCR fingerprint of Pediococcus pentosaceus HC3368 of example 2.

FIG. 6 is a graph showing the results of the tooth decay by Pediococcus pentosaceus HC3368 of example 5.

FIG. 7 is a graph showing the results of the sugar production by Pediococcus pentosaceus HC3368 of experiment 1 in example 6.

FIG. 8 is a graph of simulated tooth adhesion results of Pediococcus pentosaceus HC3368 of experiment 2 in example 6.

Fig. 9 is a graph of the self-setting effect of the pediococcus pentosaceus HC3368 of experiment 1 in example 9, and fig. 9 (a) is a graph of the self-setting effect of 2h, (b) is a graph of the self-setting effect of 4h, and (c) is a graph of the self-setting effect of 6 h.

FIG. 10 shows the effects of co-aggregation of Pediococcus pentosaceus HC3368 and pathogenic bacteria in experiment 2 of example 9, wherein (a) in FIG. 10 shows the effects of co-aggregation of Pediococcus pentosaceus HC3368 and Streptococcus mutans, (b) shows the effects of co-aggregation of Pediococcus pentosaceus HC3368 and pathogenic bacteria, (c) shows the effects of co-aggregation of Pediococcus pentosaceus HC3368 and Fusobacterium nucleatum, (d) shows the effects of co-aggregation of Pediococcus pentosaceus HC3368 and Actinobacillus viscosus, and (e) shows the effects of co-aggregation of Pediococcus pentosaceus HC3368 and Actinobacillus faciens.

FIG. 11 is a graph showing the results of the elimination of oral pathogenic biofilm by Pediococcus pentosaceus HC3368 of example 10.

FIG. 12 is a bar graph of the concentrations of pro-inflammatory cytokines for the different treatment groups of experiment 1 in example 11, where (a) is a bar graph of IL-1β concentration, (b) is a bar graph of IL-6 concentration, and (c) is a bar graph of IL-8 concentration.

FIG. 13 is a bar graph of the concentrations of pro-inflammatory cytokines for the different treatment groups of experiment 2 in example 11, where (a) is a bar graph of IL-1β concentration, (b) is a bar graph of IL-6 concentration, and (c) is a bar graph of IL-8 concentration.

Detailed Description

In order to make the technical solution of the present invention better understood by those skilled in the art, the technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.

Except as specifically noted, the inoculum solutions referred to in the embodiments of the invention were obtained as follows:

Under the aseptic condition, a proper amount of fresh pediococcus pentosaceus HC3368 bacterial liquid is taken, centrifuged for 5min at 5000 r/min, washed twice with PBS buffer solution, and then the bacterial cells are re-suspended by an equal volume of PBS buffer solution and diluted by 50 times to be used as an inoculation liquid.

Except for the specific descriptions, the Pediococcus pentosaceus HC3368 bacterial suspensions in the embodiments of the invention are all obtained as follows:

The strain is inoculated into MRS liquid culture medium according to the inoculation amount of 1 percent by volume, cultured for 24 h at 37 ℃, then the fresh bacterial liquid is centrifuged for 10 min at 8000 r/min, and the supernatant is discarded, thus obtaining the bacterial body. And (3) washing the bacterial cells twice by using PBS buffer solution (pH=7.0), and re-suspending the bacterial cells until the absorbance OD _{600 nm} reaches 0.5-0.6, thereby obtaining bacterial suspension for later use.

Except as specifically noted, the fermentation supernatants of Pediococcus pentosaceus HC3368 referred to in the embodiments of the present invention were obtained as follows:

The strain was inoculated into MRS liquid medium at an inoculum size of 1% by volume, cultured at 37℃for 24 h, and then the fresh bacterial liquid was centrifuged at 4℃for 8000 r/min for 10min to obtain fermentation supernatant.

EXAMPLE 1 isolation screening of Pediococcus pentosaceus HC3368

1. Primary screen

Sampling fresh fermented soybean sample in Yangjiang city of Guangdong in 2022 month, diluting with 100mL sterile normal saline, placing into a sterile sample bag, beating with a homogenizer, mixing, gradient diluting with 100 μl of the mixture, spreading on MRS agar medium, anaerobic culturing at 37deg.C for 48 h, and performing microscopic examination until a single colony grows out. According to the microscopic examination result, 35 potential lactobacillus strains were screened out, and the potential lactobacillus strains were named HC3350, HC3351, HC3383 and HC3384 in sequence.

2. Double screen

Preparing a MRS liquid culture medium of 1L, autoclaving at 121 ℃ for 15min, adding 3.2g pig mucosa pepsin after the culture medium is cooled, shaking for dissolving, and placing in a 37 ℃ water bath shaking table for heat preservation for 1 h to prepare the acid-resistant culture medium. And respectively inoculating 35 strains of lactobacillus obtained by primary screening into the acid-resistant culture medium according to the inoculum size of 6 percent, performing anaerobic static culture at 37 ℃ for 48 h, and taking fermentation liquor for bacterial count.

The results show that the most viable amount of HC3368 strain in 35 strains of lactobacillus is obtained after re-screening by acid-resistant culture medium, and the logarithmic value is as high as 11.03 Log ₁₀ CFU/mL, which indicates that the acid-resistant capability of HC3368 strain is the highest.

After the HC3368 strain is inoculated on MRS agar culture medium and anaerobic cultured at 37 ℃ for 24 h, as shown in figure 1, HC3368 single colony is off-white, the colony diameter is about 1.5-2.5 mm, the surface is moist and smooth, the edge is neat and opaque, and the surface is raised. After crystal violet staining, HC3368 strain was spherical, smooth, arranged singly, in clusters or in clusters, gram-positive with no sporulation (as shown in figure 2).

Example 2 identification of Pediococcus pentosaceus HC3368

1. API 50 CHL carbon Source metabolism experiment

The carbon source metabolic capacity of HC3368 strain was verified using API 50CHL reagent strip. Experimental methods and results analysis specific reference was made to the API 50CHL kit instructions. The API test results are shown in fig. 3, the ID value of the hc3368 strain and pediococcus pentosaceus is 99.9%, the T value=0.98, the carbohydrate metabolism activity is basically the same, and the identification result is excellent.

Thus, based on the results of carbon source metabolism, HC3368 strain was initially identified as Pediococcus pentosaceus (Pediococcus pentosaceus).

2. Molecular biological identification

Single colony of HC3368 strain on the plate is selected and cultured in MRS liquid medium at 37 ℃ for 24 h, then 500 mu L of fermentation liquid is taken, and genome of the strain is obtained by referring to a Tiangen bacterium genome DNA extraction kit (DP 302) and is used for subsequent molecular biological identification.

2.1 Identification of 16S rDNA Gene sequence

27F (AGAGTTTGATCCTGGCTCA, SEQ ID No. 1) and 1492R (GGTTACCTTGTTACGACTT, SEQ ID No. 2) were used as primers. The PCR amplification system was 50. Mu.L in total, and contained 5. Mu.L of 10 XPCR amplification buffer, 4. Mu.L of deoxyribonucleotides (dNTPs), 2. Mu.L of 27F upstream primer, 2.5. Mu.L of 1492R downstream primer 2. Mu. L, DNA template, 0.5. Mu.L of recombinant Taq DNA polymerase (rTaq) and 34. Mu.L of double distilled water (ddH ₂ O). Electrophoresis proves that the PCR product meets the requirements when the nucleic acid electrophoresis result is about 1500 bp.

Sequencing results showed that the 16S rDNA sequence (SEQ ID NO. 3) of HC3368 strain was as follows:

CAGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAACTTCCGTTAATTGATTATGACGTACTTGTACTGATTGAGATTTTAACACGAAGTGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCAGAAGTAGGGGATAACACCTGGAAACAGATGCTAATACCGTATAACAGAGAAAACCGCATGGTTTTCTTTTAAAAGATGGCTCTGCTATCACTTCTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCAGTGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACGTGGGTAAGAGTAACTGTTTACCCAGTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTCTTTTAAGTCTAATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGATTACTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGTAATCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAAGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTCTGACAGTCTAAGAGATTAGAGGTTCCCTTCGGGGACAGAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTCGCGAGACCGCGAGGTTAAGCTAATCTCTTAAAACCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGGGGTAACCTTTTAGGAGCTAGCCGTCTAAGGTGGGACAGATGATTAGGGTGAAGTCGTAA.

2.2 RAPD fingerprint identification

M13 (GAGGGTGGCGGTTCT, SEQ ID NO. 4) was used as a primer. A total of 20. Mu.L of RAPD reaction system was prepared, which contained 0.2. Mu.L of Taq DNA polymerase (5U/. Mu.L), 2. Mu.L of 10 Xbuffer (containing Mg ²⁺), 0.8. Mu. L, M13 primer (10. Mu.M) 1. Mu. L, DNA template, 2. Mu.L of double distilled water (ddH ₂ O) and 14. Mu.L of deoxyribonucleotide (dNTPs, 2.5 mM). 1.5% agarose gel plates were prepared, and were electrophoresed 80 and min under 100: 100V pressure-stabilizing conditions with DL2000 DNA MARKER as a result control, and finally the electrophoresis pattern was detected by using a gel imaging system, and the RAPD fingerprint pattern of HC3368 strain is shown in FIG. 4.

2.3 Rep-PCR fingerprint identification

The rep-PCR primer sequence is GTGGTGGTGGTGGTG (SEQ ID NO. 5). The rep-PCR reaction system was 20. Mu.L in total, and contained 0.2. Mu.L of recombinant Taq DNA polymerase (rTaq), 2. Mu.L of 10 XEx Taq DNA buffer (containing Mg ²⁺), 2. Mu.L of deoxyribonucleotides (dNTPs, 2.5 mM), 2. Mu.L of primer (10. Mu.M) 1. Mu. L, DNA template, and 12.8. Mu.L of double distilled water (ddH ₂ O). 1.5% agarose gel plates were prepared, and were electrophoresed 80 and min under 100: 100V pressure-stabilizing conditions using DL2000 DNA MARKER as a result control, and finally the electrophoregram was detected by using a gel imaging system, and the rep-PCR fingerprint of HC3368 strain was shown in FIG. 5.

The 16S rRNA sequence of HC3368 strain was uploaded to EzBioCloud website for alignment. The identification result of the physiological and biochemical characteristics is integrated, the HC3368 strain is determined to be a new Pediococcus pentosaceus, and the Pediococcus pentosaceus is named as Pediococcus pentosaceus HC3368.

And 2023, 05 and 15 days, preserving the pediococcus pentosaceus HC3368 to the China general microbiological culture Collection center with a preservation address of North Star Xili No. 1, 3 in the Korean region of Beijing, a preservation number of CGMCC No.27356, and classifying and naming the pediococcus pentosaceus as Pediococcus pentosaceus.

EXAMPLE 3 Pediococcus pentosaceus HC3368 physicochemical Properties

Experiment 1, determination of salinity tolerance

To 96-well plates 190. Mu.L of BSM liquid medium with 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% salt concentration was added, respectively, 3 replicates for each salt concentration, and then 10. Mu.L of inoculum was added, and wells without inoculation were used as controls. 50. Mu.L of autoclaved paraffin oil was added to each well to prevent evaporation of water during the culture. Culturing at 37 ℃ and observing whether the culture medium becomes turbid.

The results of the salinity tolerance test showed that Pediococcus pentosaceus HC3368 had a maximum tolerance salt concentration of 6%.

Experiment 2, temperature growth Range experiment

The inoculation liquid is inoculated into 10 mL MRS liquid culture medium according to 10% of inoculation amount by volume percentage, and 10 mL MRS liquid culture medium without bacteria is used as a control. Two MRS liquid culture mediums were respectively placed in 15℃constant temperature shaking incubator for 7 days, 37℃constant temperature shaking incubator, 45℃constant temperature shaking incubator and 60℃constant temperature shaking incubator for 2 days, and whether the culture solution of each MRS liquid culture medium became turbid was observed.

The results showed that the medium remained clear after 7 days of incubation at 15℃and that the medium became cloudy after 2 days of incubation at 37℃and clear after 2 days of incubation at 45℃and 60 ℃. Thus, pediococcus pentosaceus HC3368 was unable to grow at 15℃and below and 45℃and above, and grew normally at 37 ℃.

Experiment 3 determination of tolerance to Artificial gastrointestinal fluids

Accurately weighing 0.5144 g KCl、0.1225 g KH₂PO₄、2.75085 g NaCl、2.1002 g NaHCO₃、0.0203 g MgCl₂·6H₂O、0.0480 g (NH₄)₂CO₃、0.0083 g CaCl₂, to 1000: 1000 mL to obtain gastric juice buffer. 3 g pepsin is dissolved in 1000 mL gastric juice buffer solution, and the pH value is adjusted to 3.0,0.22 mu m by using a 1M HCl filter membrane for filtration and sterilization, so that simulated gastric juice is obtained, and the simulated gastric juice is prepared on site.

Accurately weighing 0.5069 g KCl、0.1089 g KH₂PO₄、2.2442 g NaCl、7.1408 g NaHCO₃、0.0067 g MgCl₂·6H₂O、0.0333 g CaCl₂, to 1000: 1000 mL to obtain intestinal buffer. Dissolving 1g trypsin and 1g bile salt in 1000 mL intestinal juice buffer solution, and filtering and sterilizing by using a filter membrane with the pH value adjusted to 8.0,0.22 mu m by using 1M NaOH to obtain simulated intestinal juice, wherein the simulated intestinal juice is prepared in an on-site manner.

The 9 mL artificial gastric juice was placed in a 37 ℃ water bath shaker to preserve heat for 1 h to simulate the human body temperature. 1 mL inoculum was added to 9 mL artificial gastric juice and incubated in a 37℃water bath shaker (200 r/min). Samples 1 mL were taken before inoculation and after incubation 2h, respectively, added to 99 mL PBS buffer, mixed by tapping with a homogenizer, 1 mL sample homogenates were drawn into sterile plates, MRS agar medium cooled to 48℃was poured into plates for about 15 mL,37℃anaerobic culture 72 h for colony counting, and two sets of replicates were performed.

The 24 mL artificial intestinal fluid was placed in a 37 ℃ water bath shaker and incubated for 1: 1h to simulate the temperature of the human body. Taking 1 mL and digesting 2h artificial gastric juice, adding into 24 mL artificial intestinal juice, and placing into a 37 ℃ water bath shaker (200 r/min) for incubation. Sample 1 mL at 3 h was added to 99 mL PBS buffer, mixed by tapping with a homogenizer, 1 mL sample homogenate was drawn into a sterile dish, MRS agar medium cooled to 48℃was poured into about 15 mL of the dish, anaerobic culture 72h at 37℃was performed for colony counting, and two sets of replicates were performed.

The viable count (Log ₁₀ CFU/mL) of Pediococcus pentosaceus HC3368 after digestion with artificial gastrointestinal fluids is shown in Table 1 below.

TABLE 1 viable count after digestion of Artificial gastrointestinal fluids (Unit: log ₁₀ CFU/mL)

As shown in Table 1, the live bacteria amount of Pediococcus pentosaceus HC3368 is reduced by 0.08Log ₁₀ CFU/mL after artificial gastric juice, and then 9.56Log ₁₀ CFU/mL after artificial intestinal juice digestion, and the total live bacteria amount is reduced by only 0.13 Log ₁₀ CFU/mL, which indicates that Pediococcus pentosaceus HC3368 has good gastric acid and bile salt resistance and can resist harsh environment in gastrointestinal tract environment.

Experiment 4 hemolytic experiment

Weighing various components of TBS basic culture medium, dissolving, autoclaving at 121deg.C for 15 min%, adding 5% sterilized defibrinated sheep blood when the culture medium is cooled to 50deg.C, mixing, and pouring into plate to obtain blood cell plate. And streaking and inoculating the Pediococcus pentosaceus HC3368 on a blood cell plate, culturing in a 37 ℃ incubator, and observing whether the Pediococcus pentosaceus HC3368 has hemolysis or not in 24-48 hours.

The results showed that the blood cell plate was unchanged, pediococcus pentosaceus HC3368 could not grow, indicating that Pediococcus pentosaceus HC3368 did not produce hemolysin, could not lyse blood cells, and was excellent in biosafety.

Experiment 5 antibiotic resistance experiment

The minimum inhibitory concentration (MIC value) of the antibiotic against pediococcus pentosaceus HC3368 was determined by a micro broth dilution method, and the specific results are shown in table 2 below.

TABLE 2 MIC values of antibiotics for Pediococcus pentosaceus HC3368 (Unit: μg/mL)

As can be seen from Table 2, pediococcus pentosaceus HC3368 is sensitive to common antibiotics such as erythromycin, streptomycin, ampicillin Lin Heke, and the like, and has good biological safety.

Experiment 6 hydrophobic cell surface test

And (3) selecting purified Pediococcus pentosaceus HC3368 colony, inoculating the Pediococcus pentosaceus HC3368 colony into an MRS liquid culture medium, and culturing for 24-48 h at 40 ℃ in a shaking way. And inoculating 1% of the inoculum size into a new MRS liquid culture medium according to the volume percentage, continuously shaking and culturing for 24-48 hours at 40 ℃, centrifuging for 10 min at 6000 Xg, collecting thalli, washing twice with sterile normal saline, and re-suspending the thalli by using a sterilized KNO ₃ solution with the concentration of 1 mL being 0.1 and M to obtain the bacterial liquid to be tested.

And (2) sucking 50 mu L of the bacteria liquid to be detected, adding the bacteria liquid to be detected into 2450 mu L of KNO ₃ solution with the concentration of 0.1M, testing the OD _{600 nm} value of the solution and marking the OD _{600 nm} value as A ₀, uniformly mixing 1.5 mL of the bacteria liquid to be detected with 500 mu L of dimethylbenzene, standing for 10 min at room temperature, carrying out vortex oscillation on the formed two-phase system for 2 min, standing for 20 min, and reforming the water phase and the organic phase. The OD _{600 nm} value was carefully aspirated and recorded as a ₁. Cell hydrophobicity was calculated as% hydrophobicity = (a ₀-A₁)/A₁ x 100%, three replicates were performed and the resulting data averaged.

The results showed that the cell surface hydrophobicity of Pediococcus pentosaceus HC3368 was 70.96% + -10.12%.

Experiment 7 determination of Aflatoxin B1 removal Capacity

Aflatoxins are secondary metabolites produced by a variety of fungi, such as aspergillus flavus and aspergillus parasiticus. The number of aflatoxins which are separated at present reaches 18, wherein, aflatoxin B1 (AFB 1) is identified as a class I cancerogenic substance by the international cancer research institution because of extremely strong cancerogenic, mutagenic and teratogenic properties, and trace aflatoxins can have harmful effects on human beings and animals. The required solution is prepared by referring to the specification of the aflatoxin B1 assay kit, and the detection operation is carried out.

The results showed that the aflatoxin B1 clearance of pediococcus pentosaceus HC3368 was 68.43% ± 6.55%.

Experiment 8 in vitro cholesterol degradation test

Accurately weighing 1 g cholesterol, dissolving in absolute ethanol, fixing volume to 100 mL, and filtering and sterilizing with 0.22 μm microporous filter membrane under aseptic condition to obtain cholesterol solution.

Weighing peptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, diammonium hydrogen citrate 2.0 g, glucose 20.0 g, tween 80 1.0 mL, sodium acetate 5.0 g, magnesium sulfate 0.1 g, manganese sulfate 0.05 g, dipotassium hydrogen phosphate 2.0 g and distilled water 1000mL, regulating pH value to 7.3,115 ℃ after dissolution, sterilizing 30min, and adding cholesterol solution to make final concentration of cholesterol be 0.1%, thus obtaining the liquid culture medium containing cholesterol.

Inoculating the inoculation liquid into a liquid culture medium containing cholesterol according to the inoculation amount of 0.1% by volume, standing at 37 ℃ for 48 h, then taking 0.2 mL bacterial liquid, adding 1.8 mL absolute ethanol, uniformly mixing, standing for 10min, centrifuging at 3000 r/min for 5min, taking supernatant, measuring the cholesterol content according to the method specified in GB 5009.128-2016 cholesterol measurement in food, and calculating the cholesterol degradation rate.

The results showed that Pediococcus pentosaceus HC3368 had an in vitro cholesterol degradation rate of 73.76% + -2.57%.

Example 4 determination of antioxidant function of Pediococcus pentosaceus HC3368

Experiment 1, determination of Pediococcus pentosaceus HC3368 ability to clear 1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH)

Taking 1 mL Pediococcus pentosaceus HC3368 bacterial suspension and 1 mL Pediococcus pentosaceus HC3368 fermentation supernatant respectively, adding 1 mL existing DPPH free radical solution with the concentration of 0.4 mM respectively, uniformly mixing, then placing the mixture at room temperature for shading reaction for 30 min, measuring absorbance A _{Sample of} of a sample at the wavelength of 517 nm, carrying out three parallel experiments, and averaging the obtained data.

In the experimental process, the mixed solution of the PBS buffer solution and the ethanol with the same volume is used as a blank for instrument zeroing, the absorbance is marked as A _{Blank space}, and the mixed solution of the PBS buffer solution and the DPPH ethanol solution with the same volume is used as a contrast, and the absorbance is marked as A _Control. DPPH radical scavenging was calculated as% = [1- (a _{Sample of}-A_{Blank space}）/A_Control ] ×100% scavenging rate, and the results are shown in table 3 below.

TABLE 3 clearance of Pediococcus pentosaceus HC3368 to DPPH radical (unit:%)

Experiment 2, determination of Pediococcus pentosaceus HC3368 ability to scavenge hydroxyl free radicals (HRS)

200. Mu.L of Pediococcus pentosaceus HC3368 suspension and 200. Mu.L of Pediococcus pentosaceus HC3368 fermentation supernatant were mixed with 100. Mu.L of sodium salicylate-ethanol solution with a concentration of 5 mM, 100. Mu.L of ferrous sulfate solution with a concentration of 5 mM and 500. Mu.L of deionized water, respectively, and then 100. Mu.L of hydrogen peroxide solution with a concentration of 3 mM was added, after 15 min in a 37℃water bath, the absorbance of the sample was measured at a wavelength of 510 nm and recorded as A _{Sample of}. Equal volumes of deionized water were used in place of the bacterial suspension or fermentation supernatant, and mixed well with 100. Mu.L of sodium salicylate-ethanol solution at a concentration of 5 mM, 100. Mu.L of ferrous sulfate solution at a concentration of 5 mM, and 500. Mu.L of deionized water, then 100. Mu.L of hydrogen peroxide solution at a concentration of 3 mM were added, each of which was subjected to a 37℃water bath of 15 min, and the absorbance of the sample was measured at a wavelength of 510 nm and designated A _{Control of}. Equal volumes of deionized water were used in place of the bacterial suspension or fermentation supernatant and hydrogen peroxide solution, and mixed with 100. Mu.L of sodium salicylate-ethanol solution at a concentration of 5 mM, 100. Mu.L of ferrous sulfate solution at a concentration of 5 mM, and 500. Mu.L of deionized water, and after a 37℃water bath of 15 min, the absorbance of the sample was measured at a wavelength of 510 nm and recorded as A _{Blank space}. HRS radical clearance was calculated as% = (a _{Sample of}-A_{Control of}）/（A_{Blank space}-A_{Control of}) ×100% clearance, and the results are shown in table 4 below.

TABLE 4 Remover of Pediococcus pentosaceus HC3368 to HRS radical (unit:%)

Experiment 3 Pediococcus pentosaceus HC3368 anti-lipid peroxidation experiment

Weighing 0.1 mL linoleic acid, 0.2 mL Tween20, 19.7 mL deionized water and 0.5 mL PBS buffer solution (pH=7.4), fully mixing the components, and uniformly stirring to obtain linoleic acid emulsion for later use.

Taking 1 mL of the prepared linoleic acid emulsion, adding 1 mL FeSO ₄ solution (1%), adding 0.5 of mL sample to be detected (bacterial suspension or fermentation supernatant of Pediococcus pentosaceus HC 3368), and gently shaking to make the mixed solution uniform. The mixture was placed in a 37 ℃ water bath and reacted at 1.5 h a with heat preservation. After the reaction, 0.2 mL TCA solution (4%) and 2 mL TBA solution (0.8%) were added to the mixture in sequence, and the mixture was heated in a water bath at 100 ℃ for 30 min. After the color development is completed, the mixed solution is rapidly cooled, and then centrifuged at 4000 r/min for 15: 15 min, so that the precipitated impurities in the system are removed. The supernatant after centrifugation was collected, and absorbance was measured at 532 nm and designated as a. Meanwhile, the above operation was repeated by substituting 0.5 mL distilled water for the sample to be measured, and the measured absorbance was designated as a ₀. Lipid peroxidation inhibition was calculated as = (a ₀-A)/A₀ x 100%) and the results are shown in table 5 below.

TABLE 5 lipid peroxidation inhibition of Pediococcus pentosaceus HC3368 (unit:%)

Example 5 evaluation of potential hazard of Pediococcus pentosaceus HC3368

Experiment 1 evaluation of acid producing ability

The acid generating capacity of Pediococcus pentosaceus HC3368 was evaluated by the end-point pH method. Pediococcus pentosaceus HC3368 was cultured in MRS liquid medium for 24 h, and the acidity of the culture broth was measured by pH meter, and Pediococcus pentosaceus CECT8330 and Porphyromonas gingivalis BNCC353909 in commercial products were used as controls. The results are shown in Table 6 below, where the endpoint pH of Pediococcus pentosaceus HC3368 was higher than that of the control strain, indicating that its acid producing ability was weaker than that of the control strain, and it was found that the use of Pediococcus pentosaceus HC3368 reduced the risk of caries caused by acid production by the strain.

TABLE 6 fermentation end pH of strains

Experiment 2, evaluation of corrosiveness

The corrosiveness of Pediococcus pentosaceus HC3368 on teeth was measured using an equivalent elemental method. Mixing the fermentation supernatant with hydroxyapatite, uniformly stirring, then incubating at 37 ℃, respectively taking equal amounts of fermentation liquid at 30 min, 1 h, 2h and 3h, centrifuging to obtain the supernatant, measuring the OD _{620 nm} value of the supernatant by an acid-molybdate spectrophotometry, wherein the OD _{620 nm} value can reflect the change of the phosphorus content in the supernatant, and further characterizing the corroded condition of the hydroxyapatite. Meanwhile, the fermentation supernatant of Porphyromonas gingivalis BNCC353909 was used as a control, and the fermentation supernatant of Porphyromonas gingivalis BNCC353909 was obtained by the same method as that of Pediococcus pentosaceus HC3368. The fermentation supernatant of 0min was used as a blank to correct the endogenous phosphorus content in the sample.

As a result, as shown in FIG. 6, the corrosion ability of Pediococcus pentosaceus HC3368 was maximized at 1h, and almost no more corrosion occurred from 1: 1 h. The corrosion effect of the porphyromonas gingivalis BNCC353909 on the hydroxyapatite is continuously enhanced, which indicates that the pediococcus pentosaceus HC3368 has weaker corrosion capability, and the damage effect on teeth is far smaller than that of oral pathogenic bacteria such as porphyromonas gingivalis.

EXAMPLE 6 evaluation of adhesion Property of Pediococcus pentosaceus HC3368

Experiment 1 evaluation of sugar production

The probiotics can enhance the self adhesion capability on the oral mucosa, the tooth surface and other parts by producing extracellular polysaccharide, and stable adhesion is a key precondition for the probiotics to successfully colonize in a host body and further play a role of probiotics. Therefore, the adhesion performance and the colonization potential of the probiotic fermented liquid in the oral cavity environment can be indirectly evaluated by measuring the content of extracellular polysaccharide in the probiotic fermented liquid.

Taking 10 mL fermentation supernatant, adding trichloroacetic acid with the concentration of 2 mL being 80% into the fermentation supernatant, mixing, placing the mixture on ice, stirring the mixture for 30 min, centrifuging the mixture to remove thalli and proteins remained in the supernatant, collecting the supernatant after centrifugation, adding three times of volume of absolute ethanol precooled at 0 ℃ in advance, then placing the mixture under a refrigerator at 4 ℃ for refrigerating for 24 h, and observing the precipitation condition of Extracellular Polysaccharide (EPS). Centrifuging the mixed solution at 10000 r/min at 4 ℃ for 15: 15 min to obtain polysaccharide precipitate, then redissolving the precipitate into a polysaccharide solution by using 10: 10 mL distilled water, and measuring the polysaccharide content in the polysaccharide solution by using a phenol-sulfuric acid method. Meanwhile, lactobacillus rhamnosus LGG in a commercially available product was used as a control.

As shown in fig. 7, the extracellular polysaccharide yield of pediococcus pentosaceus HC3368 was 106 mg/L, while the extracellular polysaccharide yield of the control strain lactobacillus rhamnosus LGG was 84 mg/L, which is significantly lower than that of pediococcus pentosaceus HC3368 provided by the present invention (P < 0.05).

Experiment 2 evaluation of dental adhesion

The absorbance of the Pediococcus pentosaceus HC3368 suspension at 600 nm wavelength was measured and recorded as OD _{600 Front part}. 10 mg sterilized hydroxyapatite powder (d=82 μm) was added to the bacterial suspension of Pediococcus pentosaceus HC3368, and after mixing, it was placed in a 37℃water bath for warm bath 1 h. After the end of the incubation, the filtrate was filtered and collected with a 0.45 μm pinhole filter, then the pinhole filter was rinsed twice with PBS buffer, and the rinsed liquid was combined with the previously collected filtrate. And then centrifuging the combined filtered liquid, collecting bacterial precipitate at the bottom of the centrifuge tube, adding 1 mL PBS buffer solution into the bacterial precipitate for resuspension, and measuring the absorbance at 600 nm wavelength, and recording as OD _{600 Rear part (S)}. Meanwhile, lactobacillus rhamnosus LGG was used as a control. The simulated dental adhesion rate is calculated as follows:

。

As shown in fig. 8, both pediococcus pentosaceus HC3368 and lactobacillus rhamnosus LGG have certain adhesive properties, and the simulated tooth adhesion rate of lactobacillus rhamnosus LGG is 36.18% which is significantly lower than 87.86% of pediococcus pentosaceus HC3368 (< 0.01). The high adhesion rate of Pediococcus pentosaceus HC3368 can enable the Pediococcus pentosaceus HC3368 to adhere to the tooth surface, and can effectively reduce the bacterial strain from being brought into the digestive tract by the effects of saliva flushing, swallowing and the like, and enhance the residence time of the bacterial strain.

Experiment 3 evaluation of cell adhesion

After recovery, culture and counting of human gingival epithelial cells C1052, they were inoculated in 6-well plates at an inoculum size of 2X 10 ⁶ cells/well and cultured in a CO ₂ incubator 24, h. The experimental group was treated by resuspension of Pediococcus pentosaceus HC3368 in logarithmic growth phase to 5X 10 ⁷ CFU/mL with MRS liquid medium, adding 1: 1mL of the suspension to the above 6-well plate with cell wall attached, and culturing in CO ₂ incubator for 2: 2 h. After the end of the incubation, the incubation was washed repeatedly three times with PBS buffer to remove non-adherent bacteria. Digestion was stopped by adding 500. Mu.L of pancreatin to 3min, followed by 1.5mL cell culture medium and repeated pipetting, and the resulting solution was collected into sterile EP tubes and subjected to 10-fold, 100-fold, 1000-fold and 10000-fold gradient dilutions in sequence for plating counts. Cell counts were performed in the same manner as in the experimental group using a 6-well plate without the addition of Pediococcus pentosaceus HC3368 suspension as a blank group. The cell adhesion ability of Pediococcus pentosaceus HC3368 was calculated according to the following formula:

Adhesion capacity (CFU/cell) =total number of bacteria adhered per culture well/total number of cells per culture well.

The result shows that the adhesion capability of Pediococcus pentosaceus HC3368 is 52.05 +/-9.39 CFU/cell, which shows that the strain has stronger adhesion capability to oral epithelial cells and is beneficial to the colonization of the oral environment.

EXAMPLE 7 antibacterial test of Pediococcus pentosaceus HC3368 against oral pathogenic bacteria

Bacterial solutions of Streptococcus mutans ATCC25175, fusobacterium nucleatum BNCC336949, actinobacillus viscosus ATCC27044, porphyromonas gingivalis BNCC353909 and Actinobacillus actinomycetes BNCC336945, which are commercially available products, were inoculated into BHI broth medium (5% bovine serum was added) in an amount of 1% by volume, respectively, and bacterial solutions of Streptococcus mutans ATCC25175, fusobacterium nucleatum BNCC336949, actinobacillus viscosus ATCC27044, porphyromonas gingivalis BNCC353909 and Actinobacillus actinomycetes BNCC336945 were collected by centrifugation 10 min at 8000 r/min after anaerobic culture at 37℃for 48 h. The bacterial cells are washed twice by PBS buffer solution (pH=7.0), then the bacterial cells are resuspended by PBS buffer solution (pH=7.0), and the initial absorbance OD _{600 nm} of the bacterial suspension is adjusted to 0.5-0.6 for standby.

The antibacterial effect of bacterial suspension and fermentation supernatant of Pediococcus pentosaceus HC3368 on the oral pathogenic bacteria is measured by adopting an oxford cup double-layer flat plate method. Preparing a BHI culture medium with agar content of 0.7%, sterilizing, and adding 0.2% mixed pathogenic bacteria liquid and shaking uniformly when the temperature of the culture medium is reduced to below 47 ℃.7 mL culture medium is poured onto a bottom agar plate, an oxford cup is placed after the culture medium is solidified, 150 mu L of Pediococcus pentosaceus HC3368 bacterial suspension or fermentation supernatant is added into the oxford cup hole, and after culturing at 37 ℃ for 48 h, the diameter of a bacteriostasis ring is measured, and the results are shown in the following table 7.

TABLE 7 antibacterial effect of Pediococcus pentosaceus HC3368 on oral pathogenic bacteria (Unit: mm)

As can be seen from Table 7, pediococcus pentosaceus HC3368 has remarkable inhibitory effect on five oral pathogenic bacteria including Streptococcus mutans, fusobacterium nucleatum, porphyromonas gingivalis, actinomyces viscosus and Actinobacillus actinomycetes, and shows a certain broad-spectrum antibacterial property. The bacterial suspension of Pediococcus pentosaceus HC3368 has better antibacterial effect than fermentation supernatant, and has most remarkable antibacterial effect on streptococcus mutans, fusobacterium nucleatum and Porphyromonas gingivalis, and the diameter of the antibacterial ring is over 20 mm.

Example 8 determination test of antibacterial substance of Pediococcus pentosaceus HC3368

It has been shown that lactic acid bacteria are capable of exerting oral health protection by producing organic acids, hydrogen peroxide, bacteriocins and adhesion inhibitors. Based on this, the present example was designed to determine the substances in Pediococcus pentosaceus HC3368 that play the primary bacteriostatic role.

And (3) regulating the pH value of the fermentation supernatant of Pediococcus pentosaceus HC3368 to 5.5-6 by using NaOH of 5M, filtering the neutralized fermentation supernatant by using a 0.22 mu m filter membrane to obtain acid-removed fermentation supernatant, and taking the acid-removed fermentation supernatant as a sample 1 to be tested.

And regulating the pH value of the fermentation supernatant of Pediococcus pentosaceus HC3368 to 5.5-6 by using 5M NaOH, adding catalase into the fermentation supernatant, uniformly mixing, and then incubating the fermentation supernatant at 37 ℃ for 30 min to further obtain the fermentation supernatant excluding H ₂O₂, and taking the fermentation supernatant as a sample 2 to be tested.

And (3) regulating the pH value of the fermentation supernatant of Pediococcus pentosaceus HC3368 to 5.5-6 by using NaOH of 5M, adding catalase, pepsin and trypsin for treatment, uniformly mixing, and then incubating at 37 ℃ for 30min to further obtain the fermentation supernatant with protein removed, and taking the fermentation supernatant as a sample 3 to be tested.

The above sample 1, sample 2 and sample 3 were used to perform antibacterial tests, and the antibacterial effect of each sample was observed on five oral pathogens, and the antibacterial test method was described in example 7. The results are shown in Table 8 below.

TABLE 8 bacteriostatic effects of different treated fermentation supernatants on oral pathogenic bacteria (Unit: mm)

Note that the letters in each column in table 8 are different to indicate significant differences (P < 0.05), and the letters are the same to indicate no significant differences (P0.05)。

As can be seen from table 8, the fermentation supernatant of pediococcus pentosaceus HC3368 still has a significant inhibitory effect on oral pathogenic bacteria after acid neutralization and removal of H ₂O₂, the size of the inhibition zone is not significantly different from that of the untreated fermentation supernatant, and the size of the inhibition zone is significantly reduced after protease treatment, which indicates that the antibacterial substance of pediococcus pentosaceus HC3368 on oral pathogenic bacteria is a protein substance, so that it is presumed that the HC3368 strain can produce protein bacteriocin to inhibit oral pathogenic bacteria, thereby playing a role in oral health protection.

EXAMPLE 9 determination of the aggregation Rate of Pediococcus pentosaceus HC3368 against oral pathogens

The stronger the self-coagulation ability of the probiotics is, the better the colonization effect of the probiotics in the oral cavity is, the thalli can not be easily washed by saliva through the self-coagulation effect, and meanwhile, the high self-coagulation effect is beneficial to improving the concentration of the probiotics in the oral cavity and promoting the survival and health function exertion of the probiotics in the oral cavity. Meanwhile, probiotics can compete with pathogenic bacteria for the binding sites on the surface of the biological membrane and compete for nutrients through the coagglutination reaction with the pathogenic bacteria, so that the probiotics lose the capability of being adsorbed on the surface of teeth. Based on the above, the self-agglutination rate of the probiotics and the co-agglutination rate of the probiotics and pathogenic bacteria are measured, and the bacteria can be used for characterizing the colonization effect of the bacterial strain.

Experiment 1, self-coagulation Rate determination

The bacterial suspension of Pediococcus pentosaceus HC3368 of 1 mL is taken and added into a 24-well plate to stand at room temperature, 100 mu L of the bacterial suspension is taken to determine the initial absorbance A ₀ at 600 nm, the absorbance A _t at 600 nm is measured at intervals of 2h sucking the upper layer of the bacterial suspension in the subsequent 6 h, and the self-agglutination rate R _{Self-supporting}=（1-A_t/A₀ multiplied by 100% is calculated, and three samples are taken in parallel. The effect of self-aggregation of Pediococcus pentosaceus HC3368 at various time points is shown in FIG. 9, and the self-aggregation rate results are shown in Table 9 below.

TABLE 9 self-clotting Rate of Pediococcus pentosaceus HC3368 (unit:%)

As can be seen from Table 9, the self-aggregation rate of Pediococcus pentosaceus HC3368 at 6 h reached 52.15%, indicating that Pediococcus pentosaceus HC3368 was able to effectively colonize the oral cavity.

Experiment 2 Co-aggregation Rate determination

100. Mu.L of Pediococcus pentosaceus HC3368 bacterial suspension was taken and its initial absorbance at 600 nm A ₀ was determined, and 100. Mu.L of bacterial suspension of pathogenic bacteria (Streptococcus mutans ATCC25175, fusobacterium nucleatum BNCC336949, actinobacillus viscosus ATCC27044, porphyromonas gingivalis BNCC353909 or Acinetobacter concomitantly Actinobacillus BNCC 336945) was taken and its initial absorbance at 600 nm B ₀ was determined.

Equal amounts of Pediococcus pentosaceus HC3368 bacterial suspension and pathogenic bacterial suspension were mixed, shaken well, and then added into 24-well plates for standing at room temperature. 100. Mu.L of the upper suspension was measured at 600 nm for absorbance A _t at intervals of 2h, and the co-aggregation ratio R _{Co-production}=[1-2A_t/(A₀+B₀ was calculated as 100% and three replicates were obtained for each sample. The coagglutination effect of Pediococcus pentosaceus HC3368 against various pathogenic bacteria at 6h is shown in FIG. 10, and the coagglutination rate results are shown in Table 10 below.

TABLE 10 Co-aggregation Rate of Pediococcus pentosaceus HC3368 with different pathogenic bacteria (unit:%)

As can be seen from Table 10, pediococcus pentosaceus HC3368 was able to cause co-aggregation with five oral pathogenic bacteria of Streptococcus mutans, porphyromonas gingivalis, fusobacterium nucleatum, actinobacillus viscosus and Actinobacillus actinomyces, and the co-aggregation effect was higher than that of Pediococcus pentosaceus HC3368 in 2h, 4 h and 6 h. Wherein, the co-aggregation effect of Pediococcus pentosaceus HC3368 on streptococcus mutans, fusobacterium nucleatum and actinobacillus conglomerates can reach more than 59%.

As can be seen, pediococcus pentosaceus HC3368 strain exhibits good self-agglutination ability within 6 h, which is beneficial for the colonization of the oral cavity. Meanwhile, the Pediococcus pentosaceus HC3368 can also generate a coagglomeration effect with streptococcus mutans, fusobacterium nucleatum and the like, so that the relative content of pathogenic bacteria in the oral cavity is effectively reduced.

EXAMPLE 10 Remover of Pediococcus pentosaceus HC3368 against oral pathogenic biofilm

The biofilms of streptococcus mutans ATCC25175, porphyromonas gingivalis BNCC353909 and fusobacterium nucleatum BNCC336949 were cultured using an orifice plate method. 300. Mu.L of BHI medium added with 2% (m/v) sucrose was added to the wells of the 24-well plate, and then the activated oral pathogenic bacteria were inoculated in an inoculum size of 2% by volume. The 24-well plate is placed under micro-anaerobic condition and cultured at 37 ℃ for 48 h, and after the culture is finished, mature biological films of pathogenic bacteria appear on the inner wall and the bottom of the well. The in-well culture solution and non-adherent cells were discarded, and washed 2 times with PBS buffer (ph=7.0). The experimental group was further co-cultured with 300. Mu.L of Pediococcus pentosaceus HC3368 fermentation supernatant in wells for 24 h, three replicates each and the equivalent amount of MRS liquid medium was used as a control group instead of Pediococcus pentosaceus HC3368 fermentation supernatant. After the incubation, the supernatant containing planktonic cells was removed while maintaining the integrity of the biofilm, followed by three washes with sterile PBS buffer and fixation with methanol 15min, and the microplate was emptied and dried 45: 45 min. The biofilm was stained with 0.1% (m/v) crystal violet for 30min and then excess stain was removed. To each well 300 μl of absolute ethanol was added and the bound crystal violet was released back into the well. Finally, the solution was transferred to a new well plate and its absorbance at 600 nm was measured.

The results are shown in fig. 11, for the streptococcus mutans biofilm, which was 73.65% reduced after co-culture with the pediococcus pentosaceus HC3368 fermentation supernatant, the experimental group had a significant difference (P < 0.01) compared to the control group, for the porphyromonas gingivalis biofilm, which was 70.00% reduced after co-culture with the pediococcus pentosaceus HC3368 fermentation supernatant, the experimental group had a significant difference (P < 0.01) compared to the control group, and for the fusobacterium nucleatum biofilm, which was 57.83% reduced after co-culture with the pediococcus pentosaceus HC3368 fermentation supernatant, the experimental group had a significant difference (P < 0.01) compared to the control group.

The biomembrane can effectively help bacteria resist the change of external environment, and is beneficial to the growth and propagation of the bacteria. Oral pathogenic bacteria exist in the oral cavity in the form of a biofilm, and when the bacterial homeostasis in the biofilm changes, oral problems such as dental caries and periodontal disease can occur. Pediococcus pentosaceus HC3368 can significantly reduce the biofilm of Streptococcus mutans, fusobacterium nucleatum and Porphyromonas gingivalis, thereby reducing the risk of dental caries and periodontal disease in the oral cavity and helping to improve oral health problems.

EXAMPLE 11 Pediococcus pentosaceus HC3368 immunoregulatory Effect on gingival epithelial cells

Experiment 1, immune modulation of Streptococcus mutans infected gingival epithelial cells by Pediococcus pentosaceus HC3368

Resuscitated passaged human gingival epithelial cells C1052 are spread in 6-well plates according to 5X 10 ⁵ cells/well, and after the cells are attached, the cells are divided into three groups, and a subsequent infection experiment is carried out.

The first group served as a control group, and no bacterial infection was performed. The second group was designated as Streptococcus mutans ATCC25175 as a suspension in DMEM medium (10% FBS) and prepared in the same manner as in example 7 to infect human gingival epithelial cell C1052 at a multiplicity of infection MOI=250. The third group was designated as the variant+probiotic group, and was infected with human gingival epithelial cell C1052 at a multiplicity of infection moi=250, calculated as the number of pathogenic bacteria, by resuspending the same volume of the bacterial suspension of streptococcus mutans ATCC25175 and the bacterial suspension of pediococcus pentosaceus HC3368 in DMEM medium (containing 10% FBS). Three parallel groups are provided for each treatment group. The three groups of cells are respectively cultured in an incubator containing 5% CO ₂ at 37 ℃ for 24: 24h, after the culture is completed, the cells are centrifuged for 20: 20 min at 1000 Xg, and the supernatant is collected for detecting cell inflammatory factors IL-1 beta, IL-6 and IL-8, and the detection steps are carried out according to the description of ELISA kits.

As shown in fig. 12, the concentrations of pro-inflammatory cytokines IL-1 beta, IL-6 and IL-8 in the supernatants of human gingival epithelial cells after infection by streptococcus mutans reached 85.16 pg/mL, 464.03 pg/mL and 1835.67 pg/mL, respectively, which were far higher than the normal gingival epithelial cells of the control group (P < 0.01), whereas after drying the pediococcus pentosaceus HC3368, the concentrations of IL-1 beta, IL-6 and IL-8 decreased to 60.47 pg/mL, 169.54 pg/mL and 1247.96 pg/mL, respectively, which were reduced by 28.99%, 63.46% and 32.02%, respectively, with a significant difference compared to the streptococcus mutans group (P < 0.01).

Experiment 2, immune modulation of F.pentosaceus HC3368 on F.nucleatum infected gingival epithelial cells

The procedure of experiment 2 was essentially the same as that of experiment 1, except that:

(1) Infection of human gingival epithelial cell C1052 alone with fusobacterium nucleatum BNCC336949 instead of streptococcus mutans ATCC25175 and this treatment group was designated fusobacterium nucleatum group;

(2) The human gingival epithelial cell C1052 was co-infected with C.pentosaceus HC3368 using F.nucleatum BNCC336949 instead of Streptococcus mutans ATCC25175, and this treatment group was designated as a nucleated+probiotic group.

As shown in fig. 13, the concentrations of pro-inflammatory cytokines IL-1 beta, IL-6 and IL-8 in the supernatants of human gingival epithelial cells after infection with fusobacterium nucleatum reached 108.49 pg/mL, 557.36 pg/mL and 1249.00 pg/mL, respectively, far higher than the normal inflammatory levels of gingival epithelial cells of the control group (< 0.01 x P), and after drying the pediococcus pentosaceus HC3368, the concentrations of IL-1 beta, IL-6 and IL-8 decreased to 55.14 pg/mL, 226.21 pg/mL and 643.96 pg/mL, respectively, reduced 49.18%, 59.41% and 48.44%, respectively, with a significant difference compared to the fusobacterium nucleatum group (< 0.01 x P).

The experimental results show that the Pediococcus pentosaceus HC3368 can effectively improve oral inflammation caused by oral pathogenic bacteria such as streptococcus mutans, fusobacterium nucleatum and the like, and is beneficial to recovery of oral health.

EXAMPLE 12 preparation of probiotic formulations Using Pediococcus pentosaceus HC3368

(1) Pediococcus pentosaceus HC3368 was inoculated into MRS liquid medium at an inoculum size of 1% by volume, and cultured at 37℃for 24 h;

(2) Centrifuging the fresh bacterial liquid at a rotational speed of 8000 r/min for 10 min, and separating supernatant and bottom bacterial bodies;

(3) Collecting the supernatant as a probiotic preparation;

(4) And collecting the bottom thalli, washing the thalli twice by using PBS buffer solution (pH=7.0), and re-suspending the thalli until the absorbance OD _{600 nm} reaches 0.5-0.6, thereby obtaining another probiotic preparation.

Although the present invention has been described in detail by way of preferred embodiments with reference to the accompanying drawings, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims.

Claims

1. Pediococcus pentosaceus HC3368 with caries preventing or treating effect and periodontal disease preventing effect is characterized in that Pediococcus pentosaceus HC3368 (Pediococcus pentosaceus) has been deposited in China general microbiological culture Collection center (China general microbiological culture Collection center) with a deposit address of North Star Xidelu No.1, 3 in the Chaoyang area of Beijing city and a deposit number of CGMCC No.27356 in month 05 of 2023.

2. A probiotic formulation prepared from pediococcus pentosaceus HC3368 as described in claim 1.

3. The probiotic preparation according to claim 2, characterized in that the probiotic preparation comprises pediococcus pentosaceus HC3368 and/or a fermentation product of pediococcus pentosaceus HC 3368.

4. The probiotic preparation according to claim 2, characterized in that the preparation method of the probiotic preparation is:

5. The probiotic preparation according to claim 2, characterized in that the preparation method of the probiotic preparation is:

6. The probiotic formulation according to claim 2, wherein the probiotic formulation is an oral care product.

7. The probiotic formulation of claim 6, wherein the oral care product is selected from any one of a dental gel, a dentifrice, a toothpaste, a mouthwash, an oral spray, a dental floss, a tooth cleaning solution, and a tooth cleaning foam.

8. Use of pediococcus pentosaceus HC3368 as claimed in claim 1 for the preparation of a probiotic preparation having the efficacy of preventing or treating dental caries and periodontal disease.

9. The use according to claim 8, wherein the probiotic preparation has an inhibitory effect on oral pathogenic bacteria including streptococcus mutans, fusobacterium nucleatum, porphyromonas gingivalis, actinomyces viscosus and/or actinomyces conglomerates.

10. The use of claim 8, wherein the probiotic formulation is capable of modulating an inflammatory immune response by pathogenic bacteria in the oral cavity, and reducing the levels of pro-inflammatory cytokines IL-1 β, IL-6 and IL-8.