CN118374413B - Saliva combined lactobacillus NHNK-612 for reducing pathogenicity of oral pathogenic bacteria, and product and application thereof - Google Patents

Abstract

The present invention relates to the field of marine microbial technology, and in particular to a saliva-associated lactobacillus NHNK-612 for reducing the pathogenicity of oral pathogens, and its products and applications. The saliva-associated lactobacillus NHNK-612 provided by the present invention is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 2024799. Experiments show that NHNK-612 has the functions of regulating virulence genes of oral pathogens, inhibiting the formation of bacterial-fungal composite biofilms of oral pathogens, competitively binding to oral pathogen fungal hyphae, agglutinating oral pathogens, and inhibiting the formation of oral flora biofilms, and can be used to prepare medicines or products that reduce the pathogenicity of oral pathogens.

Description

A salivary lactobacillus NHNK-612 strain for reducing the pathogenicity of oral pathogens and its products and applications

Technical Field

The invention relates to the technical field of marine microorganisms, in particular to salivary lactobacillus and applications thereof.

Background Art

Grasshopper shrimp often inhabit the middle and lower layers of seawater with flat terrain and silty sandy bottom in estuaries, harbors and islands with fertile water. The grasshopper shrimp in this area are characterized by uniform individuals, bright colors, high fatness and tight meat. Small shrimps are added with salt and fermented into sticky shrimp paste, which is a common seasoning and folk food in coastal areas. The fermentation process involves a series of complex biochemical reactions to form a unique flavor and improve nutritional value. The microbial community is closely related to the fermentation matrix, showing a complex relationship between microorganisms, including partial harm symbiosis, parasitism, symbiosis, mutualism and competition. The wide variety of fermentation microorganisms in the marine industry play a very important role in inhibiting pathogenic microorganisms.

Biofilms are composed of a collection of microorganisms, mainly bacteria, but also fungi, viruses, protozoa and other microorganisms. They attach to the surface of the contact object and embed in the extracellular matrix produced by themselves, forming a large number of bacterial aggregation membrane-like objects. Bacterial biofilm is a life phenomenon that bacteria adapt to the natural environment and is conducive to survival. It is formed by the accumulation of microorganisms and their secretions and shows high resistance to antibiotics.

Mutans Streptococcus is a caries culprit, but it is usually stationary and restricted by saliva, but studies have found that they can bind to Candida albicans hyphae to move and reproduce at the same time, so that a microbial mass grows larger and larger. Mutans Streptococcus can attach to any part of Candida albicans, including Candida albicans itself, and even to the polysaccharides on the surface of Candida albicans. In addition to the wide range of movement, this intricate cell mass is also very resilient. Studies have also confirmed that the composite biofilms produced by bacterial-fungal collaboration are larger and more numerous than those formed by a single species. Individual bacteria can be easily washed away from teeth, while microbial masses are more likely to adhere to teeth and are more resistant to toothbrushes and antibacterial agents.

Veillonella is an important pathogen of periodontitis. It is a native oral bacterium in the tongue coating and has a high ability to attach to the tongue. It has been identified as the main producer of hydrogen sulfide ( _H2S ) and is also the most common anaerobic pathogen in chronic maxillary sinusitis. Streptococcus and Veillonella are the earliest oral colonizers and typical commensal bacteria, and they jointly participate in the early formation of oral biofilm. A large number of studies have shown that the symbiotic imbalance of Streptococcus and Veillonella is not only closely related to oral diseases such as caries and periodontal disease, but also can break through or invade the digestive barrier to achieve distal colonization. It has become a new potential biomarker for predicting the occurrence, development and prognosis of various systemic diseases.

Marine microorganisms have developed complex molecular adaptations to cope with these harsh conditions, affecting their primary and secondary metabolic pathways. This has led to the evolution of unique physiological characteristics and metabolic processes, and marine microorganisms are more likely to synthesize structurally unique enzymes and secondary metabolites than terrestrial microorganisms. Exploring the application of marine microorganisms has important practical significance in expanding the value of the marine industry.

Summary of the invention

In view of this, the present invention provides saliva-associated Lactobacillus and its application, which has the functions of regulating virulence genes of oral pathogens, inhibiting the formation of bacterial-fungal composite biofilms of oral pathogens, competitively binding to oral pathogen fungal hyphae, agglutinating oral pathogens, and inhibiting the formation of oral flora biofilms, and can be used to prepare medicines or products that reduce the pathogenicity of oral pathogens.

In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

The invention provides a strain of salivary lactobacillus (Ligilactobacillus salivarius) for reducing the pathogenicity of oral pathogens, the strain is named NHNK-612, and the preservation number is CCTCC NO: M 2024799.

The present invention also provides a product prepared based on the above-mentioned Ligilactobacillus salivarius, including a fermentation product or an inactivated bacterial cell of the above-mentioned Ligilactobacillus salivarius;

The preparation method of the fermentation product is as follows: the saliva and Lactobacillus salivarius are inoculated into a culture medium, cultured at 37°C for 46-50 hours to obtain a fermentation liquid, centrifuged to obtain a supernatant, filtered at a precision of 0.22 μm, and the obtained filtrate is the fermentation product;

The preparation method of the inactivated bacteria is as follows: inoculating the saliva-combined lactobacillus (Ligilactobacillus salivarius) into a culture medium, culturing at 37°C for 46-50 hours to obtain a fermentation liquid, centrifuging to obtain a precipitate, washing the precipitate, inactivating at 100-130°C for 10-30 minutes, and obtaining the inactivated bacteria.

The present invention also provides the use of the above-mentioned Ligilactobacillus salivarius or the above-mentioned product in preparing a product for reducing or lowering the pathogenicity of oral pathogens.

The present invention also provides the use of the above-mentioned saliva-associated lactobacillus (Ligilactobacillus salivarius) or the above-mentioned product in reducing or lowering the pathogenicity of oral pathogens.

In some specific embodiments of the present invention, the reduction or lowering of the pathogenicity of oral pathogens described in the above application includes at least one of the following:

(A1), regulating virulence genes of oral pathogens;

(A2) Inhibit the growth or proliferation of oral pathogens;

(A3), inhibiting the formation of oral pathogen biofilm;

(A4), competitive binding to fungal hyphae in oral pathogens;

(A5), agglutination of oral pathogens;

(A6) inhibit the proliferation of oral flora;

(A7), inhibiting the formation of oral flora biofilm;

The oral pathogens include one or more of Staphylococcus hominis, Staphylococcus aureus, Staphylococcus haemolyticus, Streptococcus mutans, Veillonella Atypica or Candida albicans.

In some specific embodiments of the present invention, the oral pathogen virulence gene described in the above application includes one or more of the virulence genes of Streptococcus mutans, Candida albicans or Staphylococcus aureus subsp. aureus;

The mutans streptococcus virulence genes include one or more of gtfC, gbpB, gbpC, ftf or luxS;

The Candida albicans virulence gene includes one or more of ALS1, ALS3, BGL2 or HWP1;

The virulence genes of Staphylococcus aureus subspecies aureus include one or more of icaA, agrA or fnbA.

In some specific embodiments of the present invention, the oral pathogenic bacteria biofilm described in the above application includes an oral pathogenic bacteria-fungus composite biofilm;

The oral pathogen bacteria-fungus composite biofilm includes: one or more of: Staphylococcus aureus-Candida albicans composite biofilm, Staphylococcus aureus subspecies aureus-Candida albicans composite biofilm, Staphylococcus haemolyticus-Candida albicans composite biofilm, Streptococcus mutans-Candida albicans composite biofilm or atypical Veillonella-Candida albicans composite biofilm.

In some specific embodiments of the present invention, the competitive binding to oral fungal hyphae described in the above application includes binding to hyphae of Candida albicans and interfering with the binding of the hyphae with other oral bacteria.

In some specific embodiments of the present invention, the agglutinated oral pathogens described in the above application include one or more of agglutinated Candida albicans, atypical Veillonella, Streptococcus mutans, Staphylococcus aureus subspecies aureus, Staphylococcus hominis or Staphylococcus haemolyticus.

The present invention also provides a product for reducing or lowering the pathogenicity of oral pathogens, comprising the above-mentioned Ligilactobacillus salivarius or the above-mentioned product, and acceptable adjuvants or auxiliary agents.

In some specific embodiments of the present invention, the above-mentioned product includes one or both of the following:

(B1), the above-mentioned strains and/or inactivated bacteria of Ligilactobacillus salivarius;

(B2) fermentation/secretion products of the above saliva combined with Lactobacillus salivarius.

The present invention also provides a medicine, comprising the above-mentioned Ligilactobacillus salivarius or the above-mentioned product, and acceptable auxiliary materials or adjuvants.

The present invention also provides the use of the above-mentioned Ligilactobacillus salivarius in the preparation of a product for regulating the virulence gene of oral pathogens, wherein the virulence gene for regulating oral pathogens comprises at least one of the following:

mutans Streptococcus virulence genes gtfC, gbpB, gbpC, ftf, and luxS;

Candida albicans virulence genes ALS1, ALS3, BGL2, and HWP1;

Virulence genes icaA, agrA, and fnbA of Staphylococcus aureus subspecies aureus.

The present invention also provides the use of the above-mentioned saliva combined with Lactobacillus salivarius in the preparation of a product for inhibiting the formation of oral pathogenic bacteria-fungus composite biofilms, wherein the oral pathogenic bacteria-fungus composite biofilm inhibiting the formation of the biofilm comprises at least one of Staphylococcus aureus-Candida albicans, Staphylococcus aureus subspecies aureus-Candida albicans, Staphylococcus haemolyticus-Candida albicans, Streptococcus mutans-Candida albicans and atypical Veillonella-Candida albicans composite biofilms.

The present invention also provides the use of the above-mentioned saliva combined with Lactobacillus salivarius inactivated bacteria in the preparation of a product for competitively binding oral pathogenic fungal hyphae, wherein the competitively binding oral pathogenic fungal hyphae binds to Candida albicans hyphae and interferes with the binding effect of its hyphae with other bacteria.

The present invention also provides the use of the above-mentioned saliva combined with inactivated Lactobacillus salivarius in the preparation of a product for agglutinating oral pathogens, wherein the agglutinating oral pathogens include at least one of Candida albicans, atypical Veillonella, Streptococcus mutans, Staphylococcus aureus subspecies aureus, Staphylococcus hominis, and Staphylococcus haemolyticus.

The present invention also provides the use of the saliva combined with the inactivated bacteria of Lactobacillus salivarius in the preparation of a product for reducing oral flora biofilm.

The present invention also provides the use of the above-mentioned saliva-associated lactobacillus (Ligilactobacillus salivarius) in the preparation of a product for reducing the pathogenicity of oral pathogens, wherein the conditions for reducing the pathogenicity of oral pathogens include at least one of regulating virulence genes of oral pathogens, inhibiting bacterial-fungal composite biofilms of oral pathogens, competitively binding to fungal hyphae of oral pathogens, agglutinating oral pathogens, and reducing oral flora biofilms.

In some specific embodiments of the present invention, the product described in the above application is a medicine or a product for reducing the pathogenicity of oral pathogens.

In some specific embodiments of the present invention, the above application also includes inhibiting the formation of oral pathogenic bacteria biofilm, and the inhibition of oral pathogenic bacteria biofilm includes inhibiting at least one of Staphylococcus aureus, Staphylococcus haemolyticus, Streptococcus mutans, atypical Veillonella and Candida albicans.

In some specific embodiments of the present invention, the above-mentioned application of inhibiting the proliferation of oral flora is to reduce the growth rate of oral flora.

In some specific embodiments of the present invention, the above-mentioned application of inhibiting oral flora biofilm formation is to reduce the oral flora biofilm formation rate.

The present invention also provides a method for reducing or lowering the pathogenicity of oral pathogens, comprising the step of applying the above-mentioned saliva-associated lactobacillus (Ligilactobacillus salivarius).

In some specific embodiments of the present invention, the reduction or lowering of the pathogenicity of oral pathogens described in the above application includes at least one of the following:

The present invention also provides a method for regulating virulence genes of oral pathogens, comprising the step of administering the above-mentioned saliva-associated lactobacillus (Ligilactobacillus salivarius).

The present invention also provides a method for inhibiting the growth or proliferation of oral pathogens, comprising the step of applying the above saliva-lactobacillus (Ligilactobacillus salivarius).

The present invention also provides a method for inhibiting the formation of oral pathogenic bacteria biofilm, comprising the step of applying the above saliva-associated lactobacillus (Ligilactobacillus salivarius).

The present invention also provides a method for competitively binding fungal hyphae in oral pathogens, comprising the step of applying the above-mentioned saliva-associated lactobacillus (Ligilactobacillus salivarius).

The present invention also provides a method for agglutinating oral pathogens, comprising the step of applying the above saliva-associated lactobacillus (Ligilactobacillus salivarius).

The present invention also provides a method for inhibiting the proliferation of oral flora, comprising the step of applying the above-mentioned saliva-associated lactobacillus (Ligilactobacillus salivarius).

The present invention also provides a method for inhibiting the formation of oral flora biofilm, comprising the step of applying the above-mentioned saliva-associated lactobacillus (Ligilactobacillus salivarius).

The saliva combined with lactobacillus and application thereof of the present invention have the following effects:

In vitro experiments show that the saliva-combined lactobacillus NHNK-612 of the present invention has the effect of inhibiting the expression of glucosyltransferase SI gene gtfC, glucose binding protein B gene gbpB, glucose binding protein C gene gbpC, fructosyltransferase gene ftf, and autoinducer-2 production protein gene luxS related to Streptococcus mutans, and the relative expression amount of the genes is downregulated to 0.01-0.83 times.

In vitro experiments show that the saliva combined with Lactobacillus NHNK-612 of the present invention has the effect of inhibiting the expression of cell surface glycoprotein genes ALS1/ALS3, cell wall β-1,3-glucan transferase gene BGL2, and hyphae cell wall protein gene HWP1 related to Candida albicans, and the relative expression of the genes is downregulated to 0.05-0.89 times.

In vitro experiments show that the saliva combined with Lactobacillus NHNK-612 of the present invention has the effect of inhibiting the expression of intercellular adhesion protein icaA, auxiliary gene regulatory factor a agrA, and fibronectin precursor fnbA related to Staphylococcus aureus subspecies aureus, and the relative expression of the genes is downregulated to 0.44-0.81 times.

In vitro experiments show that the saliva combined with Lactobacillus NHNK-612 of the present invention has the effect of inhibiting the formation of biofilms of Staphylococcus aureus, Staphylococcus aureus aureus subspecies, Staphylococcus haemolyticus, Streptococcus mutans, atypical Veillonella, and Candida albicans, as well as the formation of composite biofilms of Staphylococcus aureus-Candida albicans, Staphylococcus aureus aureus subspecies-Candida albicans, Staphylococcus haemolyticus-Candida albicans, Streptococcus mutans-Candida albicans, and atypical Veillonella-Candida albicans, and the biofilm formation rate is reduced to 18.68%~86.85%.

In vitro experiments show that the saliva combined with Lactobacillus NHNK-612 of the present invention has the effect of agglutinating oral pathogens such as Candida albicans, atypical Veillonella, Streptococcus mutans, Staphylococcus aureus subspecies aureus, Staphylococcus hominis, and Staphylococcus haemolyticus, and the agglutination rate can reach 15.79% to 44.00% after acting with the pathogens for 30 minutes.

In vitro experiments show that the saliva combined with Lactobacillus NHNK-612 of the present invention can effectively inhibit the formation of Candida albicans hyphae, and the hyphae formation rate is as low as 11.73% to 39.87%.

In vitro experiments show that the saliva combined with the Lactobacillus NHNK-612 fermentation product of the present invention has the effect of inhibiting the proliferation of oral flora, and the growth rate of oral flora is reduced to 32.01%.

In vitro experiments show that the saliva combined with Lactobacillus NHNK-612 of the present invention has the effect of inhibiting the formation of oral flora biofilm, and the oral flora biofilm formation rate is reduced to 31.05% to 63.15%.

In summary, NHNK-612 has the functions of regulating virulence genes of oral pathogens, inhibiting the formation of bacterial-fungal composite biofilms of oral pathogens, competitively binding to fungal hyphae of oral pathogens, agglutinating oral pathogens, and inhibiting the formation of oral flora biofilms. It can be used to prepare medicines or products that reduce the pathogenicity of oral pathogens.

Biological Deposit Description

Biological material: NHNK-612, classification name: Ligilactobacillus salivarius NHNK-612, deposited in China Center for Type Culture Collection on April 25, 2024, the address of the collection center is: Wuhan University, China; the collection number is CCTCC NO: M 2024799.

The NHNK-612 described in the present invention is the strain with the above-mentioned deposit number of CCTCC NO: M 2024799.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

FIG1 shows the MRS plate colony image and Gram staining image of saliva-associated Lactobacillus NHNK-612;

FIG2 shows the screening results of saliva combined with Lactobacillus to inhibit the growth of pathogens;

FIG3 shows the agglutination effect of saliva combined with inactivated Lactobacillus NHNK-612 and pathogenic bacteria;

FIG. 4 shows saliva combined with inactivated Lactobacillus NHNK-612 and Candida albicans mycelium.

DETAILED DESCRIPTION

The present invention discloses saliva combined lactobacillus and application thereof, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to realize. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

The saliva-associated Lactobacillus strain NHNK-612 of the present invention is derived from naturally fermented shrimp paste in Qingdao and is identified as saliva-associated Lactobacillus (Ligilactobacillus salivarius) by 16S rDNA. The strain is Gram-positive and short rod-shaped under a microscope; it grows on an MRS plate to form a round colony with a smooth, translucent surface, white in color, and neat edges; it grows uniformly turbid in an MRS liquid culture medium, and the bacteria form white precipitation after long-term placement, and the optimum growth temperature is 37°C.

Ligilactobacillus salivarius NHNK-612, depository: China Center for Type Culture Collection, address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, deposit date: April 25, 2024, deposit number: CCTCC NO: M 2024799.

Furthermore, the saliva-associated Lactobacillus NHNK-612 provided by the present invention is in the form of sterilization or fermentation product (i.e., supernatant) in the application described in the present invention, and the derivative form is preferably selected from: metabolites, metabolic biological products, prebiotics, cell walls and components thereof, extracellular polysaccharides, and compounds containing immunogenic components, preferably selected from: fermentation products, inactivated bacteria.

It should be understood that the expression "one or more of..." includes individually each of the objects recited after the expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and/or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

The use of any and all examples or exemplary language such as "for example" or "including" herein is intended only to better illustrate the present invention and is not intended to limit the scope of the present invention. Any language in this specification should not be construed as indicating that any unclaimed element is essential to the practice of the present invention.

In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

The present invention will be further described below in conjunction with embodiments:

Example 1: Isolation of NHNK-612

The naturally fermented shrimp paste from Qingdao was shaken and mixed, then diluted 10 times with physiological saline. After mixing again, 100 μL of the dilution was spread on MRS culture medium and placed in an anaerobic bag. After constant temperature culture at 37°C for 48 h, white colonies were picked and repeatedly streaked and screened until a uniform single colony was obtained, which was named NHNK-612.

Gram staining microscopy: strain NHNK-612 is a Gram-positive colony, which appears as a short rod under a microscope; it grows on an MRS plate and forms white, smooth, round, semi-transparent round colonies with neat edges; it grows evenly in MRS liquid culture medium, and the bacteria form white precipitates after long-term storage. See Figure 1.

Example 2: Nucleic acid identification of NHNK-612

1. 16S rDNA gene sequence analysis:

Single colonies were picked and placed in MRS liquid medium, cultured overnight at 37°C, centrifuged at 8000 rpm for 1 min to collect the bacteria, and operated according to the instructions of the Gram-positive bacteria DNA extraction kit. The primers used were bacterial 16S sequencing universal primers 27F and 1492R, and the PCR amplification system was 20 μL. The PCR amplification program was 95°C pre-denaturation for 5 min, 94°C for 15 s, 57°C for 15 s, 72°C for 1 min, 35 cycles; 72°C extension for 10 min.

2. Results

The PCR product was sequenced and compared with the standard sequence published in the GenBank database for homology (BLASTN), and the NHNK-612 strain was found to be Ligilactobacillus salivarius. Its nucleotide sequence is as follows:

GGCTGGCTCCTTGCGGTTACCCCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGACATGCTGATTCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAACGGCTTAAGAGATTAGCTAAACCTCGCGGTCTCCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATG ACTTGACGTCGTCCCCACCTTCCTCCGGGTTTGTCACCGGCAGTCTCGCCAGAGTGCCCAACTTAATGCTGGCAACTGACAACAAGGGTTGCGC TCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCACTTTGTCCCCGAAGGGAAAGCCTAATCTCTTAGGTGGTCAAAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTGGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTATTGCGTTAGCTGCGGCACTGAAGGGCGG AAACCCTCCAACACCTAGCATTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTACCCACGCTTTCGAACCTCAGCGTCAG TTACAGACCAGAGAGCCGCTTTCGCCACTGGTGTTCTTCCATATATCTACGATTTCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGTCTTCCAGTTTCCAATGCACTACTCCGGTTAAGCCGAAGGCTTTCACATCAGACTTAAAAGACCGCCTGCGTTCCCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTGCTGG TTAGATACCGTCATCGAATGAACAGTTACTCTCACTCGTGTTCTTCTCTAACAACAGAGTTTTACGATCCGAAGACCTTCTTCACTCACGCGGCGTTGCT CCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAACCTCTCAGTTCGGCTACGTATCATCACCTTGGTAGGCCGTTACCCCACCAACTAGTTAATACGCCGCGGGTCCATCTAAAAGCGATAGCAGAACCATCTTTCATCTAAGGATCATGCGATCCTTAGAGATATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTT CTTTTAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCACTCAACTTCTTACGGTGAATGCAAGCATTCGGTGTAAGAAAGTTTCGTCGACTG (SEQ ID NO: 1).

Example 3: Experiment on the inhibition of oral pathogen proliferation by saliva combined with Lactobacillus

1. Preparation of saliva combined with lactobacillus fermentation products

The saliva was combined with different strains of Lactobacillus NHNK-612, C-1, C-2, C-3, C-4, C-5, C-6, C-7, C-8, C-9, C-10, C-11, C-12, C-13, C-14, C-15, C-16, C-17, C-18, and C-19 at a ratio of 2%, respectively, inoculated into fresh MRS medium, cultured at 37°C for 48 h, adjusted to _OD600 = 1.0 with MRS, then centrifuged at 5000 g for 10 min, and the supernatant was taken and filtered through a 0.22 μm sterile filter membrane to obtain the fermentation product.

2. Cultivation of oral pathogens

Mutans Streptococcus CGMCC 1.2499 was inoculated into BHI medium at a ratio of 1%. Staphylococcus aureus subsp. aureus CGMCC 1.8721 was inoculated into BHI medium at a ratio of 1%. Atypical Veillonella DSM 20739 was inoculated into Veillonella medium at a ratio of 1%. Candida albicans CGMCC 2.4550 was inoculated into Sabouraud medium at a ratio of 1%. Staphylococcus haemolyticus CGMCC 1.540 was inoculated into BHI medium at a ratio of 1%. Staphylococcus hominis CGMCC 1.493 was inoculated into BHI medium at a ratio of 1%. All the above pathogens were cultured at 37°C with shaking for 24 h and adjusted to OD ₆₀₀ = 1.0 with the original culture medium for use.

3. Experiment on the inhibition of proliferation of common oral pathogens by saliva combined with lactobacillus fermentation products

In the experimental group, 4 ml of oral pathogen culture medium (BHI medium/Veillonella medium/Sabouraud medium) and 1 ml of saliva combined with Lactobacillus fermentation product were added to a centrifuge tube. In the control group, an equal volume of MRS liquid culture medium was added and the oral pathogen culture liquid was inoculated at a 1% inoculation rate. The cells were cultured at 37°C for 24 hours and the absorbance at OD=600 nm was measured. The results are shown in Figure 2.

The results showed that the 20 strains of saliva combined with lactobacillus had different degrees of inhibitory effect on the growth of different pathogens. NHNK-612 also had a strong inhibitory effect on the proliferation of Streptococcus mutans, Staphylococcus aureus subspecies aureus, atypical Veillonella, Candida albicans, Staphylococcus haemolyticus, and Staphylococcus hominis. It has a broad-spectrum antibacterial effect and can be used for further research and development against oral pathogens.

Example 4: Experiment on the inhibition of virulence gene expression of Streptococcus mutans by NHNK-612

1. Preparation of NHNK-612 fermentation products and inactivated bacteria

A single colony of saliva-associated Lactobacillus NHNK-612 was selected and placed in MRS liquid medium, cultured at 37°C for 48 h, adjusted to OD ₆₀₀ = 1.0 with PBS, centrifuged at 5000 rpm for 10 min, and the supernatant was obtained, and then filtered with a 0.22 μm filter membrane to obtain a sterile fermentation product. The bacterial precipitate was washed twice with PBS solution, and the bacterial body was resuspended with PBS solution to OD ₆₀₀ = 1.0. The inactivated bacterial body was obtained by high-pressure sterilization at 121°C for 15 min.

2. Experiment on inhibiting the expression of virulence genes of Streptococcus mutans

Cryopreserved tubes of Streptococcus mutans CGMCC 1.2499 were inoculated into fresh brain heart infusion broth (BHI) at a ratio of 1%, and cultured at 37°C for 24 h. Take 3 mL of cultured Streptococcus mutans, add 1 mL of NHNK-612 fermentation product or inactivated bacteria (the control group added an equal volume of PBS), 1 mL of fresh BHI, and culture at 37°C for 24 h. After the culture, centrifuge at 8000rpm for 1 min to obtain the bacteria, extract total RNA according to the instructions of the kit, detect RNA concentration and purity, and reverse transcribe it into cDNA. Using 16S as the internal reference gene, qPCR was used to detect the expression of luxS, gbpB, gtfC, ftf and gbpC genes. The relative expression fold of the control group gene F=1, and the F value of each sample was calculated using the 2 ^-ΔΔCT method.

Formula: F=2 ^-ΔΔCT , where:

△ _CTexperiment = _CTexperiment -CT _{internal reference (experiment);}

△CT _control = CT _control - CT _{internal reference (control);}

△△CT=△CT _{experimental-} △CT _control.

The results are shown in Tables 1 and 2.

Table 1: NHNK-612 fermentation products reduce the expression of virulence genes of Streptococcus mutans

Table 2: NHNK-612 inactivated bacteria reduces the expression of virulence genes of Streptococcus mutans

The results showed that the fermentation products and inactivated bacteria of NHNK-612 had the effect of reducing the expression of virulence genes of Streptococcus mutans.

Example 5: Experiment on the inhibition of virulence gene expression of Candida albicans by NHNK-612

1. Preparation of NHNK-612 fermentation products and inactivated bacteria

The preparation method is as in Example 4.

2. Experiment on inhibiting the expression of virulence genes of Candida albicans

The CGMCC 2.4550 cryopreserved tube of Candida albicans was inoculated into fresh Sabouraud medium at a ratio of 1%, and cultured at 37°C for 24 h. Take 3 mL of the cultured Candida albicans liquid, add 1 mL of NHNK-612 fermentation product or inactivated bacteria (the control group added an equal volume of PBS), 1 mL of fresh Sabouraud medium, and cultured at 37°C for 24 h. After the culture, centrifuge at 8000 rpm for 1 min to obtain the bacteria, extract total RNA according to the instructions of the kit, detect RNA concentration and purity, reverse transcribe it into cDNA, use TDH1 as the internal reference gene, and use qPCR to detect the expression of ALS1, HWP1, ALS3 and BGL2 genes. The relative expression multiple of the control group gene F=1, and the F value of each sample is calculated by the 2 ^-ΔΔCT method, and the calculation method is referred to Example 3.

The results are shown in Table 3.

Table 3: NHNK-612 fermentation products reduce the expression of virulence genes in Candida albicans

Table 4: NHNK-612 inactivated bacteria reduces the expression of virulence genes in Candida albicans

The results showed that the fermentation products and inactivated bacteria of NHNK-612 had the effect of reducing the expression of virulence genes of Candida albicans.

Example 6: Experiment on the inhibition of virulence gene expression of Staphylococcus aureus subsp. aureus by NHNK-612

1. Preparation of NHNK-612 fermentation products and inactivated bacteria

The preparation method is as in Example 4.

2. Experiment on inhibiting the expression of virulence genes of Staphylococcus aureus subspecies aureus

The Staphylococcus aureus subspecies aureus CGMCC 1.8721 cryopreservation tube was inoculated into fresh BHI at a ratio of 1% and cultured at 37°C for 24 h. Take 3 mL of the cultured Staphylococcus aureus subspecies aureus bacterial solution, add 1 mL of NHNK-612 fermentation product or inactivated bacteria (the control group added an equal volume of PBS), 1 mL of fresh BHI, and culture at 37°C for 24 h. After the culture was completed, the bacteria were centrifuged at 8000 rpm for 1 min, and the total RNA was extracted according to the instructions of the kit. After the RNA concentration and purity were detected, it was reverse transcribed into cDNA, and gyrB was used as the internal reference gene. qPCR was used to detect the expression of icaA, agrA and fnbA genes. The relative expression multiple of the control group gene F=1, and the F value of each sample was calculated by the 2 ^-ΔΔCT method, and the calculation method was referred to Example 3.

The results are shown in Table 5.

Table 5: NHNK-612 fermentation products reduce the expression of virulence genes of Staphylococcus aureus subsp. aureus

Table 6: NHNK-612 inactivated bacteria reduces the expression of virulence genes of Staphylococcus aureus subsp. aureus

The results showed that the fermentation products and inactivated bacteria of NHNK-612 had the effect of inhibiting the expression of virulence genes of Staphylococcus aureus subsp. aureus.

Example 7: Experiment on the inhibition of pathogenic bacteria biofilm formation by NHNK-612

1. Preparation of NHNK-612 fermentation products and inactivated bacteria

Pick a single colony of saliva-associated Lactobacillus NHNK-612 in MRS liquid medium, culture it at 37°C for 48 h, adjust it to OD ₆₀₀ = 1.0 with MRS, centrifuge it at 5000 rpm for 10 min, take the supernatant, and then filter it with a 0.22 μm filter membrane to obtain a sterile fermentation product. After the bacterial precipitate is washed twice with PBS solution, the bacteria are resuspended with PBS solution to OD ₆₀₀ = 1.0. Autoclave at 121°C for 15 min to obtain inactivated bacteria.

2. Preparation of pathogenic bacteria solution

Single colonies of pathogens: Staphylococcus aureus CGMCC 1.493, Staphylococcus aureus subsp. aureus CGMCC 1.8721, Staphylococcus haemolyticus CGMCC 1.540, and Streptococcus mutans CGMCC 1.2499 were picked and inoculated into BHI liquid culture medium, cultured at 37°C overnight with shaking, and adjusted to _OD600 = 0.3 with BHI liquid culture medium after the culture was completed; single colonies of atypical Veillonella DSM20739 were picked and inoculated into Veillonella liquid culture medium, cultured anaerobically at 37°C for 24 h, and adjusted to _OD600 = 0.3 with Veillonella liquid culture medium after the culture was completed; single colonies of Candida albicans CGMCC 2.4550 were picked and inoculated into Sabouraud liquid culture medium, cultured at 37°C overnight with shaking, and adjusted to _OD600 = 0.3 with Sabouraud liquid culture medium after the culture was completed.

3. Experiment on inhibition of pathogenic bacteria biofilm

100 μL of pathogenic bacteria solution was added to a 96-well plate. 100 μL of NHNK-612 fermentation product or inactivated bacteria was added to the experimental group, and the control group was replaced with an equal volume of MRS medium or PBS, respectively. Three parallels were set up for each group and cultured at 37°C for 24 h. After the culture was completed, the supernatant was discarded, and 100 μL of sterile PBS was added to each well for washing twice. Then, 100 μL of 4% paraformaldehyde fixative was added to each well and fixed at room temperature for 30 min. The fixative was discarded, and 100 μL of crystal violet was added to each well and stained at room temperature for 30 min. After staining, the plates were washed twice with sterile PBS and dried, and 100 μL of anhydrous ethanol was added to each well. After standing for 1 min, the absorbance at 600 nm was measured, and the biofilm formation rate was calculated according to the formula.

The calculation formula and results are as follows:

Table 7: NHNK-612 fermentation products inhibit pathogenic bacterial biofilm formation

Table 8: NHNK-612 inactivated bacteria inhibits pathogenic bacteria biofilm formation

The results showed that the fermentation products and inactivated bacteria of NHNK-612 had the effect of inhibiting the biofilm formation of pathogenic bacteria.

Example 8: Experiment on the inhibition of pathogenic bacteria-fungus composite biofilm formation by NHNK-612

1. Preparation of NHNK-612 fermentation products and inactivated bacteria

The preparation method is similar to that of Example 7.

2. Preparation of pathogenic bacteria solution

The preparation method is similar to that of Example 7.

3. Experiment on inhibition of pathogenic bacteria-fungus composite biofilm

100 μL of Candida albicans liquid was mixed with 100 μL of other pathogenic bacteria liquid and added to 96-well plates. 100 μL of NHNK-612 fermentation product or inactivated bacteria was added to the experimental group, and the control group was replaced with an equal volume of MRS medium or PBS. Three parallels were set up for each group and cultured at 37°C for 24 h. The fixation, staining and determination methods of the well plate were referred to Example 6.

The calculation formula and results are as follows:

Table 9: NHNK-612 fermentation products inhibit bacterial-fungal composite biofilm formation

Table 10: NHNK-612 inactivated bacteria inhibits bacterial-fungal composite biofilm formation

The results showed that NHNK-612 fermentation products and inactivated bacteria could inhibit the formation of pathogenic bacteria-fungus composite biofilm.

Example 9: NHNK-612 agglutination of pathogenic bacteria experiment

1. Preparation of NHNK-612 inactivated bacteria

The preparation method is similar to that of Example 7.

2. Preparation of pathogenic bacteria solution

The culture methods of Candida albicans, atypical Veillonella, Streptococcus mutans, Staphylococcus aureus, Staphylococcus hominis, and Staphylococcus haemolyticus were referred to Example 7. After the culture was completed, the supernatant was discarded by centrifugation at 5000 rpm for 10 min, and then washed twice with sterile PBS buffer, and then resuspended with sterile PBS buffer to adjust _OD600 = 1.0.

3. Agglutination

The pathogen solution was mixed with the NHNK-612 inactivated bacteria solution in a volume ratio of 1:1. The pathogen plus PBS was used as a control. The flocculation state was observed after standing for 30 min.

4. Index determination

At 30 minutes after the reaction, samples of NHNK-612 alone, pathogens alone, and the mixed reaction liquid of NHNK-612 and pathogens were collected. The sampling range was the top 50 μL of the liquid surface, and the samples were transferred to a 96-well plate to measure the absorbance at OD = 600nm. At the same time, the agglutination precipitate was stained with Gram stain to observe the agglutination state of the bacteria.

Calculation formula:

Agglutination rate (%) = [(Ax + Ay) - 2Amix] / (Ax + Ay) × 100%;

Note: Ax: _OD600 value measured at the reaction time for lactic acid bacteria alone; Ay: _OD600 value measured at the reaction time for pathogens alone; Amix: _OD600 value measured at the reaction time after lactic acid bacteria and pathogens were mixed.

The agglutination effect of NHNK-612 inactivated bacteria on pathogens is shown in Table 11.

Table 11: Agglutination of pathogens by NHNK-612 inactivated bacteria

The agglutination state of the bacteria is shown in FIG3 , and the results in combination with those in Table 11 show that after 30 min of reaction, the NHNK-612 inactivated bacteria can effectively agglutinate and capture Candida albicans, atypical Veillonella, Streptococcus mutans, Staphylococcus aureus, Staphylococcus hominis and Staphylococcus hemolyticus.

Example 10: Experiment on the combination of NHNK-612 inactivated bacteria and Candida albicans hyphae

1. Preparation of NHNK-612 inactivated bacteria

The preparation method is similar to that of Example 7.

2. Preparation of Candida albicans mycelium

The method for culturing Candida albicans was referred to Example 7. After the culture was completed, the cells were centrifuged at 5000 rpm for 10 min to obtain the cells. The cells were resuspended with fetal bovine serum (FBS) and adjusted to OD = 0.1, and cultured at 37°C for 3 h to obtain mycelium, which was washed twice with PBS, and after centrifugation to remove the supernatant, the mycelium was adjusted to OD ₆₀₀ = 1.0 with PBS.

3. Experiment on inactivated bacteria combined with Candida albicans mycelium

Add 0.7 mL of Candida albicans mycelium and 0.7 mL of NHNK-612 bacteria into the test tube, vortex thoroughly and let stand. After 1 h, a white precipitate appears in the test tube. Gently aspirate the precipitate and stain it with Gram stain. Observe and photograph it under a microscope with a 100x oil objective.

The results are shown in Figure 4. There is a significant size difference between the inactivated NHNK-612 bacteria and the mycelia of Candida albicans, and the inactivated NHNK-612 bacteria and the mycelia of Candida albicans can be distinguished with the naked eye. The results show that the inactivated NHNK-612 bacteria can significantly bind to the hyphae of the mycelia of Candida albicans, and have a competitive inhibition effect on the binding of other pathogens to the mycelia of Candida albicans, interfering with the synergistic effect of bacteria and fungi.

Example 11: Experiment on the inhibition of Candida albicans hyphae formation by NHNK-612

1. Preparation of NHNK-612 fermentation products and inactivated bacteria

The preparation method is similar to that of Example 7.

2. Induction of hyphae morphology of Candida albicans

The method for culturing Candida albicans is referred to Example 7. After the culture is completed, the cells are centrifuged at 5000 rpm for 10 min to obtain the cells. The cells are resuspended with fetal bovine serum (FBS) and adjusted to OD ₆₀₀ = 0.1.

3. Experiment on the inhibition of Candida albicans hyphae formation by NHNK-612

In the experimental group, 100 μL of FBS-resuspended Candida albicans was added to a 96-well plate, and 100 μL of NHNK-612 fermentation product or inactivated bacteria was added. In the control group, an equal volume of MRS medium or PBS was used instead. Three parallels were set up for each group and cultured at 37°C for 2 h. After the culture, the culture medium was discarded, and the plate was washed once with 70% alcohol, once with 0.25% SDS solution, and washed 3 times with sterile water, and then stained with 0.1% crystal violet solution for 30 min. After staining, it was washed once with 0.25% SDS solution and washed 3 times with sterile water. The biofilm at the bottom was observed after the plate was naturally dried. 200 μL of 40 mmol/L HCL-isopropanol solution and 50 μL of 0.25% SDS solution were added to each well, and the absorbance at OD=600 nm was measured after standing at room temperature for 1 min. The calculation formula and results are shown in Table 12.

Table 12: NHNK-612 inhibits Candida albicans hyphae formation

The results showed that NHNK-612 fermentation products and inactivated bacteria could effectively inhibit the hyphae formation of Candida albicans.

Example 12: Experiment on the inhibition of oral flora proliferation by NHNK-612 fermentation product

1. Preparation of NHNK-612 fermentation products

The preparation method is similar to that of Example 7.

2. Collection and cultivation of oral flora

Dental plaque of volunteers was scraped with a sterile cotton swab and inoculated into BHI broth medium. The culture was anaerobically placed at 37°C for 24 hours to obtain oral bacterial fluid.

3. Experiment on the inhibition of oral flora proliferation by NHNK-612 fermentation products

In the experimental group, 4 ml of BHI and 1 ml of NHNK-612 fermentation product were added to a centrifuge tube, and in the control group, an equal volume of MRS liquid culture medium was added. The oral bacterial culture fluid was inoculated at a 1% inoculation rate and cultured at 37°C for 24 hours. The absorbance at OD=600nm was measured. The results are shown in Table 13.

Table 13: NHNK-612 fermentation products inhibit the proliferation of oral flora

The results showed that the fermentation products of NHNK-612 had the effect of inhibiting the proliferation of oral flora.

Example 13: Experiment on the inhibition of oral flora biofilm formation by NHNK-612

1. Preparation of NHNK-612 fermentation products and inactivated bacteria

The preparation method is similar to that of Example 7.

2. Collection and cultivation of oral flora

The preparation method is similar to that of Example 12.

3. Experiment on the inhibition of oral flora biofilm formation by NHNK-612

The cultured oral bacterial suspension OD ₆₀₀ was adjusted to 0.3 with BHI liquid medium. 100 μl of oral bacterial suspension was added to each well of the experimental group, and 100 μl of NHNK-612 fermentation product or inactivated bacteria was added. The control group was replaced with an equal volume of MRS medium or PBS, and each group was set up with 3 parallels. Cultured at 37 degrees for 24 hours, Gram staining was performed after the culture was completed according to the method described in Example 6, and the absorbance at 600 nm was measured. The calculation formula and results are shown in Table 14.

Table 14: NHNK-612 inhibits oral flora biofilm formation

The results showed that both NHNK-612 fermentation products and inactivated bacteria could reduce the formation of oral flora biofilm.

The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims (8)

1. A salivary lactobacillus (Ligilactobacillus salivarius) for reducing pathogenicity of pathogenic bacteria in oral cavity is characterized in that the preservation number is CCTCC NO: M2024799.

2. A product made on the basis of lactobacillus salivarius (Ligilactobacillus salivarius) as claimed in claim 1 characterised in that it is a fermentation product or an inactivated cell of said lactobacillus salivarius (Ligilactobacillus salivarius);

the preparation method of the fermentation product comprises the following steps: inoculating the saliva combined lactobacillus (Ligilactobacillus salivarius) into a culture medium, culturing for 46-50 hours at 37 ℃ to obtain a fermentation liquor, centrifuging to obtain a supernatant, and filtering with the accuracy of 0.22 mu m to obtain a filtrate which is the fermentation product;

The preparation method of the inactivated bacteria comprises the following steps: inoculating the saliva combined lactobacillus (Ligilactobacillus salivarius) into a culture medium, culturing for 46-50 h at 37 ℃ to obtain fermentation liquor, centrifuging to obtain precipitate, cleaning the precipitate, and inactivating at 100-130 ℃ for 10-30 min to obtain the inactivated bacteria.

3. Use of lactobacillus salivarius (Ligilactobacillus salivarius) as claimed in claim 1 or a product as claimed in claim 2 in the manufacture of a product for reducing pathogenicity of oral pathogenic bacteria;

The reducing oral pathogenic bacteria pathogenicity comprises at least one of:

(A1) Inhibiting growth or proliferation of pathogenic bacteria in the oral cavity;

(A2) Inhibiting oral pathogenic bacteria biofilm formation;

(A3) Competing for binding to fungal hyphae in oral pathogens;

(A4) Agglutinating oral pathogenic bacteria;

The oral pathogenic bacteria include one or more of human staphylococcus (Staphylococcus hominis), staphylococcus aureus (Staphylococcus aureus) subspecies aureous, staphylococcus hemolyticus (Staphylococcus haemolyticus), streptococcus mutans (Streptococcus mutans), veillonella atypical (Veillonella Atypica) or Candida albicans.

4. The use according to claim 3, wherein the oral pathogenic biofilm comprises an oral pathogenic bacteria-fungi composite biofilm;

The oral pathogenic bacteria-fungi composite biological film comprises: one or more of a human staphylococcus-candida albicans composite biological film, a staphylococcus aureus subspecies aureoviridae-candida albicans composite biological film, a lysostaphylococcus-candida albicans composite biological film, a streptococcus mutans-candida albicans composite biological film or an atypical veillonella-candida albicans composite biological film.

5. The use according to claim 3, wherein said competing for binding of fungal hyphae in oral pathogenic bacteria comprises binding to candida albicans hyphae, interfering with the binding of said hyphae to other oral bacteria.

6. Use of lactobacillus salivarius (Ligilactobacillus salivarius) as claimed in claim 1 or a product as claimed in claim 2 in the manufacture of a product for reducing pathogenicity of oral pathogenic bacteria;

the reducing the pathogenicity of an oral pathogenic bacteria comprises down-regulating an oral pathogenic bacteria virulence gene;

The oral pathogen virulence genes comprise one or more of streptococcus mutans virulence genes, candida albicans virulence genes or staphylococcus aureus subspecies aureobasidium virulence genes;

The streptococcus mutans virulence genes include one or more of gtfC, gbpB, gbpC, ftf or luxS;

the candida albicans virulence gene comprises one or more of ALS1, ALS3, BGL2 or HWP 1;

The staphylococcus aureus subspecies aureoviridae virulence genes comprise one or more of icaA, agrA or fnbA.

7. A product for reducing pathogenicity of oral pathogens comprising saliva according to claim 1 in combination with lactobacillus (Ligilactobacillus salivarius) or a product according to claim 2, and acceptable adjuvants or adjuvants.

8. Pharmaceutical product, characterized in that it comprises a salivary lactobacillus (Ligilactobacillus salivarius) in combination according to claim 1 or a preparation according to claim 2, and acceptable adjuvants or auxiliaries.