CN118345015B

CN118345015B - Fermented lactobacillus mucilaginosus and application thereof in preparation of oral health improving products

Info

Publication number: CN118345015B
Application number: CN202410780432.2A
Authority: CN
Inventors: 陈奕兴; 崔庆宇; 赵兰慧; 谭磊磊; 冯海霞; 宁长春; 殷冀煜
Original assignee: Qingdao Nuohe Nuokang Biotechnology Co ltd
Current assignee: Qingdao Nuohe Nuokang Biotechnology Co ltd
Priority date: 2024-06-18
Filing date: 2024-06-18
Publication date: 2024-09-03
Anticipated expiration: 2044-06-18
Also published as: CN118345015A

Abstract

The invention belongs to the field of marine microorganisms, and provides fermented lactobacillus mucilaginosus and application thereof in preparation of oral health products, wherein the fermented lactobacillus mucilaginosus NHNK-606 (Limosilactobacillus fermentum NHNK-606) is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2024419 and is obtained from intestinal tracts of blue-green-island perches, and the inventor finds that the strain has the functions of regulating oral pathogen virulence genes, inhibiting oral pathogen bacteria-fungus composite biological membranes, competing to combine oral pathogen fungus hyphae, aggregating oral pathogen and reducing oral flora biological membranes, and can be used for preparing products for reducing oral pathogen pathogenicity, improving oral health products and the like.

Description

Fermented lactobacillus mucilaginosus and application thereof in preparation of oral health improving products

Technical Field

The invention belongs to the field of marine microorganisms, and provides a lactobacillus mucilaginosus strain and application thereof in preparation of products for improving oral health.

Background

In the fish intestinal microbiota, bacteria (aerobic, facultative anaerobic and obligate anaerobic) are the main colonising microorganisms, which play a very important role in the growth process of fish, and their equilibrium state is related to the health of the fish body. A wide variety of microorganisms in the intestinal tract of fish play a very important role in inhibiting pathogenic microorganisms, and can protect host fish from being affected by the pathogenic microorganisms.

Biofilms consist of collections of microorganisms, mainly bacteria, but also fungi, viruses, protozoa and other microorganisms. They adhere to the surface of the contact and become embedded in the self-produced extracellular matrix, forming a large number of bacterial aggregate membrane samples. Bacterial biofilms are a life phenomenon in which bacteria are formed by the accumulation of microorganisms and their secretions to adapt to natural environments and are beneficial to survival, exhibiting high resistance to antibiotics.

Mutans streptococcus is the leading cause of dental caries, but is usually stationary and subject to saliva limitation, but studies have found that they can move in combination with candida albicans hyphae while also multiplying resulting in an increasingly larger microbiota. Streptococcus mutans can be attached to any location on the surface of Candida albicans, including Candida albicans itself, or even polysaccharides on the surface of Candida. In addition to the wide range of movement, this intricate cell pellet is also very flexible, and studies have also demonstrated that bacterial-fungal cooperation produces a greater number of complex biofilms than those produced by a single species. Individual bacteria are readily washed away from the teeth, while microbiota is more readily attached to the teeth and is more resistant to toothbrushes and bacteriostatic agents.

Veillonella is an important causative agent of periodontitis, a indigenous oral bacterium in the tongue coating, and a high ability to adhere to the tongue, has been identified as the primary producer of hydrogen sulfide, and is also the most common anaerobic causative agent in chronic maxillary sinusitis. Streptococcus (Streptococcus) and Vellonella (Vellonella) are the earliest colonising bacteria and typical commensal bacteria in the oral cavity, together involved in early biofilm formation in the oral cavity. A large number of researches show that the symbiotic disorder of streptococcus and veillonella is closely related to dental diseases such as caries, periodontal disease and the like, and can break through or invade the digestive barrier to realize distal colonization, so that the streptococcus and veillonella symbiotic disorder becomes a new potential biomarker for predicting occurrence, development and prognosis of various systematic diseases.

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

Disclosure of Invention

Aiming at the blank existing in the prior art, the invention provides a fermented lactobacillus mucilaginosus and application thereof, wherein the fermented lactobacillus mucilaginosus NHNK-606 (Limosilactobacillus fermentum NHNK-606) has a preservation number of CCTCC NO: M2024419, and is preserved in China center for type culture collection (China center) for 3-7 of 2024.

The inventor firstly wipes the belly surface of a fish body from a sea water cage culture base of Qingdao, cuts off the stomach, pylorus blind sac and front middle intestine section of the fish body by using 75% alcohol, removes the content, washes with sterile physiological saline for 2 times, scrapes mucus on the inner wall of each organ, adds the mucus into the sterile physiological saline, shakes the mixture uniformly by vortex vibration, coats the mixture in an MRS solid culture medium and places the MRS solid culture medium in an anaerobic bag, picks up white bacterial colonies after culturing for 48 hours at the constant temperature of 37 ℃, repeatedly streaks and screens until uniform single bacterial colonies are obtained, is named NHNK-606, and is identified as fermented mucus lactobacillus (Limosilactobacillus fermentum) by 16S rDNA. The strain is gram positive and is in a short rod shape under a microscope; the bacteria grow on an MRS flat plate, so that round colonies with smooth and semitransparent surfaces can be formed, the bacteria are white, and the edges are tidy; the strain grows in a MRS liquid culture medium in a uniform turbidity way, and the strain is in a white precipitate after long-term placement, and the optimal growth temperature is 37 ℃.

The inventor reserves NHNK-606 with China Center for Type Culture Collection (CCTCC) No. M2024419.

The inventors further studied the above strain NHNK-606, and found that it has the following uses and beneficial effects:

In vitro experiments show that the fermentation lactobacillus mucilaginosus NHNK-606 has the functions of inhibiting the expression of the streptococcus mutans related glucosyltransferase SI gene gtfC, the glucose binding protein B gene gbpB, the glucose binding protein C gene gbpC, the fructosyltransferase gene ftf and the self-inducer-2 protein generation gene luxS, and the relative expression quantity of the genes is reduced to 0.06-0.74 times.

In vitro experiments show that the fermentation lactobacillus mucilaginosus NHNK-606 has the functions of inhibiting the expression of candida albicans related cell surface glycoprotein genes ALS1/ALS3, cell wall internal beta-1, 3-glucan transferase genes BGL2 and hypha cell wall protein genes HWP1, and the relative expression quantity of the genes is reduced to 0.28-0.71 times.

In vitro experiments show that the fermentation lactobacillus mucilaginosus NHNK-606 has the function of inhibiting the expression of intercellular adhesion proteins icaA, auxiliary gene regulatory factors a agrA and fibronectin precursors fnbA related to staphylococcus aureus golden subspecies, and the relative expression quantity of genes is reduced to 0.50-0.84 times.

In vitro experiments show that the fermented lactobacillus mucilaginosus NHNK-606 has the effect of inhibiting the formation of human staphylococcus, staphylococcus aureus subspecies aureons, lysostaphylococcus, streptococcus mutans, atypical veillonella, candida albicans biomembrane, human staphylococcus-candida albicans, staphylococcus aureus subspecies aureons-candida albicans, lysostaphylococcus-candida albicans, streptococcus mutans-candida albicans and atypical veillonella-candida composite biomembrane, and the biomembrane formation rate is reduced to 28.05% -89.08%.

In vitro experiments show that the fermented lactobacillus mucilaginosus NHNK-606 has the functions of agglutinating oral pathogenic bacteria candida albicans, atypical veillonella, streptococcus mutans, staphylococcus aureus subspecies aureons and human staphylococcus, and the agglutination rate can reach 28.03-52.26% after 30min of the action with the pathogenic bacteria.

In vitro experiments show that the fermentation lactobacillus mucilaginosus NHNK-606 can effectively inhibit the formation of candida albicans hyphae, and the hyphae formation rate is as low as 10.43% -51.37%.

In vitro experiments show that the fermentation product of the lactobacillus mucilaginosus NHNK-606 has the function of inhibiting the proliferation of oral flora, and the growth rate of the oral flora is reduced to 52.97%.

In vitro experiments show that the fermented lactobacillus mucilaginosus NHNK-606 has the function of inhibiting the formation of oral cavity flora biomembrane, and the formation rate of the oral cavity flora biomembrane is reduced to 22.34% -44.07%.

Based on the above experimental results, the inventors also provided applications of the lactobacillus mucilaginosus, in particular applications in preparing products for improving oral health, further including but not limited to:

1. Use of a product for the preparation of a gene for modulating virulence of an oral pathogen, said gene for modulating virulence of an oral pathogen comprising at least one of the genes gtfC, gbpB, gbpC, ftf, luxS for virulence of streptococcus mutans; at least one of candida albicans virulence genes ALS1, ALS3, BGL2 and HWP 1; at least one of the staphylococcus aureus subspecies aureoviridis virulence genes icaA, agrA, fnbA.

2. Use of a biofilm product for inhibiting oral pathogenic bacteria including at least one of human staphylococci (Staphylococcus hominis), staphylococcus aureus (Staphylococcus aureus), staphylococcus haemolyticus (Staphylococcus haemolyticus), streptococcus mutans (Streptococcus mutans), atypical veillonella (Veillonella Atypica) and Candida albicans (Candida albicans).

3. The application of the composite biological film product for inhibiting the formation of oral pathogenic bacteria and fungi comprises at least one of human staphylococcus-candida albicans, staphylococcus aureus subspecies aureoviridae-candida albicans, staphylococcus hemolyticus-candida albicans, streptococcus mutans-candida albicans and atypical veillonella-candida albicans composite biological film.

4. The application of the preparation of the product of the mycelium of the competing and combining oral pathogenic fungi, wherein the mycelium of the competing and combining oral pathogenic fungi is the mycelium of the combining candida albicans, and the combination effect of the mycelium and other bacteria is interfered; or inhibiting candida albicans hypha formation by reducing candida albicans hypha formation rate.

5. Use of an agglomerated oral pathogenic bacteria product comprising at least one of candida albicans, atypical veillonella, streptococcus mutans, staphylococcus aureus subspecies aureons, and human staphylococcus.

6. Use in the preparation of a reduced oral microbiota biofilm product.

In the above application, the lactobacillus mucilaginosus NHNK-606 is present in a sterilized form, or in a fermentation product (i.e., supernatant) form, or in a derivative form, wherein the derivative form is selected from the group consisting of: metabolites, metabolic biological products, probiotics, cell walls and components thereof, extracellular polysaccharides and compounds containing immunogenic components; preferably selected from fermentation products or inactivated bacteria.

The inventor carries out biological preservation on lactobacillus mucilaginosus NHNK-606, and the preservation information is as follows:

preservation time: 2024 3 and 7 days

Preservation unit name: china center for type culture Collection

Preservation number: CCTCC NO: M2024419

Deposit unit address: chinese, wuhan, university of Wuhan

The classification names are lactobacillus mucilaginosus NHNK-606 (Limosilactobacillus fermentum NHNK-606).

Drawings

FIG. 1 is a drawing of MRS plate colonies of Lactobacillus fermentum NHNK-606 of the present invention;

FIG. 2 is a gram stain of Lactobacillus mucilaginosus NHNK-606 according to the present invention;

FIG. 3 is a bar graph showing the screening of Lactobacillus fermentum NHNK-606 for inhibiting the growth of pathogenic bacteria in accordance with the present invention;

FIG. 4 is a schematic representation of the aggregation of inactivated and pathogenic bacteria of Lactobacillus fermentum NHNK-606 in accordance with the present invention;

FIG. 5 is a photograph showing the combination of inactivated Lactobacillus mucilaginosus NHNK-606 with Candida albicans mycelium in the present invention.

Detailed Description

The following examples describe lactobacillus mucilaginosus and their use. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention. The test materials adopted by the invention are all common commercial products and can be purchased commercially, and the inoculation proportion is volume ratio except for special description in the following examples; the invention is further illustrated by the following examples.

Isolation and characterization of examples 1 NHNK-606:

The method comprises the steps of wiping the belly surface of a fish body by using 75% alcohol, cutting off the stomach, pylorus blind sac and front midgut section of the fish body by using a sterile appliance, removing contents, flushing with sterile normal saline for 2 times, scraping mucus on the inner wall of each organ, adding the mucus into the sterile normal saline, uniformly shaking by vortex oscillation, coating the mucus on an MRS solid culture medium, placing the slurry in an anaerobic bag, culturing at the constant temperature of 37 ℃ for 48 hours, and repeatedly marking and screening white bacterial colonies until uniform single bacterial colonies are obtained, and the bacterial colonies are named NHNK-606.

Gram staining microscopy: strains NHNK-606 are gram-positive colonies, and are short-rod-shaped under a microscope; the white semitransparent circular colony with smooth and round surface can be formed by growing on an MRS flat plate, and the edge is neat; the bacteria can grow uniformly in the MRS liquid culture medium, and can be white precipitated after long-term storage, as shown in figures 1 and 2.

NHNK-606 nucleic acid identification

1. 16S rDNA gene sequence analysis:

Single colonies were picked up in MRS liquid medium, cultured overnight at 37℃and centrifuged at 8000 rpm for 1min to collect the colonies, which were then subjected to the procedure according to the instructions of the gram-positive DNA extraction kit. The primers were bacterial 16S sequencing universal primers 27F and 1492R, and the PCR amplification system was 20. Mu.L. The PCR amplification procedure was 95℃for 5min,94℃for 15s,57℃for 15s,72℃for 1min,35 cycles; extending at 72℃for 10min.

2. Results

The PCR products were sequenced and compared for homology (BLASTN) with published standard sequences in the GenBank database to yield NHNK-606 strain Lactobacillus fermentum (Limosilactobacillus fermentum).

Example 2 fermentation of lactobacillus mucilaginosus inhibition of oral pathogenic bacteria proliferation assay:

1. Preparation of fermentation products of fermented lactobacillus mucilaginosus

The inventor separates 20 strains of fermentation lactobacillus from different marine organisms, inoculates the 20 strains of fermentation lactobacillus (NHNK-606, B-1, B-2, B-3, B-4, B-5, B-6, B-7, B-8, B-9, B-10, B-11, B-12, B-13, B-14, B-15, B-16, B-17, B-18 and B-19) into fresh MRS culture medium according to the proportion of 1 percent (V/V) after the freezing glycerol tube returns to the temperature, cultures for 48 hours at 37 ℃, adjusts the MRS to OD ₆₀₀ =1.0, then centrifugates for 10min at 5000 g to obtain the supernatant, and passes through a 0.22 mu m sterile filter membrane to obtain the corresponding fermentation product of 20 strains of fermentation lactobacillus.

2. Culture of oral pathogenic bacteria

Commercial streptococcus mutans CGMCC 1.2499 was inoculated into BHI medium at a ratio of 1%. Staphylococcus aureus subspecies CGMCC 1.8721 is inoculated in a BHI culture medium according to the proportion of 1 percent. Atypical veillonella DSM 20739 was inoculated with Yu Weirong coccoid medium at a rate of 1%. Candida albicans CGMCC 2.4550 is inoculated in a sand culture medium according to the proportion of 1 percent. The staphylococcus hemolyticus CGMCC 1.540 is inoculated into a BHI culture medium according to the proportion of 1 percent. Human staphylococcus CGMCC 1.493 is inoculated into BHI culture medium according to the proportion of 1 percent. The pathogenic bacteria were cultured at 37℃for 24 hours with shaking and the original medium was adjusted to OD ₆₀₀ = 1.0 for use.

3. Experiment for inhibiting proliferation of common pathogenic bacteria in oral cavity by fermenting lactobacillus mucilaginosus fermentation product

The experimental group takes 4 milliliters of oral pathogenic bacteria culture medium and 1 milliliter of fermentation product of fermentation lactobacillus mucilaginosus, the control group is added with an equal volume of MRS liquid culture medium, oral pathogenic bacteria liquid is inoculated according to the inoculation amount of 1 percent, the static culture is carried out for 24 hours at 37 ℃, and the light absorption value at the OD=600 nm is measured, and the result is shown in figure 3.

The result shows that 20 strains of fermented lactobacillus mucilaginosus have different degrees of inhibition on the growth of different pathogenic bacteria, wherein NHNK-606 have stronger inhibition proliferation effects on streptococcus mutans, staphylococcus aureus subspecies aureons, atypical veillonella, candida albicans, lysostaphin and human staphylococcus, have broad-spectrum antibacterial effects, and can be used for further research and development on oral pathogenic bacteria.

Examples 3 NHNK-606 inhibition of mutans streptococcus virulence gene expression experiments:

1. NHNK-606 preparation of fermentation products and inactivated bacteria

Single colony of lactobacillus mucilaginosus NHNK-606 is selected and placed in MRS liquid culture medium, and is subjected to static culture for 48 hours at 37 ℃, PBS is used for adjusting to OD ₆₀₀ =1.0, centrifugation is carried out at 5000rpm for 10min, supernatant is obtained, and then a 0.22 mu m filter membrane is used for filtering, so that a sterile fermentation product is obtained.

After the cell pellet was washed twice with PBS solution, the cell was resuspended to OD ₆₀₀ =1.0 with PBS solution. Sterilizing at 121deg.C for 15min to obtain inactivated thallus.

2. Experiment for inhibiting virulence gene expression of streptococcus mutans

The streptococcus mutans CGMCC 1.2499 frozen stock is inoculated into fresh brain-heart infusion Broth (BHI) according to the proportion of 1 percent, and is cultured for 24 hours at 37 ℃ in a shaking way. Taking 3mL of the cultured streptococcus mutans bacterial liquid, adding 1mL NHNK-606 fermentation products or inactivated bacterial bodies (the control group is added with equal volume of PBS), 1mL of fresh BHI, and carrying out shake culture at 37 ℃ for 24h. After the culture is finished, centrifuging at 8000rpm for 1min to obtain thalli, extracting total RNA according to the instruction of a kit, detecting the concentration and purity of the RNA, reversely transcribing the RNA into cDNA, taking 16S as an internal reference gene, and detecting the expression of luxS, gbpB, gtfC, ftf and gbpC genes by adopting qPCR. The relative expression fold f=1 for the control group genes, and the F value for each sample was calculated using the 2 ^-ΔΔCT method.

The formula: f=2 ^-ΔΔCT, wherein:

△CT_Experiment=CT_Experiment-CT_{Internal reference （ Experiment ）;}

△CT_Control=CT_Control-CT_{Internal reference （ Control ）;}

△△CT=△CT_Experiment-△CT_{Control .}

The results are shown in tables 1 and 2 below:

TABLE 1

TABLE 2

The results show that NHNK-606 fermentation products and inactivated bacteria have the function of reducing the expression of the virulence genes of the streptococcus mutans.

Examples 4 NHNK-606 inhibition of candida albicans virulence gene expression experiments:

1. NHNK-606 fermentation product and preparation of inactivated thallus

The preparation is described in example 3.

2. Experiment for inhibiting candida albicans virulence gene expression

Inoculating candida albicans CGMCC 2.4550 cryopreservation tube into fresh Saccharum medium according to the proportion of 1%, and standing and culturing at 37 ℃ for 24 hours. Taking 3mL of cultured candida albicans liquid, adding 1mL NHNK-606 fermentation products or inactivated thalli (equal volume of PBS is added into a control group), and carrying out static culture for 24 hours at 37 ℃ by 1mL of fresh sand culture medium. After the culture is finished, centrifugation is carried out at 8000rpm for 1min to obtain thalli, total RNA is extracted according to the instruction of a kit, the concentration and purity of the RNA are detected, reverse transcription is carried out to obtain cDNA, TDH1 is used as an internal reference gene, and qPCR is adopted to detect the expression of ALS1, HWP1, ALS3 and BGL2 genes. The relative expression fold f=1 for the control group gene was calculated for each sample F value using the method of 2 ^-ΔΔCT, and the calculation method was referred to in example 3.

The results are shown in tables 3 and 4 below:

TABLE 3 Table 3

TABLE 4 Table 4

The results show that NHNK-606 fermentation products and inactivated thalli have the effect of reducing the expression of the virulence genes of candida albicans.

Examples 5 NHNK-606 inhibition of staphylococcus aureus subspecies aureoviridae virulence gene expression experiments:

1. NHNK-606 fermentation product and preparation of inactivated thallus

The preparation is described in example 3.

2. Experiment for inhibiting virulence gene expression of staphylococcus aureus golden subspecies

The staphylococcus aureus subspecies CGMCC 1.8721 freeze-preserving tube is inoculated to fresh BHI according to the proportion of 1 percent, and the mixture is subjected to stationary culture for 24 hours at 37 ℃. Taking 3mL of cultured staphylococcus aureus subspecies aureoviridis bacterial liquid, adding 1mL NHNK-606 fermentation products or inactivated bacterial bodies (equal volume PBS is added into a control group), 1mL of fresh BHI, and standing and culturing at 37 ℃ for 24h. After the culture is finished, centrifuging at 8000rpm for 1min to obtain thalli, extracting total RNA according to the instruction of a kit, detecting the concentration and purity of the RNA, reversely transcribing the RNA into cDNA, taking gyrB as an internal reference gene, and detecting the expression of the ica A, agrA and fnbA genes by adopting qPCR. The relative expression fold f=1 for the control group gene was calculated for each sample F value using the method of 2 ^-ΔΔCT, and the calculation method was referred to in example 3.

The results are shown in tables 5 and 6 below:

TABLE 5

TABLE 6

The results show that NHNK-606 fermentation products and inactivated bacteria have the effect of inhibiting the expression of virulence genes of staphylococcus aureus subspecies aureons.

Examples 6 NHNK-606 inhibition of pathogenic biofilm formation experiments:

1. NHNK-606 fermentation product and preparation of inactivated thallus

Single colony of lactobacillus mucilaginosus NHNK-606 is selected and placed in MRS liquid culture medium, and is subjected to static culture for 48 hours at 37 ℃, the MRS is adjusted to OD ₆₀₀ =1.0, centrifugation is carried out at 5000rpm for 10min, and the supernatant is obtained, and then filtration is carried out by using a 0.22 mu m filter membrane, so that a sterile fermentation product is obtained.

2. Preparation of pathogenic bacteria liquid

Pathogenic bacteria are prepared by the following steps: the method comprises the steps of respectively picking single colonies of human grape ball CGMCC 1.493, staphylococcus aureus golden subspecies CGMCC 1.8721, staphylococcus hemolyticus CGMCC 1.540 and streptococcus mutans CGMCC 1.2499, inoculating the single colonies to a BHI liquid culture medium, vibrating at 37 ℃ for overnight culture, and adjusting OD ₆₀₀ = 0.3 by using the BHI liquid culture medium after the culture is finished; the atypical veillonella DSM 20739 is picked up to be inoculated into Yu Weirong type of a liquid culture medium for anaerobic culture for 24 hours at 37 ℃, and after the culture is finished, the veillonella liquid culture medium is used for adjusting OD ₆₀₀ =0.3; the candida albicans CGMCC 2.4550 is selected to be inoculated into a Saccharomycetes liquid culture medium, shake is carried out at 37 ℃ for overnight culture, and OD ₆₀₀ =0.3 is regulated by the Saccharomycetes liquid culture medium after the culture is finished.

3. Biological film experiment for inhibiting pathogenic bacteria

100 Mu L of pathogenic bacteria liquid is added into a 96-well plate, 100 mu L NHNK-606 fermentation products or inactivated bacteria are added into an experimental group, an control group is replaced by an equal volume of MRS culture medium or PBS respectively, 3 groups are parallel, and the culture is carried out for 24 hours at 37 ℃. After the incubation was completed, the supernatant was discarded, and 100. Mu.L of sterile PBS was added to each well and washed twice, followed by adding 100. Mu.L of 4% paraformaldehyde fixing solution to each well, and fixing at room temperature for 30min. The fixative was discarded, 100. Mu.L of crystal violet was added to each well and stained at room temperature for 30min. After the dyeing is finished, the biological membrane is washed twice by using sterile PBS and dried, 100 mu L of absolute ethyl alcohol is added into each hole, the obtained product is stood for 1min, the light absorption value at 600nm is measured, and the formation rate of the biological membrane is calculated according to a formula.

The calculation formula and the results are shown in tables 7 and 8 below:

TABLE 7 NHNK-606 fermentation products inhibiting pathogenic biofilm formation

Table 8 NHNK-606 inactivated cells inhibiting pathogenic biofilm formation

The results show that NHNK-606 fermentation products and inactivated bacteria have the effect of inhibiting the formation of pathogenic bacteria biological films.

Examples 7 NHNK-606 inhibition of pathogenic bacteria bacterial-fungal composite biofilm formation experiments:

1. NHNK-606 fermentation product and preparation of inactivated thallus

The preparation is described in example 6.

2. Preparation of pathogenic bacteria liquid

The preparation is described in example 6.

3. Experiment for inhibiting pathogenic bacteria-fungi composite biological film

100 Mu L of candida albicans liquid is mixed with 100 mu L of other pathogenic bacteria liquid and added into a 96-well plate respectively, 100 mu L NHNK-606 fermentation products or inactivated bacteria are added into an experimental group, an control group is replaced by an equal volume of MRS culture medium or PBS respectively, 3 groups are parallel, and the culture is carried out for 24 hours at 37 ℃. The method of fixing, staining and measuring the well plate is described in example 6.

The calculation formula and the results are shown in tables 9 and 10 below:

TABLE 9 NHNK-606 fermentation products inhibiting the formation of bacterial-fungal composite biofilm

Table 10 NHNK-606 inactivated cells inhibiting formation of bacterial-fungal composite biofilm

The results show that NHNK-606 fermentation products and inactivated thalli can inhibit the formation of a pathogenic bacteria-fungi composite biological film.

Examples 8 NHNK-606 agglutination pathogen assay:

1. Preparation of NHNK-606 inactivated cells

The preparation is described in example 6.

2. Preparation of pathogenic bacteria liquid

Reference is made to example 7 for a method of culturing candida albicans, atypical veillonella, streptococcus mutans, staphylococcus aureus and human staphylococcus. After the incubation was completed, the supernatant was discarded by centrifugation at 5000rpm for 10min, followed by washing twice with sterile PBS buffer, and then resuspended in sterile PBS buffer to adjust OD ₆₀₀ = 1.0.

3. Agglutination

Mixing pathogenic bacteria liquid and NHNK-606 inactivated bacteria according to a volume ratio of 1:1, adding PBS as a control into the pathogenic bacteria, standing for 30min, and observing flocculation.

4. Index measurement

At the time of reaction for 30min, the reaction solution of NHNK-606 alone, the pathogen alone and the mixed reaction solution of NHNK-606 and the pathogen were sampled in a range of 50 μl of the uppermost layer of the liquid surface, and transferred to a 96-well plate after pipetting, and the absorbance at od=600 nm was measured. Meanwhile, the aggregated precipitate was stained with a gram-type dye and then observed for the state of bacterial aggregation. As shown in fig. 4.

The calculation formula is as follows:

aggregation ratio (%) = [ (ax+ay) -2Amix ]/(ax+ay) ×100%;

Note that: ax, OD ₆₀₀ values were measured at reaction time for NHNK-606 alone; ay, OD ₆₀₀ value is measured by single pathogenic bacteria in the reaction time; amix OD ₆₀₀ values were measured at the reaction time after mixing NHNK-606 with the pathogen.

The aggregation of NHNK-606 inactivated cells against pathogenic bacteria is shown in Table 11 below:

TABLE 11

The results show that NHNK-606 inactivated cells can effectively agglutinate and capture candida albicans, atypical veillonella, streptococcus mutans, staphylococcus aureus and staphylococcus hominis after 30min of reaction. Further realizing physical contact with pathogenic bacteria, thereby exerting NHNK-606 functions of down regulating virulence genes of the pathogenic bacteria, reducing pathogenicity, inhibiting formation of biological films of the pathogenic bacteria and reducing the colonization capability of the pathogenic bacteria.

Examples 9 NHNK-606 inactivated bacteria combined with candida albicans hypha experiments:

1. Preparation of NHNK-606 inactivated cells

The preparation is described in example 6.

2. Preparation of candida albicans mycelium

Reference is made to example 6 for a method of Candida albicans culture. After the completion of the culture, the cells were obtained by centrifugation at 5000rpm for 10 minutes. The mycelia were resuspended in Fetal Bovine Serum (FBS) and adjusted to OD ₆₀₀ =0.1, incubated at 37 ℃ for 3 hours to obtain mycelia, washed twice with PBS, centrifuged to remove the supernatant, and adjusted to OD ₆₀₀ =1.0 with PBS.

3. Experiment of combining inactivated bacteria with candida albicans mycelium

0.7Ml of candida albicans mycelium and 0.7ml of NHNK-606 cells were added to the test tube, left to stand after being vortexed sufficiently, white precipitate appeared in the test tube after 1h, and the precipitate was gently sucked out and stained with a gram stain, observed with a microscope 100-fold oil microscope and photographed. As shown in fig. 5.

NHNK-606 inactivated bacteria and candida albicans mycelium have obvious size difference, and NHNK-606 inactivated bacteria and candida albicans mycelium can be distinguished by naked eyes. The results show that NHNK-606 inactivated bacteria can obviously bind to the mycelium part of candida albicans mycelium, have competitive inhibition effect on other pathogenic bacteria binding to candida albicans mycelium, and interfere with the synergism of bacteria and fungi.

Examples 10 NHNK-606 inhibit candida albicans hypha formation experiments:

1. NHNK-606 fermentation product and preparation of inactivated thallus

The preparation is described in example 6.

2. Candida albicans mycelium morphology induction

Reference is made to example 6 for a method of Candida albicans culture. After the completion of the culture, the cells were obtained by centrifugation at 5000rpm for 10 minutes. The cells were resuspended in Fetal Bovine Serum (FBS) and OD ₆₀₀ = 0.1 was adjusted.

3. NHNK-606 test for inhibiting the formation of candida albicans hyphae

The experimental group takes 100 mu L of FBS resuspended candida albicans liquid, adds 100 mu L NHNK-606 fermentation products or inactivated thalli into a 96-well plate, and the control group is replaced by an equal volume of MRS culture medium or PBS respectively, and three groups are arranged in parallel and are cultured for 2 hours at 37 ℃. After the completion of the culture, the medium was discarded, washed 1 time with 70% alcohol, 1 time with 0.25% SDS solution, 3 times with sterile water, and then stained with 0.1% crystal violet solution for 30min. After the dyeing is finished, the membrane is washed by 0.25% SDS solution for 1 time and sterile water for 3 times, and the biological membrane at the bottom is observed after the pore plate is naturally dried. 200. Mu.L of 40mmol/L HCl-isopropanol solution and 50. Mu.L 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 the results are shown in the following table 12:

TABLE 12 NHNK-606 inhibition of candida albicans hyphae formation

The results show that NHNK-606 fermentation products and inactivated bacteria are effective in inhibiting candida albicans hyphae formation.

Examples 11 NHNK-606 fermentation product inhibition oral flora proliferation experiments:

1. NHNK-606 preparation of fermentation products

The preparation is described in example 6.

2. Collection and culture of oral flora

Volunteer dental calculus was scraped with a sterile cotton swab and inoculated into BHI broth medium for anaerobic stationary culture at 37deg.C for 24h.

3. NHNK-606 fermentation product inhibition oral cavity flora proliferation experiment

The experimental group was prepared by adding 4 ml of BHI and 1 ml of NHNK-606 fermentation product into a centrifuge tube, adding an equal volume of MRS liquid medium into a control group, inoculating oral bacteria liquid according to an inoculum size of 1%, standing and culturing at 37 ℃ for 24 hours, and measuring the absorbance at od=600 nm, and the results are shown in table 13 below:

TABLE 13 NHNK-606 fermentation products inhibiting oral flora proliferation

The results show that NHNK-606 fermentation products have the effect of inhibiting the proliferation of oral flora.

Examples 12 NHNK-606 inhibit oral flora biofilm formation experiments:

1. NHNK-606 fermentation product and preparation of inactivated thallus

The preparation is described in example 6.

2. Collection and culture of oral flora

The preparation is described in example 11.

3. NHNK-606 experiments to inhibit oral flora biofilm formation

The cultured oral bacterial suspension OD ₆₀₀ was adjusted to 0.3 with BHI broth. 100 mu L of oral bacterial suspension is added into each hole of the experimental group, 100 mu LNHNK-606 fermentation products or inactivated bacteria are additionally added, the control group is replaced by an equal volume of MRS culture medium or PBS respectively, and 3 groups are arranged in parallel. After incubation at 37℃for 24 hours, gram staining was performed according to the method described in example 6 and absorbance at 600nm was measured.

The calculation formula and the results are shown in the following table 14:

Table 14 NHNK-606 inhibits oral flora biofilm formation

The results show that NHNK-606 fermentation products and inactivated bacteria can reduce the formation of oral cavity flora biomembrane.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. A strain of fermented lactobacillus mucilaginosus (Limosilactobacillus fermentum) NHNK-606 is characterized in that the collection number is CCTCC NO: M2024419, and the strain is preserved in China center for type culture Collection (China) for 3 months and 7 days of 2024.

2. Use of lactobacillus fermentum NHNK-606 according to claim 1 for the preparation of a product for improving oral health, said product being an inactivated thallus of lactobacillus fermentum NHNK-606.

3. Use of fermented lactobacillus mucilaginosus NHNK-606 according to claim 2 for the preparation of a product for improving oral health, wherein the product is a product for modulating oral pathogen virulence genes including at least one of candida albicans virulence genes ALS1, ALS3, BGL2, HWP 1; at least one of the staphylococcus aureus subspecies aureoviridis virulence genes icaA, agrA, fnbA.

4. Use of lactobacillus mucilaginosus NHNK-606 according to claim 2 for the preparation of a product for improving oral health, wherein the product is an oral pathogenic biofilm inhibiting product, and wherein the oral pathogenic bacteria include at least one of human staphylococci, staphylococcus aureus, staphylococcus haemolyticus, atypical veillonella and candida albicans.

5. Use of fermented lactobacillus mucilaginosus NHNK-606 in the preparation of a product for improving oral health according to claim 2, wherein the product is an oral pathogenic bacteria-fungus composite biofilm inhibiting product comprising at least one of human staphylococcus-candida albicans, staphylococcus aureus subspecies aurus-candida albicans, staphylococcus haemolyticus-candida albicans, streptococcus mutans-candida albicans and atypical veillonella-candida composite biofilm.

6. Use of fermented lactobacillus mucilaginosus NHNK-606 according to claim 2 for the preparation of a product for improving oral health, wherein the product is a competing binding product for oral pathogenic fungus hyphae, which are candida albicans hyphae.

7. Use of fermented lactobacillus mucilaginosus NHNK-606 in the preparation of a product for improving oral health according to claim 2, wherein the product is a product for the preparation of an agglomerated oral pathogenic bacteria comprising at least one of candida albicans, veillonella atypical, staphylococcus aureus subspecies aureons, staphylococcus hominis.