CN116622548B - Streptococcus crest capable of preventing and/or treating periodontitis and application thereof - Google Patents

Abstract

The application provides streptococcus cristatus capable of preventing and/or treating periodontitis and application thereof. The streptococcus cristatus is named as streptococcus cristatus (Streptococcus cristatus) CA119 and has been preserved in the China center for type culture collection (CCTCC NO) located in university of armed Han in China at 12 months of 2022: m20221855. The strain has the advantages of high growth rate, good environmental tolerance, capability of producing various digestive enzymes, strong sugar fermentation capability and high safety, can inhibit the main pathogenic bacteria of periodontitis, namely Porphyromonas gingivalis and Fusobacterium nucleatum, has good periodontitis resistance effect, has a protective effect on colon inflammation caused by the pathogenic bacteria of periodontitis, and provides a new thought for preventing and treating periodontitis.

Description

Streptococcus crest capable of preventing and/or treating periodontitis and application thereof

Technical Field

The application relates to the field of microorganisms, in particular to streptococcus cristatus capable of preventing and/or treating periodontitis and application thereof.

Background

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Periodontitis is a chronic inflammation of periodontal supporting tissues caused by periodontal pathogenic bacteria, is one of the most common oral diseases, not only affects periodontal tissues and induces alveolar bone absorption to cause tooth loss, but also seriously affects whole body health, and has been studied to prove that the periodontitis is related to various systemic diseases such as cardiovascular diseases, digestive tract diseases, premature labor, respiratory diseases and the like. Research shows that periodontitis is closely related to oral flora, and the oral environment is a micro-ecological system consisting of a plurality of microorganisms, and the microorganisms are mutually symbiotic and balanced to each other to jointly maintain the balance of oral micro-ecology, so that once the oral micro-ecology is disturbed, periodontitis can be initiated.

Porphyromonas gingivalis (Porphyromonas gingivalis) is a gram-negative oral anaerobic bacterium, is one of the most dominant bacteria in the lesion part of chronic periodontitis, participates in the pathogenesis of periodontitis and is also currently accepted periodontal pathogenic bacterium. Although Porphyromonas gingivalis is less abundant in the oral microbiota, it can act synergistically with other microorganisms to disrupt host microbiota homeostasis, induce inflammatory responses in periodontal tissues and inhibit host clearance of pathogenic bacteria.

Fusobacterium nucleatum (Fusobacterium nucleatum) is an invasive gram-negative anaerobic Fusobacterium, and is the most abundant Fusobacterium at infection sites such as supragingival plaque, subgingival plaque and periodontal pocket of periodontitis. Studies have shown that the rate of detection of fusobacterium nucleatum is positively correlated with the extent of lesions and destruction of periodontal tissue, which is the "second complex" of periodontal pathogenic bacteria, a core bacterium closely correlated with periodontitis. With the gradual penetration of research in recent years, the influence of clostridium nucleatum on periodontitis is gradually gaining attention.

Periodontitis is mainly treated to control plaque, eliminate inflammation, restore periodontal tissue function, prevent the progression of disease and prevent recurrence. The most commonly used methods at present are mechanical and pharmaceutical treatments, the most effective of which is subgingival scraping and root planing (scaling and root planing, SRP). However, the bacterial plaque on the affected part cannot be thoroughly removed by simple mechanical treatment, the site after treatment can be subjected to re-implantation of periodontal pathogenic bacteria in a short period, the operation is difficult, the reaction is easy to cause, and certain limitation exists. The drug treatment of periodontitis usually adopts antibiotic drugs such as amoxicillin, cefalexin, metronidazole and the like to eliminate periodontal pathogenic bacteria, but the local or systematic use of the antibiotic drugs can reduce the colonization of symbiotic bacteria while effectively controlling the growth of oral periodontal pathogenic bacteria, destroy the normal oral flora microecology and cause the increasingly serious drug resistance of bacteria.

Disclosure of Invention

The invention provides a streptococcus cristatus strain which has the advantages of high growth rate, good environmental tolerance, capability of producing various digestive enzymes, strong sugar fermentation capacity and high safety, can inhibit the main pathogenic bacteria of periodontitis, namely Fusobacterium nucleatum and Porphyromonas gingivalis, has good anti-periodontitis effect and has a protective effect on colon inflammation caused by the pathogenic bacteria of periodontitis.

Specifically, the invention provides the following technical scheme.

In a first aspect of the present invention, there is provided a strain of Streptococcus crest (Streptococcus cristatus) CA119 (hereinafter also referred to simply as strain CA 119) which has been deposited on month 12.02 of 2022 with the China center for type culture collection, having a accession number CCTCC NO: m20221855.

The strain CA119 is a gram-positive bacterium and is in a sphere shape under an electron microscope; the community forms are semitransparent small dots, and the edges are tidy and smooth. The strain can produce amylase but not protease, and has higher cellulase activity. Whether the strain can produce a series of digestive enzymes such as amylase, protease, cellulase and the like is a key whether the strain can improve the growth performance of a host, and the produced digestive enzymes have the potential of promoting the digestion capability of the host and improving the growth performance.

The strain CA119 provided by the invention has the capability of fermenting sorbitol, sucrose, mannitol, maltose, salicin, glucose and xylose, but cannot ferment raffinose, and has strong sugar fermentation capability. Different bacteria have different sugar decomposing capacities, and metabolized products are different, and the bacterial strain can play the roles of reducing the pH of the feed, inhibiting pathogenic bacteria, improving the palatability of the feed, reducing the raising cost and the like by using organic acid, vitamins and other metabolites produced after the bacterial strain has the sugar fermentation capacity.

Probiotics should possess the ability to combat adverse environments of the body, including enzymes, pH, temperature, etc. when used as a pharmaceutical product, and should also possess colonisation and adhesion capabilities, including self-aggregation and hydrophobic properties. The normal oral temperature is about 1 ℃ higher than that of a human body, the temperature is maintained between 37.5 and 38.5 ℃, and the probiotic preparation is influenced by temperature change in the preparation process, so that the strain needs to have certain heat resistance, and saliva of the oral cavity contains lysozyme with certain concentration, so that bacteria including escherichia coli can be inhibited or even killed, and the strain used as the oral probiotic preparation needs to be capable of tolerating the lysozyme with certain concentration. The strain CA119 provided by the invention grows rapidly, can enter the logarithmic growth phase after 2 hours of culture, and can enter the growth stabilization phase after 8 hours of culture; the strain has strong heat resistance, acid resistance, bile salt resistance and lysozyme resistance, can survive in an environment with the temperature of 70 ℃ and the bile salt concentration of 0.5%, the survival rate still exceeds 50% in an environment with the pH value of 3, and the survival rate still approaches 90% when the lysozyme concentration is 1.0 mg/mL. In addition, the strain CA119 has higher self-condensation and hydrophobicity, and has an inhibiting effect on the main pathogenic bacteria of periodontitis, namely Porphyromonas gingivalis and Fusobacterium nucleatum. The strain provided by the invention has good environmental adaptability, can survive and colonize in oral cavity and gastrointestinal tract environment, and provides a foundation for the strain to play a role in the oral cavity and gastrointestinal tract environment.

In the embodiment of the invention, the tooth Zhou Tuma strain CA119 can obviously relieve the food intake and daily gain reduction caused by periodontitis, reduce the relative expression amount of IL-6, IL-1 beta, IL-18, COX-2, iNOS and MCP-1 in gingival tissues, up-regulate the ratio of OPG to RANKL, inhibit the increase of oxidative stress indexes and the decrease of antioxidant stress indexes in serum, obviously reduce the alveolar bone absorption value, and reduce periodontal tissue injury and colon inflammatory reaction caused by periodontitis pathogenic bacteria.

In addition, the safety problem of probiotics is also an important consideration, the strain CA119 disclosed by the invention is sensitive to various antibiotics, the probability of resistance gene transfer is low, the hemolysis test is negative, and the safety is high.

In a second aspect of the invention, there is provided a culture of Streptococcus crest comprising a substance obtained by culturing Streptococcus crest (Streptococcus cristatus) CA119 in a microbial culture medium.

In a third aspect of the invention, the invention provides a microbial agent comprising streptococcus cristae (Streptococcus cristatus) CA119 and/or a metabolite thereof, or a culture thereof, as claimed in claim 1.

In a fourth aspect of the invention, the invention provides a composition comprising streptococcus cristae (Streptococcus cristatus) CA119 and/or a metabolite thereof, or a culture thereof, as claimed in claim 1.

The composition of the invention has the use of at least one of the following:

1) Reducing periodontal tissue inflammatory response;

2) Inhibiting differentiation and maturation of osteoclasts in periodontal tissue;

3) Reducing oxidative stress and improving the capacity of resisting oxidative stress;

4) Can reduce alveolar bone resorption;

5) Can reduce the damage of periodontal tissue;

6) Reducing irritation of periodontitis pathogenic bacteria to colon tissue.

In a fifth aspect of the invention, the invention provides a product comprising streptococcus cristae (Streptococcus cristatus) CA119 and/or a metabolite thereof, or a culture thereof, or a composition comprising the foregoing;

in the present invention, the product has a use as described in at least one of the following:

1) Reducing periodontal tissue inflammatory response;

2) Inhibiting differentiation and maturation of osteoclasts in periodontal tissue;

3) Reducing oxidative stress and improving the capacity of resisting oxidative stress;

4) Can reduce alveolar bone resorption;

5) Can reduce the damage of periodontal tissue;

6) Reducing irritation of periodontitis pathogenic bacteria to colon tissue.

In embodiments of the invention, products include pharmaceuticals, commodity products, feeds, and probiotics.

In some embodiments of the invention, the pharmaceutical product may be a chewable tablet, a lozenge, an effervescent tablet, a spray, an oral patch, an oral rinse, and other common dosage forms, and the like.

In some embodiments of the invention, the daily chemical product such as a cosmetic product includes, but is not limited to, an oral hygiene cosmetic product such as a toothpaste, mouthwash, dentifrice, or the like.

The product of the invention can be prepared from streptococcus cristatus (Streptococcus cristatus) CA119 and/or a metabolite thereof, or a culture thereof (or a composition containing streptococcus cristatus (Streptococcus cristatus) CA119 and/or a metabolite thereof) and a proper auxiliary material. The product may be in solid form, liquid form, semi-solid form, gel, aerosol form, and the like.

Some embodiments of the invention include methods of producing the product, which methods comprise mixing an active substance according to the invention with suitable excipients, by any suitable method, generally by uniformly mixing the active substance with a liquid and/or finely divided solid excipient in the desired proportions, and then, if desired, shaping the resulting mixture into the desired shape. The auxiliary materials can play roles in filling, binding, disintegrating and lubricating, and sometimes, coloring, flavoring, and aesthetic effects, and the common auxiliary materials such as starches, sugars, celluloses (such as microcrystalline cellulose), inorganic salts (such as inorganic calcium salts), and the like.

In a sixth aspect of the invention, the invention provides the use of Streptococcus crest (Streptococcus cristatus) CA119 and/or a metabolite thereof or a culture thereof in the manufacture of an antibacterial agent.

Wherein the antibacterial agent is capable of inhibiting at least Porphyromonas gingivalis and/or Fusobacterium nucleatum.

In a seventh aspect of the invention, the invention provides the use of streptococcus cristatus (Streptococcus cristatus) CA119 and/or a metabolite thereof or a culture thereof in the manufacture of a product for the prevention and treatment of periodontitis. Wherein the product comprises medicines, daily chemicals, feeds and microecologics.

In an eighth aspect of the invention, the invention provides the use of streptococcus cristatus (Streptococcus cristatus) CA119 and/or a metabolite thereof or a culture thereof, in the manufacture of a product for the prevention and treatment of colitis; wherein the colitis is colitis caused by periodontitis pathogenic bacteria; the product comprises medicines, daily chemicals, feeds and microecologics.

In a ninth aspect of the invention, the invention provides a method enabling use as set out in at least one of the following 1) to 6): 1) Reducing periodontal tissue inflammatory response; 2) Inhibiting differentiation and maturation of osteoclasts in periodontal tissue; 3) Reducing oxidative stress and improving the capacity of resisting oxidative stress; 4) Can reduce alveolar bone resorption; 5) Can reduce the damage of periodontal tissue; and, 6) reducing irritation of periodontitis pathogens to colon tissue; the method comprises administering to the subject an effective amount of streptococcus cristatus (Streptococcus cristatus) CA119 and/or a metabolite thereof or a product comprising streptococcus cristatus (Streptococcus cristatus) CA119 and/or a metabolite thereof. The product comprises medicines, daily chemicals, feeds and microecologics.

In an embodiment of the invention, any of the above uses 1) to 5) is to be achieved, preferably in an oral administration, such as oral (solid, liquid), spray, spread, such as in some embodiments, the product is to be applied in a suitable form (such as a lozenge, mouthwash) contained in the mouth, such as by spraying the product in a suitable form (such as an oral spray) onto the oral cavity, in particular the teeth (in particular the periodontal), such as by fitting or applying the product in a suitable form (such as an oral patch or paste) onto the oral cavity, in particular the teeth (in particular the periodontal), such as by orally administering the product, and so on.

In a tenth aspect of the invention, the invention provides a method of preventing periodontitis comprising administering to a subject an effective amount of streptococcus cristatus (Streptococcus cristatus) CA119 and/or a metabolite thereof or a product comprising streptococcus cristatus (Streptococcus cristatus) CA119 and/or a metabolite thereof. The product comprises medicines, daily chemicals, feeds and microecologics, and particularly refers to products which can be orally used.

In embodiments of the invention, the product is preferably administered orally, such as orally (solid, liquid), spray, spread, such as in some embodiments, by including the product in the mouth in a suitable form, such as a lozenge, mouthwash, such as by spraying the product in a suitable form, such as an oral spray, particularly on teeth (particularly periodontal), such as by fitting or applying the product in a suitable form, such as an oral patch or paste, particularly on teeth (particularly periodontal), such as by oral administration, and the like.

And, in an eleventh aspect thereof, the present invention provides a method of preventing and treating colitis, comprising administering to a subject an effective amount of streptococcus cristatus (Streptococcus cristatus) CA119 and/or a metabolite thereof or a product comprising streptococcus cristatus (Streptococcus cristatus) CA119 and/or a metabolite thereof. The product can be a product comprising medicines, daily chemicals, feeds and microecologics, in particular a product which can be orally taken.

In the present invention, the term "subject" refers to an animal, such as a mammal (including a human), that has been or will be the subject of treatment, observation or experiment. The methods described herein may be used for therapeutic and/or veterinary applications in humans.

In the present invention, the "effective amount" refers to an amount by which administration of the streptococcus cristatus (Streptococcus cristatus) CA119 and/or a metabolite thereof, or a product comprising the streptococcus cristatus (Streptococcus cristatus) CA119 and/or a metabolite thereof, of the present invention, is capable of eliciting a biological or medical response in a subject and is sufficient to effectively alleviate an undesirable state, condition, disorder or the like of the body. The effective amount may be determined by taking into account self knowledge, the state of the art, and routine to those skilled in the art, such as by in vivo, in vitro, or ex vivo experimentation, or demonstrating the amount.

Compared with the prior art, the invention has the advantages that:

the invention provides a streptococcus cristatus (Streptococcus cristatus) CA119 which has the advantages of rapid growth, low requirement on growth environment, capability of growing in high temperature, high acid and other environments, good cholate and lysozyme resistance, sensitivity to various antibiotics, high self-condensation and hydrophobicity, inhibition effect on the main pathogenic bacteria of periodontitis, namely Porphyromonas gingivalis and Fusobacterium nucleatum, capability of reducing inflammatory reaction of periodontal tissues, inhibiting differentiation and maturation of osteoclast in periodontal tissues, reducing oxidative stress, improving antioxidant stress capability, reducing alveolar bone absorption, reducing injury of periodontal tissues, reducing irritation of periodontitis pathogenic bacteria to colon tissues, and protection effect on colonic inflammation caused by periodontitis pathogenic bacteria.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute an undue limitation to the application. Embodiments of the present application are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows the morphological characteristics of strain CA119, wherein A is a photograph of a colony on a medium; B-C is a scanning electron microscope observation photograph; D-E is a transmission electron micrograph.

FIG. 2 shows the growth curve of strain CA 119.

Figure 3 shows the survival of strain CA119 at different temperature treatments.

Figure 4 shows the survival of strain CA119 at different pH conditions.

Figure 5 shows the survival of strain CA119 at different bile salt concentrations.

Figure 6 shows the viability of strain CA119 at different lysozyme concentrations.

FIG. 7 shows average daily feed intake and daily gain monitoring results for rats, wherein A is average daily feed intake and B is average daily gain; in contrast to the Control group, ^*** P<0.001; in contrast to the Model group, ^## P<0.01， ^### P<0.001; in contrast to the group of OPMs, ^× P<0.05， ^×× P<0.01。

FIG. 8 shows the results of real-time fluorescent quantitative PCR, wherein A is the relative expression level of pro-inflammatory cytokines and B is the relative expression level of COX-2, iNOS, MCP-1; c is a real-time fluorescent quantitative PCR result heat map; in contrast to the Control group, ^*** P<0.001; in contrast to the Model group, ^# P<0.05， ^## P<0.01， ^### P<0.001; in contrast to the group of OPMs, ^× P<0.05， ^×× P<0.01。

fig. 9 shows detection of the expression levels of OPG, RANKL in gingival tissue, where a is the expression level of OPG, B is the expression level of RANKL, and C is the ratio of OPG to RANKL expression level. In contrast to the Control group, ^*** P<0.001; in contrast to the Model group, ^# P<0.05， ^## P<0.01， ^### P<0.001; in contrast to the group of OPMs, ^×× P<0.01。

FIG. 10 shows serum oxidative stress index changes; in contrast to the Control group, ^*** P<0.001; in contrast to the Model group, ^# P<0.05， ^### P<0.001; in contrast to the group of OPMs, ^× P<0.05， ^×× P<0.01。

FIG. 11 shows a Micro-CT image of rat maxillary molars and their surrounding bone tissue.

Fig. 12 shows the alveolar bone resorption values of rats.

FIG. 13 shows a pathological section of rat alveolar bone tissue, wherein A-F are periodontal tissue between the first and second molars; a-f are periodontal tissue at the root bifurcation of the second molar; FM is the first molar; SM is second molar; AB is alveolar bone; CEJ is cementum-glaze junction; ABC is the alveolar bone crest; the red box is periodontal disease.

Fig. 14 shows a pathological section of colon tissue of a rat, in which black arrows are pathological regions.

Detailed Description

The present application is further illustrated below in conjunction with specific embodiments. It should be understood that these examples are illustrative only of the present application and are not intended to limit the scope of the present application. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The reagents or materials used in this application are all commercially available in conventional manners, and unless specifically indicated otherwise, are all used in conventional manners in the art or according to the product specifications. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present application. The preferred methods and materials described herein are presented for illustrative purposes only.

Example 1Isolation and identification of strains

1 sample collection and processing

Oral samples of adult healthy beagle dogs in vinca were collected. The oral samples are divided into four parts, saliva, gingival crevicular fluid, plaque and tonsillar mucus.

(1) Saliva collection: flushing the subject's mouth with sterile physiological saline to remove food debris; placing cotton swab in saliva collecting tube under tongue, soaking cotton swab, taking out, centrifuging at 1500 Xg for 5min.

(2) Gingival crevicular fluid collection: the tooth surface and surrounding gums of the test are rubbed with a sterile cotton swab, the front end of the moisture-absorbing paper tip is gently inserted from the entrance of the gingival sulcus until the tip is stopped for 20s after slight resistance, and the moisture-absorbing fingertip is taken out and placed in the GAM culture medium.

(3) Plaque collection: after food residues in the tested oral cavity are removed by using sterile physiological saline, taking sterile cotton to isolate dampness, slightly scraping the bacterial plaque on the gingiva by using a sterile spoon, and placing the bacterial plaque in a GAM culture medium.

(4) Tonsil mucus collection: the sterile cotton swab is soaked by sterile physiological saline, gently rubbed around tonsil, and prevented from touching parts except tonsil as much as possible during operation.

2 isolation and identification of probiotics

2.2.1 isolated culture of the Strain

2.2.1.1 dilution culture

The collected oral samples are diluted by 4000 times, 40000 times and 400000 times respectively by sterile PBS buffer solution, 180 mu L of bacterial solutions with different dilution times are sucked and placed on the surfaces of all solid flat culture mediums, the solid flat culture mediums are uniformly smeared by a disposable L-shaped coating rod, and the solid flat culture mediums are reversely buckled in an anaerobic workstation and a microorganism incubator for culturing for 48 hours at 37 ℃.

2.2.1.2 enrichment culture

500. Mu.L of the oral sample was drawn with a 1mL syringe and added to a blood enrichment flask, and the flask was incubated at 37℃for 72 hours at 220 rpm. And taking a sample after enrichment, and carrying out dilution culture.

2.2.1.3 separation and purification

Taking a solid plate culture medium after 48 hours of culture, respectively picking colonies with different shapes, sizes, colors and textures in an anaerobic workstation and an ultra-clean workstation, inoculating the colonies on a fresh solid plate culture medium by a continuous streaking method, and culturing the colonies in the anaerobic workstation and a microorganism incubator at 37 ℃ for 24 hours in an inverted manner. The purified single colonies are inoculated into corresponding liquid culture media respectively, and cultured for 18 hours at 37 ℃ and 220 rpm.

2.2.1.4 sequencing identification of the Strain

And (3) carrying out 1% agarose gel electrophoresis inspection on the PCR product, selecting the PCR product with the target band as the electrophoresis result, sending the PCR product to an Anhui general biological System Co., ltd for sequencing, and searching and comparing the sequence result of the 16S rRNA obtained by sequencing through EzBioCloud (Prokaryote Identification (EzBioCloud. Net)).

The homology of the 16S rRNA full-length gene comparison of a strain is less than 98.65%, the strain is streptococcus cristatus (Streptococcus cristatus, also can be abbreviated as S.cristatus) and named as CA119, the strain CA119 is picked up by a disposable sterile inoculating loop in an anaerobic workstation and is transferred into a BHI liquid culture medium, the strain CA119 is inoculated on the BHI slant culture medium in a continuous streaking mode, the strain CA119 is cultured for 16 hours at 37 ℃, and the strain CA119 is sent to China center for patent preservation, and the preservation number is CCTCC NO: m20221855.

2.2.1.5 gram staining of strains

Inoculating the strain CA119 on a BHI solid flat-plate culture medium by using a disposable sterile inoculating loop in a streaking mode, and culturing for 12 hours at 37 ℃ in an anaerobic workstation; dropping a drop of sterile double distilled water on a glass slide, picking a small amount of colonies, uniformly coating on water drops, drying and fixing on an alcohol lamp flame, adding ammonium oxalate crystal violet solution for dyeing for 1min, lightly flushing with the sterile double distilled water, adding new iodine solution after the smear is dried, standing for 1min, washing with water, dropping 95% ethanol solution for decoloring for about 15s, washing with water, adding safranine for counterstaining for 1min, washing with water, and performing oil microscopic examination after the smear is dried. The color of the thallus is red and is gram-negative bacteria, and the color of the thallus is purple and is gram-positive bacteria.

Gram staining was performed on strain CA119 to observe that the strain was purple and was a gram positive bacterium.

2.2.1.6 morphological observations of strains

a) Colony morphology

Bacterial strain CA119 is inoculated on BHI solid flat-plate culture medium by a disposable sterile inoculation ring in a streaking way, and is placed in an anaerobic workstation for culturing for 12 hours at 37 ℃ to observe colony morphology.

b) Scanning and transmission electron microscope observation

The strain CA119 is picked by a disposable sterile inoculating loop and is transferred into a BHI liquid culture medium, the culture is carried out for 12 hours at 37 ℃ and 220rpm, the bacterial liquid is taken at 8000rpm/min, the centrifugation is carried out for 3 minutes, the supernatant is discarded, the sterile PBS buffer is added for three times, 2.5 percent glutaraldehyde is added for fixation, and the obtained mixture is sent to a Hangzhou Haake Biotechnology Co Ltd for Scanning (SEM) and Transmission (TEM) electron microscope observation.

The colony morphology, scanning (SEM) and Transmission (TEM) electron microscope observation of the strain CA119 are shown in FIG. 1. Colony bodies of the strain CA119 on the BHI solid culture medium are semitransparent small dots, and the edges are neat and smooth; the strain CA119 under the electron microscope is in a sphere shape and has a capsular structure.

Example 2Evaluation of CA119 Probiotics of Streptococcus crest

Preparing a bacterial liquid to be tested: inoculating a strain to be tested on a BHI solid flat-plate culture medium by a disposable sterile inoculation ring in a streaking mode, placing the strain in an anaerobic workstation for culturing for 12 hours at 37 ℃, picking single colonies from the solid flat-plate culture medium, inoculating the strain to be tested into a Hengager tube filled with 15mL of BHI liquid culture medium, culturing for 12 hours at 37 ℃ and 220rpm, and taking the cultured bacterial liquid as the bacterial liquid to be tested.

1 Strain growth curve

200 mu L of bacterial liquid to be detected is respectively sucked, inoculated into a plurality of Hengaget tubes filled with 10mL of BHI liquid culture medium, cultured at 37 ℃ and 220rpm, any tube of bacterial liquid is respectively taken after 2 nd, 4 th, 6 th, 8 th, 10 th, 12 th, 16 th, 24 th, 36 th and 48 th hours, 1mL of bacterial liquid is sucked, and the light absorption value of the bacterial liquid with the wavelength at 600nm is measured, and each group is repeated three times.

The strain growth status was monitored continuously for 48h after strain inoculation, and the growth curve drawn with GraphPad Prism 9 is shown in fig. 2. Wherein, the strain CA119 enters the logarithmic growth phase after 2 hours of culture and enters the stationary phase after 8 hours of culture.

2 Heat resistance measurement

Respectively sucking 200 mu L of bacterial liquid to be detected for 12h, inoculating the bacterial liquid to be detected into Hungate tubes filled with 10mL of BHI culture medium, respectively placing the inoculated Hungate tubes into water baths at 37 ℃, 40 ℃, 50 ℃, 60 ℃ and 70 ℃ for incubation for 20min, transferring the Hungate tubes to a constant-temperature shaking table at 37 ℃ after incubation, culturing at 220rpm for 8h, respectively sucking 1mL of bacterial liquid, measuring the absorbance value of the bacterial liquid at 600nm, taking the bacterial liquid treated at 37 ℃ as a control, calculating the survival rate, and repeating each group for three times.

The viability of strain CA119 was measured by heat resistance test at different temperatures and the results are shown in FIG. 3. With the increase of the temperature, the survival rate of the strain is reduced, but the strain can still survive in a high-temperature environment at 70 ℃, and has stronger heat resistance.

3 acid resistance measurement

The pH value of the BHI liquid culture medium is respectively adjusted to 7,6,5,4,3 by using a concentrated hydrochloric acid solution, 10mL of the BHI liquid culture medium is respectively packaged in Hengaget tubes, the BHI liquid culture medium is immediately capped after high-purity nitrogen is filled, and the BHI liquid culture medium is sterilized at 121 ℃ for 15min and cooled for later use. 1mL of the bacterial liquid to be detected which is cultivated for 12h is respectively sucked, inoculated into Hengaget tubes with different gradient pH values, cultivated for 12h at 37 ℃ and 220rpm, the light absorption value of the bacterial liquid with the wavelength of 600nm is respectively sucked for 1mL of the bacterial liquid, the bacterial liquid with the pH value of 7 is used as a control, and the survival rate is calculated and repeated three times for each group.

The acid resistance measurement results are shown in FIG. 4. With the decrease of the pH value, the survival rate of the strain CA119 is reduced, but the strain CA119 can still survive under the low pH condition, the survival rate of the strain CA119 at the pH value of 3 is still more than 50%, and the acid resistance is high.

4 determination of bile salt resistance

Preparing BHI liquid culture medium with bile salt concentration of 0%,0.1%,0.3% and 0.5%, respectively, subpackaging 10mL of the liquid culture medium into Hengaget tubes, immediately capping after filling high-purity nitrogen, sterilizing at 121deg.C for 15min, and cooling. 1mL of bacterial liquid to be detected which is cultivated for 12h is respectively sucked and inoculated into Hengaget tubes with different bile salt concentrations, the bacterial liquid is cultivated for 10h at 37 ℃ and 220rpm, the light absorption value of the bacterial liquid with the wavelength of 600nm is respectively sucked and detected, the bacterial liquid without bile salt is used as a control, and the survival rate is calculated, and each group is repeated for three times.

The results of the bile salt tolerance test are shown in FIG. 5, the survival rate of the strain CA119 is affected with the increase of the bile salt concentration, and the strain CA119 can still survive and has the strongest bile salt tolerance at the bile salt concentration of 0.5%.

5 oral lysozyme tolerance assay

The lysozyme freeze-dried powder is dissolved in sterile double distilled water to prepare a lysozyme solution with the concentration of 50mL/mg, and the lysozyme solution is filtered and sterilized by a sterile filter membrane with the concentration of 0.22 mu m for standby. The lysozyme concentration in the BHI liquid culture medium is respectively regulated to 0, 0.2, 0.4, 0.6, 0.8 and 1.0mg/mL by using a lysozyme solution in an anaerobic workstation, the culture is carried out for 10 hours at 37 ℃ and 220rpm, 1mL of bacterial liquid is respectively sucked for measuring the absorbance value of the bacterial liquid with the wavelength of 600nm, the bacterial liquid without adding lysozyme is used as a control, the survival rate is calculated, and each group is repeated three times.

The results of the oral lysozyme resistance assay are shown in FIG. 6. With the increase of the lysozyme concentration, the survival rate of the strain CA119 is not obviously reduced, and when the lysozyme concentration is 1.0mg/mL, the survival rate of the strain CA119 is still close to 90%, and the lysozyme resistance is stronger. The saliva of the oral cavity contains lysozyme with a certain concentration, so that bacteria including escherichia coli can be inhibited or even killed, the oral cavity probiotic bacterial strain can endure the lysozyme with a certain concentration, and the growth of the bacterial strain CA119 is not influenced by the lysozyme basically, so that the oral cavity probiotic bacterial strain can survive and colonize the oral cavity well.

6 enzyme production ability assay

The strain to be tested is inoculated on BHI solid plate culture medium, and is subjected to activation culture for 12 hours at 37 ℃.

3.7.1 amylase assay

BHI solid plate medium containing 0.25% soluble starch was prepared for use. Taking activated test bacteria by using a disposable sterile inoculation ring point, transferring the test bacteria to a BHI solid flat plate culture medium containing soluble starch, inverting the test bacteria to a microorganism incubator for culturing for 24 hours at 37 ℃, uniformly spraying a Russell iodine solution on the surface of the culture medium for dyeing, measuring the diameter (D) of each colony and the diameter (D) of a transparent ring, and judging the amylase production capacity of the strain according to the ratio (D/D) of the transparent ring to the colony diameter, wherein each group is repeated three times.

3.7.2 protease assay

15g of skim milk powder was added to 100mL of double distilled water, autoclaved at 115℃for 20min, and 10% by volume was added to BHI medium to prepare a solid plate for use. Taking activated test bacteria by using disposable sterile bacteria-inoculating loop points, transferring the test bacteria to a BHI solid flat-plate culture medium containing skimmed milk powder, placing the test bacteria in an anaerobic workstation for culturing for 24 hours at 37 ℃, measuring the diameter (D) of each colony and the diameter (D) of a transparent ring, and judging the protein production capacity of the strain according to the ratio (D/D) of the transparent ring and the colony diameter, wherein each group is repeated three times.

3.7.3 cellulase assay

The activated test bacteria are transferred into a BHI liquid culture medium by a disposable sterile inoculating loop, cultured for 12 hours at 37 ℃ and 220rpm, and the absorbance of the bacterial liquid at the wavelength of 600nm is regulated to be 0.5 by a fresh BHI culture medium. Sucking 100 μl of the bacterial liquid to be detected into a 1.5mL centrifuge tube, centrifuging at 1200Xg at 4deg.C for 2min, discarding the supernatant, adding 1mL cellulase extract, performing ultrasonic crushing under ice bath for 6min, centrifuging at 8000 Xg at 4deg.C for 10min, collecting the supernatant, and detecting cellulase activity of the strain with Cellulase (CL) activity detection kit, wherein each group is repeated three times.

The results of the enzyme productivity measurement are shown in Table 1. Strain CA119 produced amylase but not protease.

The strain CA119 has higher cellulase activity, and the CL activity value of the strain CA119 is 0.16U/10 through detection ⁴ cell。

Whether the strain can produce a series of digestive enzymes such as amylase, protease and cellulase is a key whether the strain can improve the growth performance of a host, and the strain CA119 can simultaneously produce the amylase and the cellulase and has the potential of promoting the digestion capability of the host and improving the growth performance.

TABLE 1

7 determination of sugar fermentation Capacity

The method comprises the steps of respectively wiping and sterilizing raffinose, sorbitol, sucrose, mannitol, maltose, salicin, glucose and xylose bacteria trace biochemical identification tubes with alcohol cotton in an anaerobic workstation, starting the tubes, respectively inoculating activated test bacteria into each bacteria trace biochemical identification tube by using a disposable sterile inoculating loop, carrying out anaerobic fermentation at 37 ℃ for 24 hours, observing the color change of the reagent in the biochemical identification tubes, judging the sugar fermentation capacity of each strain, and repeating each group for three times. The test result shows that the strain CA119 has the capacity of fermenting sorbitol, sucrose, mannitol, maltose, salicin, glucose and xylose, but cannot ferment raffinose, and has strong sugar fermentation capacity.

Different bacteria have different sugar decomposing capacities, the metabolized products are also different, and partial probiotics exert the organic acid, vitamin and other metabolites produced after the sugar fermentation capacity, so that the effects of reducing the pH of the feed, inhibiting pathogenic bacteria, improving or improving the palatability of the feed, reducing the raising cost and the like can be achieved. The strain CA119 can ferment various saccharides, alcohols and glycosides, and has certain utilization potential in the aspect of feed fermentation.

8 antibiotic susceptibility assay

Transferring activated test bacteria into a BHI liquid culture medium by using a disposable sterile bacteria-inoculating ring, culturing at 37 ℃ and 220rpm for 12 hours, sucking 150 mu L of bacteria liquid to be tested on the BHI solid flat culture medium, uniformly smearing by using a disposable L-shaped coating rod, placing 30 different antibiotic drug-sensitive paper sheets on the flat culture medium at intervals, placing in an anaerobic workstation, culturing at 37 ℃ for 18 hours, measuring the diameter of a bacteriostasis ring around each drug-sensitive paper sheet, judging the drug resistance of the strain according to the CLIS standard and combining the actual situation, and repeating each group for three times. The criteria are shown in Table 2 and the results are shown in Table 3.

The strain CA119 is resistant to 6 antibiotics such as oxacillin, penicillin and ceftazidime, moderately sensitive to 13 antibiotics such as cefuroxime sodium, cefazolin and compound neonomine, highly sensitive to 11 antibiotics such as ampicillin, gentamicin and norfloxacin, and has low probability of resistant gene transfer.

TABLE 2 criteria for drug sensitive inhibition zones

TABLE 3 antibiotic susceptibility assay results

9 hemolysis assay

Preparing a BHI solid culture medium, sterilizing at 121 ℃ for 15min, adding 5% of sterile defibrinated sheep blood when the temperature of the culture medium is reduced to 50 ℃, and shaking and mixing uniformly to prepare the BHI blood plate. The activated test bacteria are transferred to a BHI blood plate by using a disposable sterile bacteria-inoculating loop point, clostridium perfringens (Clostridium perfringens) is used as a positive control, the test bacteria are placed in an anaerobic workstation in an inverted mode for culturing for 24 hours at 37 ℃, whether hemolysis loops appear around colonies or not is observed, and each group is repeated three times. Determination criteria: alpha-hemolysis is the generation of grass green hemolytic rings, typically conditional pathogenic bacteria; beta-hemolysis is to generate transparent hemolytic ring, most of which are pathogenic bacteria, which are easy to cause various diseases; gamma-hemolysis is a non-hemolytic ring, does not produce hemolysin, and is generally non-pathogenic.

Clostridium perfringens is used as a positive control, the strain CA119 does not generate hemolytic rings, and as a result, the clostridium perfringens does not produce cytohemolysin, and the clostridium perfringens has higher safety.

10. Evaluation of in vitro bacteriostatic ability

10.1 Cell surface property measurement

10.1.1 surface hydrophobicity detection

Transferring the strain to be tested into BHI liquid culture medium with a disposable sterile inoculating loop, culturing at 37deg.C and 220rpm for 20h, centrifuging for 7min, discarding supernatant, adjusting absorbance of bacterial suspension at 600nm with sterile PBS buffer to about 0.8, and recording as A; and respectively adding three volumes of dimethylbenzene and chloroform into each bacterial suspension, mixing uniformly by vortex, standing at room temperature for 40min, absorbing the layered water phase, measuring the light absorption value of the light absorption value at 600nm by taking PBS buffer solution as a reference, marking as B, calculating the hydrophobicity according to a formula, and repeating each group for three times.

Cell surface hydrophobicity (%) = (1-B/a) ×100% (1.1)

10.1.2 self-agglutination Capacity detection

Adding 4mL of the bacterial suspension with the adjusted concentration into a 5mL centrifuge tube, vortex mixing uniformly, standing at room temperature for 6 hours, absorbing the upper bacterial suspension, measuring the light absorption value of the upper bacterial suspension with the wavelength of 600nm, marking as C, calculating the self-agglutination rate according to a formula, and repeating each group for three times.

Self-aggregation ratio (%) = (1-C/a) ×100% (1.2)

10.1.3 Co-agglutination Capacity detection

Inoculating Porphyromonas gingivalis and Fusobacterium nucleatum into WCA liquid culture medium, culturing at 37 ℃ for 24h at 220rpm, centrifuging for 7min, discarding supernatant, adjusting the absorbance of bacterial suspension at the wavelength of 600nm to about 0.8 by using sterile PBS buffer, respectively absorbing 4mL of bacterial suspension and pathogenic bacterial suspension after the adjustment of concentration, fully mixing, standing for 6h at room temperature, absorbing upper bacterial suspension, measuring the absorbance of bacterial suspension at the wavelength of 600nm, recording as D, calculating the co-agglutination rate according to a formula, and repeating each group for three times.

Co-aggregation ratio (%) = (1-D/A). Times.100% (1.3)

The surface hydrophobicity, self-cohesiveness and co-cohesiveness of strain CA119 were measured, respectively. The strain CA119 has hydrophobic effect on chloroform and dimethylbenzene, and the hydrophobic rate is 38.09% and 37.05% respectively; the self-agglutination rate is higher and is close to 80%; in the coagglutination test with Porphyromonas gingivalis, the coagglutination rate of the strain CA119 was 90.34%, and in the coagglutination test with Fusobacterium nucleatum, the coagglutination rate was 76.97%.

The hydrophobic capacity and the self-coagulation capacity of the strain are positively correlated, and the self-coagulation reaction of the strain can enable the strain to be gathered in a large amount in the oral cavity so as to form a biological film, so that the adhesion capacity of the strain in the oral cavity is improved, and the strain is beneficial to exerting the beneficial effect. The strain CA119 has stronger hydrophobicity and self-coagulation property, and can be better planted in the oral cavity.

10.2 in vitro bacteriostasis test

The antibacterial ability of the test bacteria to the main pathogenic bacteria of periodontitis, porphyromonas gingivalis (P.gingivalis) and Fusobacterium nucleatum (F.nuciferatum), was measured by a double-layer agar diffusion perforation method.

10.2.1 Single layer agar plates

2% nutrient agar medium was prepared, autoclaved at 121℃for 15min, 10mL of each sterile plate was poured, and cooled to serve as the bottom layer.

10.2.2 fermentation supernatant

Inoculating the strain to be tested on BHI solid plate culture medium by using disposable sterile inoculating loop in anaerobic workstation, culturing at 37deg.C for 12 hr, picking single colony from solid plate culture medium, transferring into BHI liquid culture medium, culturing at 37deg.C and 220rpm for 18 hr, centrifuging at 1200rpm and 4deg.C for 10min, collecting supernatant, filtering with 0.22 μm sterile filter membrane, and storing at 4deg.C for use.

10.2.3 pathogenic bacteria

Taking porphyromonas gingivalis and fusobacterium nucleatum which are main pathogenic bacteria of periodontitis as pathogenic indicator bacteria, fully dissolving pathogenic indicator bacteria freeze-dried powder by using sterile double distilled water in an anaerobic workstation, absorbing 200 mu L of the freeze-dried powder on an FSA solid flat plate culture medium, uniformly smearing the freeze-dried powder by using a disposable L-shaped coating rod, culturing the freeze-dried powder at 37 ℃ for 24 hours, picking single bacterial colonies by using a disposable sterile bacteria-inoculating ring, transferring the single bacterial colonies into a WCA liquid culture medium, culturing the single bacterial colonies at 220rpm for 24 hours at 37 ℃ to obtain pathogenic indicator bacteria liquid.

10.2.4 bacteriostasis test

Preparing FSA solid culture medium, autoclaving at 121deg.C for 15min, cooling to 50deg.C, adding 5% sterilized defibrinated sheep blood, mixing, adding Porphyromonas gingivalis and Clostridium nucleatum bacteria liquid at 40deg.C, and regulating final concentration of pathogenic bacteria to 1×10 ⁷ CFU/mL, mixing well, placing a sterile oxford cup with the diameter of 6mm on a single-layer agar plate uniformly, taking 20mL of FSA culture medium containing pathogenic indicator bacteria, slowly adding the FSA culture medium into the plate, cooling and solidifying the culture medium, taking out the oxford cup by using sterile forceps to form a 6mm round hole, sucking 120 mu L of fermentation supernatant of test bacteria, adding the fermentation supernatant into the round hole, performing anaerobic culture at 37 ℃ for 24 hours, observing and measuring the size of a bacteriostasis zone around the round hole, and repeating each group for three times.

As described above, the antibacterial effect of the strain on Porphyromonas gingivalis (P.gingivalis) and Fusobacterium nucleatum (F.nuceleatum), the main etiology of periodontitis, was examined by double-layer agar diffusion perforation. The strain CA119 has certain inhibition effects on porphyromonas gingivalis (the diameter of a inhibition zone is 10.4+/-0.5 mm) and fusobacterium nucleatum (the diameter of the inhibition zone is 18.1+/-0.4 mm), wherein the inhibition effect on the fusobacterium nucleatum is obvious.

10.2.5 bacterial whole genome analysis

a) Bacterial assembly results

The sequencing result is subjected to data quality evaluation and short sequence assembly, and the genome of the strain CA119 is obtained by splicing, and the result shows that the total length of the genome of the strain CA119 is 2,144,611bp, the length of N50 is 487,491bp, the length of L50 is 3bp, the length of N75 is 250,245 bp, the length of L75 is 4bp, and the G+C content is 44.22%.

b) Secondary metabolites

The potential bacteriostatic secondary metabolite-producing gene cluster in strain CA119 was subjected to mining analysis. There are 3 potential bacteriostatic secondary metabolite-encoding gene clusters annotated on their genome, and no gene clusters similar thereto are known at present. This shows that the bacterial strain CA119 has a certain degree of inhibition effect on the main pathogenic bacteria of periodontitis, namely Porphyromonas gingivalis and Fusobacterium nucleatum, and the antibacterial capacity of the bacterial strain CA119 can be related to the novel compounds generated by the biosynthesis gene cluster.

Example 3Research on action of streptococcus cristatus CA119 on preventing and treating periodontitis

1. The experimental method comprises the following steps:

1 animal Experimental design arrangement

Experimental animals: the laboratory animals used in this study were SPF-grade SD rats 5 weeks old weighing 150-160g purchased from Gibbs laboratory animal technology Co., ltd.

The rats were randomly and equally divided into 6 groups of 48 SPF-class 5-week-old female SD rats in an environment with a 12h light/dark cycle and a temperature of 22-24 ℃. Namely: control group (Control); model sets (models); applying a probiotic group (OP); a gastric lavage probiotic Group (GP); a spread control group (OPM); gastric lavage control Group (GPM). The experiment was performed for a total of 40d, during which all rats were given sterile sugar water with 10% (m/m) sucrose, and after the start of the experiment, all rats were given 1mg/mL kanamycin in their drinking water to reduce the interference of the oral local flora for 7d. Each group of rats was treated as follows:

(1) Model group, OPM group, GPM group: starting at experiment 10d, each rat inoculated with periodontitis pathogenic bacteria by periodontal smear was administered 5×10 ⁹ CFU Porphyromonas gingivalis (P.gingivalis ATCC 33277) and 5X 10 ⁹ CFU Fusobacterium nucleatum (F.nuleatum ATCC 10953), prohibited from drinking water for 1h after each inoculation, and inoculated every two daysSeed once until the end of the experiment (40 d).

(2) OP group, OPM group: rats were given 4×10 in periodontal spread ⁹ CFU streptococcus cristae CA119, prohibited from drinking water for 1h after each application, once daily until the end of the experiment.

(3) GP group, GPM group: 4X 10 rats were given by gavage ⁹ CFU streptococcus cristae CA119, once daily, until the end of the experiment.

(4) Control: equal amounts of sterile PBS buffer were administered.

2 food intake and weight changes

The rats were dosed daily at regular intervals, and the remaining rats were monitored and recorded for body weight and daily intake and daily gain at the end of the experiment.

3 quantitative analysis of periodontal tissue

3.1 extraction of Total RNA from tissues

(1) After euthanizing the rats, 100mg of gingival tissue of the rats is collected by a sterile scalpel, placed into an enzyme-free 1.5mL centrifuge tube containing magnetic beads and 900 mu LTrizol, fully homogenized, stood for 5min at room temperature and centrifuged for 5min at 12000 Xg;

(2) Centrifuging, transferring the supernatant to a new 1.5ml centrifuge tube, adding 200 μl of chloroform, swirling for 15s, standing for 10min, centrifuging at 12000×g at 4deg.C for 15min;

(3) Centrifuging, collecting supernatant, adding equal volume of isopropanol into supernatant, mixing, standing for 10min, centrifuging at 4deg.C for 10min,

(4) Centrifuging, removing supernatant, adding 1mL 75% ethanol solution to wash precipitate, centrifuging at 7500 Xg at 4deg.C for 5min,

(5) Centrifuging, removing supernatant, reversely buckling on absorbent paper, adding DEPC water to dissolve RNA after drying, and measuring concentration by using a spectrophotometer.

3.2 reverse transcription of RNA

Preparing RT reaction liquid according to the requirements of primeScrip RT reagent Ki kit instruction; reacting at 37deg.C for 15min, reacting at 85deg.C for 5s, packaging, and storing at-80deg.C.

TABLE 4 reverse transcription system

3.3Real Time PCR reaction

Using TB Green Premix Ex Taq II kit reaction system to prepare mixed solution: 12.5. Mu.L of TB Green Premix Ex Taq II, 1. Mu.L of each of the upstream primer and the downstream primer, 2. Mu.L of template DNA and 25. Mu.L of the total system were added to the enzyme-free PCR tube, and the reaction conditions were as follows: 95 ℃ for 30s; and (3) detecting Ct values of each hole by using a real-time fluorescence quantitative PCR instrument after three holes are formed in each sample in 95 ℃,5s,60 ℃,45s and 40 cycles, and analyzing mRNA relative expression amounts of genes IL-6, IL-1 beta, IL-18, TNF-alpha, COX-2, iNOS and MCP-1 related to inflammation by using GAPDH as an internal reference gene.

4 periodontal tissue Osteoprotegerin (OPG), osteoclast differentiation factor (RANKL) expression level

After euthanizing the rats, 100mg of gingival tissues of the rats are collected by a sterile scalpel, placed into a 1.5mL centrifuge tube containing magnetic beads and 900 mu L of PBS, fully homogenized, centrifuged at 3000rpm and 4 ℃ for 20min, and the supernatant is collected and diluted 5 times by a diluent to be used as a sample to be tested; adding 10 mu L of a sample to be detected and 50 mu L of a standard substance into an enzyme-labeled coating plate in an ELISA kit, incubating for 30min at 37 ℃, washing the enzyme-labeled coating plate with a washing solution for 5 times, adding 50 mu L of an enzyme-labeled reagent, incubating for 30min at 37 ℃, repeatedly washing the enzyme-labeled coating plate for 5 times, respectively adding 50 mu L of a developing solution A, B into each hole, developing at 37 ℃ for 10min after uniformly mixing, adding 50 mu L of a stopping solution into each hole, and measuring the light absorption value of the wavelength at 450 nm.

5. Oxidative stress index detection

5.1 Serum was collected

Before the rat is killed, the heart is used for taking blood by a sterile injector, the collected blood is placed at 37 ℃, and is kept stand for 1h, after the blood is coagulated and layered, the blood is centrifuged at 3000rpm and 25 ℃ for 10min, the supernatant is collected, the supernatant is centrifuged at 12000rpm and 4 ℃ for 15min, the supernatant is sucked and split-packed into a 2.0mL sterile centrifuge tube, and the supernatant is transferred to a-80 ℃ for preservation.

5.2 index detection

The serum oxidation index detection kit is used, and a colorimetric method is adopted for detecting the large sizeMalondialdehyde (MDA) and hydrogen peroxide (H) in mouse serum ₂ O ₂ ) The content of Myeloperoxidase (MPO), glutathione peroxidase (GSH-PX), superoxide dismutase (T-SOD) and total antioxidant capacity (T-AOC) is measured.

6 alveolar bone microstructure scanning

After euthanizing the rat, separating the maxilla of the rat by using a sterile scalpel, fixing the maxilla in 4% paraformaldehyde for 48 hours, washing the maxilla by using a 75% ethanol solution, storing the maxilla in the 75% ethanol solution at 4 ℃, sending the maxilla to Hangzhou Haoke biotechnology limited company, and performing X-ray scanning imaging on the maxilla sample of the rat by using Micro-CT (Micro Computed Tomography, a microcomputer tomography technology); the specific scanning parameters are as follows: voltage, 46kV; current, 75 μa; scanning accuracy, 9 μm; the number of layers, 400; camera mode, high resolution. Alveolar bone resorption was evaluated by measuring the distance from the alveolar bone crest to the enamel-bone junction at the mesial and distal positions of the second molar of the upper jaw.

7 periodontal tissue pathology analysis

(1) Placing the separated rat maxilla tissue in 4% paraformaldehyde for fixing for 48 hours, soaking the rat maxilla tissue in double distilled water for 60 minutes, replacing the double distilled water every 20 minutes, placing the tissue in a glass beaker, adding 25 times of PLANK-Rychlo decalcification liquid for decalcification treatment, replacing the decalcification liquid every two days, and detecting the decalcification degree of the tissue by adopting a physical needling method;

(2) Soaking with double distilled water for 60min after decalcification, changing double distilled water every 20min, soaking with G3430-decalcification alkali treatment solution for 40min for deacidification, changing treatment solution every 20min, and washing under running water for 18h;

(3) Dehydrating the tissue sample in ethanol solutions of 70% (2 h), 80% (2 h), 85% (12 h), 90% (2 h), 95% (3 h) and 100% (2 h), putting the tissue sample into xylene for transparency for 3min, and sequentially immersing the tissue sample into wax I, wax II and wax III at 58 ℃ for 30min respectively; paraffin embedding, longitudinally slicing with the thickness of 4 mu m, and baking at 70 ℃ for 1.5h;

(4) Sequentially placing slices into xylene I and xylene II for 12min respectively, sequentially soaking in ethanol solution with concentration of 100%, 95%, 80% and 70% for 45s, collecting hematoxylin, dyeing for 3.5min, soaking in double distilled water, placing into dilute ammonia water for 2min, soaking in double distilled water, dyeing for 6min with eosin, soaking in double distilled water, sequentially placing into ethanol solution with concentration of 80%, 95% and 100% for 15s, and drying in oven at 60deg.C;

(5) The slides were sealed with a cover slip by adhesive, and the histopathological lesions were observed under a microscope.

8 colon tissue pathology analysis

After the isolated rat colon tissue was fixed in 4% paraformaldehyde, the procedure was the same as 2.7 (no decalcification, alkali treatment was required). The pathological lesions of the colon tissue and inflammatory cell infiltration were observed under a microscope.

9 data analysis

The experimental data adopts IBM SPSS Statistics to carry out statistical analysis on the data, adopts a t-test method to analyze the significance of the difference between two groups, and adopts Tukey test in One-way Anova to compare the difference between groups of the data; experimental results are expressed as mean ± standard deviation. Graphics were drawn using GraphPad Prism 9.

2. Experimental results

1 food intake and weight variation

The effect of periodontitis on daily feed and body weight of rats was recorded during the experiment, and the average daily feed and daily gain of rats were calculated, and the results are shown in fig. 7. The average daily intake and daily gain of Model rats was significantly reduced (P < 0.001) compared to Control rats; the average daily intake and daily gain of OPM rats was significantly increased (P < 0.01) compared to Model group, but the average daily intake and daily gain of streptococcus cristatus (GPM) rats with periodontitis was not significantly affected. Demonstrating that dental Zhou Tuma streptococcus cristatus CA119 can alleviate the reduction of food intake and weight gain caused by periodontitis.

2 quantitative analysis of periodontal tissue

The relative expression levels of some of the pro-inflammatory cytokines, COX-2, iNOS, MCP-1 in the gingival tissue of the rat were examined by real-time fluorescent quantitative PCR, and the results are shown in FIG. 8. The relative expression levels of IL-6, IL-1. Beta., IL-18, TNF-. Alpha., COX-2, iNOS, MCP-1 were significantly increased (P < 0.001) in the gingival tissue of the Model group rats as compared with the Control group; compared with the Model group, the relative expression amount of IL-6, IL-1 beta, IL-18, COX-2, iNOS and MCP-1 in gingival tissues of the OPM group rats is obviously reduced (P < 0.05), the relative expression amount of TNF-alpha is not obviously changed, and the relative expression amount of proinflammatory cytokines in gingival tissues of the rats suffering from periodontitis can be reduced to a certain extent by using the streptococcus chebulus CA119 (GPM), but the difference is not obvious. It was shown that dental Zhou Tuma Streptococcus crest CA119 reduced periodontal inflammatory response.

3 periodontal tissue OPG (osteoprotegerin), RANKL (osteoclast differentiation factor) expression level

Results of detection of OPG, RANKL levels in rat gingival tissue using ELISA are shown in figure 9. The effect of the ratio of OPG and RANKL in gingival tissue on OPG and RANKL expression in and of streptococcus chebulus CA119 (GPM) was not significant, indicating that dental Zhou Tuma streptococcus chebulus CA119 was able to inhibit the differentiation and maturation of osteoclasts in periodontal tissue.

4 oxidative stress index analysis

The related index of oxidative stress in rat serum is detected by adopting a colorimetric method, and the result is shown in figure 10. MDA, H in the serum of Model rats compared to Control group ₂ O ₂ The MPO level is significantly increased (P<0.001 T-SOD, GSH-PX, T-AOC, and significantly reduced levels (P)<0.001 A) is provided; MDA, H in serum of OPM group rats compared to Model group ₂ O ₂ Significantly reduced MPO levels (P<0.001 T-SOD and T-AOC levels are significantly elevated (P)<0.05 But GSH-PX level changes are not significant, H in serum of GPM group rats ₂ O ₂ Significantly reduced MPO levels (P<0.05 Significantly elevated T-AOC levels (P<0.05 But has significant differences compared with OPM group (P<0.05 No other index was significantly improved. Indicating that the streptococcus cristatus CA119 can relieve oxidative stress and extractHigh antioxidant stress capability and tooth Zhou Tuma effect higher than gastric lavage.

5 alveolar bone microstructure analysis

The alveolar bone resorption was evaluated by observation analysis of rat maxillary molar and its surrounding bone tissue by in vivo Micro-computed tomography (Micro-CT), and the results are shown in FIGS. 11 and 12. Alveolar bone resorption values were significantly increased in Model rats compared to Control group (P < 0.001); the alveolar bone resorption values were significantly reduced (P < 0.01) in the OPM group of rats compared to the Model group, whereas the effects of streptococcus cristatus CA119 (GPM) on alveolar bone resorption caused by periodontitis were insignificant. It was shown that Streptococcus crest CA119 of tooth Zhou Tuma reduced alveolar bone resorption and provided protection for periodontal support.

6 periodontal tissue pathological analysis

By HE staining, the alveolar bone tissue pathology of rats was analyzed, as shown in FIG. 13, and periodontal ligament fiber arrangement disorder, fibrosis and dissolution occurred in rats in Model group compared with Model group, and the linear distance from cementum-enamel junction (CEJ) to Alveolar Bone Crest (ABC) was significantly increased; the periodontal ligament fibers of the OPM group rats were aligned well, fibrosis and dissolution were not evident, the distance of cementum-enamel bond to the alveolar bone crest was significantly reduced, but no significant improvement was observed in the GPM group, compared with the Model group. Demonstrating that dental Zhou Tuma Streptococcus crest CA119 can reduce periodontal tissue damage.

7 colon tissue pathology analysis

Histopathological analysis of the rat colon was performed as shown in fig. 14. It was observed that the colon of Model rats showed massive inflammatory cell infiltration into the basal portion of the crypt and various degrees of bleeding; inflammatory cell infiltration and bleeding points were significantly reduced in the rat colon in OPM and GPM groups compared to Model groups. Indicating that periodontal smearing and gastric lavage of streptococcus cristatus CA119 can reduce irritation of periodontitis pathogenic bacteria to colon tissues.

The foregoing description is only a preferred embodiment of the present application, and is not intended to limit the present application, but although the present application has been described in detail with reference to the foregoing embodiment, it will be apparent to those skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or that equivalents may be substituted for part of the technical features thereof. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims (14)

1. A strain of streptococcus cristatus, designated streptococcus cristatus (Streptococcus cristatus) CA119, which has been deposited at the chinese collection of typical cultures at university of armed chinese in china at 12 months 2 of 2022, with a deposit number of CCTCC NO: m20221855.

2. A culture of streptococcus cristae comprising the substance obtained by culturing the streptococcus cristae of claim 1 in a microbial medium;

the substance is supernatant, and the specific acquisition steps are as follows:

inoculating the strain to be tested on BHI solid plate culture medium by using disposable sterile inoculating loop in anaerobic workstation, culturing at 37deg.C for 12 hr, picking single colony from solid plate culture medium, transferring into BHI liquid culture medium, culturing at 37deg.C and 220rpm for 18 hr, centrifuging at 1200rpm and 4deg.C for 10min, collecting supernatant, filtering with 0.22 μm sterile filter membrane, and storing at 4deg.C for use.

3. A microbial agent comprising the streptococcus cristatus of claim 1, or the culture of claim 2.

4. A composition comprising the streptococcus cristatus of claim 1, or the culture of claim 2.

5. The composition of claim 4, wherein the product has a use of at least one of:

1) Reducing periodontal tissue inflammatory response;

2) Inhibiting differentiation and maturation of osteoclasts in periodontal tissue;

3) Reducing oxidative stress and improving the capacity of resisting oxidative stress;

4) Can reduce alveolar bone resorption;

5) Can reduce the damage of periodontal tissue;

6) Reducing irritation of periodontitis pathogenic bacteria to colon tissue.

6. A product comprising the streptococcus cristatus of claim 1, or the culture of claim 2, or the composition of claim 4 or 5.

7. The product of claim 6, wherein the product comprises a pharmaceutical product, a commodity product, a feed product, and a microecological formulation.

8. The product according to claim 7, characterized in that it has a use according to at least one of the following:

1) Reducing periodontal tissue inflammatory response;

2) Inhibiting differentiation and maturation of osteoclasts in periodontal tissue;

3) Reducing oxidative stress and improving the capacity of resisting oxidative stress;

4) Can reduce alveolar bone resorption;

5) Can reduce the damage of periodontal tissue;

6) Reducing irritation of periodontitis pathogenic bacteria to colon tissue.

9. Use of streptococcus cristae according to claim 1 or a culture according to claim 2 for the preparation of an antibacterial agent.

10. The use according to claim 9, wherein the antibacterial agent is capable of inhibiting porphyromonas gingivalis and/or fusobacterium nucleatum.

11. Use of streptococcus cristae according to claim 1 or a culture according to claim 2 for the preparation of a product for the prevention and treatment of periodontitis.

12. Use of streptococcus cristae according to claim 1 or a culture according to claim 2 for the preparation of a product for the prevention and treatment of colitis.

13. The use according to claim 12, wherein the colitis is colitis caused by pathogenic bacteria of periodontitis.

14. The use according to claim 11 or 12, wherein the products comprise pharmaceuticals, daily chemicals, feed and micro-ecological agents.