Lactobacillus zeae and application thereof

Abstract

The invention relates to the technical field of microorganisms, and discloses a lactobacillus zeae strain and an application thereof, and the preservation number of the lactobacillus zeae strain is CGMCC (China General Microbiological Culture Collection Center) NO.26771. The lactobacillus zeae strain has the advantages that the lactobacillus zeae strain can be used for preparing a lactobacillus zeae strain; the lactobacillus zeae provided by the invention has the effects of inhibiting the activity of pathogenic bacteria, reducing inflammatory factors, inhibiting alveolar bone resorption and regulating digestive tract flora.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to a strain of Lactobacillus zeaxanthin and its applications. Background Technology

[0002] The homeostasis of the oral microbiota plays a crucial role in maintaining health. Oral problems are often associated with infections caused by oral pathogens, such as Streptococcus mutans, Candida albicans, Fusobacterium nucleatum, and Porphyromonas gingivalis. Disruption of the oral microbiota balance can lead to various oral infectious diseases. Furthermore, oral pathogens can not only cause oral infections but also reach other tissues through the digestive tract or bloodstream, causing digestive diseases and even systemic diseases.

[0003] Common methods for preventing and treating oral health issues include the use of fluoride products and antibiotics. However, the toxic side effects of fluoride and the adverse reactions of antibiotics limit their application. Therefore, probiotics have increasingly been used in the prevention and treatment of oral problems in recent years. Various probiotics present in the oral cavity can maintain oral health through co-aggregation to form barriers, adhere to epithelial cells and compete for nutrients to inhibit pathogen colonization and growth, produce antibacterial substances such as hydrogen peroxide and bacteriostatic agents, and regulate immune responses. Furthermore, the metabolites of probiotics also have certain bactericidal effects. Therefore, probiotics play an important role in the prevention and treatment of oral diseases such as dental caries and periodontal disease. However, there are currently few reports of oral probiotic strains in China, and subsequent industrialization faces many challenges. For example, before applying probiotics to oral diseases, a series of steps are required, including precise and efficient screening of bacterial species or strains, clarifying their genetic information and probiotic mechanisms, and conducting functional evidence-based studies. Furthermore, current probiotic products suffer from severe homogenization, and there is insufficient research on the efficacy evaluation and mechanisms of action of screened probiotics. Moreover, research on oral probiotics lags significantly behind research on gut microbiota. Therefore, in-depth research on bacterial species and even strains related to oral diseases is necessary. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems existing in the prior art and provide a strain of Lactobacillus zeatus and its applications. This Lactobacillus zeatus and its metabolites have high antibacterial activity, can reduce inflammatory factors, inhibit alveolar bone resorption, and regulate the intestinal flora.

[0005] To achieve the above objectives, the first aspect of the present invention provides a strain of Lacticaseibacillus zeae, the preservation number of which is CGMCCNO.26771.

[0006] A second aspect of the present invention provides a microbial agent containing *Lactobacillus zealis* and excipients as described above.

[0007] A third aspect of the present invention provides the use of *Lactobacillus zedoaria* or the bacterial agent or metabolites of *Lactobacillus zedoaria* as described above in the preparation of a medicament for the prevention and / or treatment of digestive tract diseases.

[0008] The fourth aspect of the present invention provides the use of *Lactobacillus zedoaria* or the bacterial agent or metabolites of *Lactobacillus zedoaria* as described above in the preparation of a medicament for reducing inflammatory factors.

[0009] The fifth aspect of the present invention provides the use of *Lactobacillus zei* or the bacterial agent or metabolites of *Lactobacillus zei* as described above in the preparation of a medicament for inhibiting alveolar bone resorption.

[0010] The sixth aspect of the present invention provides the use of *Lactobacillus zedoaria* or the bacterial agent or metabolites of *Lactobacillus zedoaria* as described above in the preparation of a medicament for regulating the gut microbiota.

[0011] The *Lactobacillus zei* of this invention is characterized by its resistance to lysozyme, salt tolerance, and high self-aggregation rate. The *Lactobacillus zei* and its metabolites of this invention have bactericidal effects, reduce inflammatory factors, inhibit alveolar bone resorption, and regulate the gut microbiota. Therefore, the *Lactobacillus zei* of this invention can be used to prepare drugs for the prevention and / or treatment of digestive tract diseases.

[0012] Biological Preservation

[0013] The *Lactaseibacillus zeae* of this invention, classified and named (Latin name), was deposited on March 9, 2023, at the China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101) (abbreviation of depositary institution: CGMCC), with accession number CGMCCNo. 26771. Attached Figure Description

[0014] Figure 1 The results show the self-aggregation rate and co-aggregation rate of different strains. (A) is the self-aggregation rate, (B) is the co-aggregation rate with Fusobacterium nucleatum, and (C) is the co-aggregation rate with Porphyromonas gingivalis. Different lowercase letters in the figure represent significant differences between groups, p < 0.05, where ** indicates p < 0.01.

[0015] Figure 2The results show the tolerance of different strains. (A) is the 0.1 g / L lysozyme environment, (B) is the 1 wt% NaCl environment, (C) is the 2 wt% NaCl environment, and (D) is the 4 wt% NaCl environment. In the legend, control- represents the control group that does not contain lysozyme or NaCl.

[0016] Figure 3 The results are Micro-CT analysis of alveolar bone in rats treated in each group. (A) is a representative Micro-CT image of rats in each group, and (B) is the amount of alveolar bone resorption. Different lowercase letters in the figure represent significant differences between groups, p<0.05.

[0017] Figure 4 The figures show the levels of inflammatory factors in the gingival tissue of rats in each treatment group. (A) represents the TNF-α level, and (B) represents the IL-6 level. Different lowercase letters in the figure represent significant differences between groups, p < 0.05.

[0018] Figure 5 This is a bar chart showing the community distribution of oral flora at the phylum level in rats treated in each group.

[0019] Figure 6 This is a circular diagram showing the species distribution of oral microbiota at the genus level in each treatment group of rats.

[0020] Figure 7 This is a heatmap of the oral microbiota distribution at the genus level in each treatment group of rats.

[0021] Figure 8 The results are PCoA analysis of the oral microbiota of rats in each treatment group at the genus level.

[0022] Figure 9 This is a LEfSe multilevel species chromatographic tree diagram of the oral microbiota of rats in each treatment group.

[0023] Figure 10 This is a table showing the LDA discrimination results of the LEfSe multilevel species of oral microbiota in rats treated in each group (threshold is 3.5).

[0024] Figure 11 The Spearman correlation heatmap shows the correlation between genus-level pathological indicators and oral microbiota values ​​of TNF-α, IL-6, and ABL in rats treated in each group and oral microbiota.

[0025] Figure 12 This is a graph showing the rat genus-level ABL and PCoA ordination regression results for each treatment group.

[0026] Figure 13 This is a bar graph of the gut microbiota of rats in each treatment group at the phylum level.

[0027] Figure 14 This is a columnar section of the gut microbiota of rats in each treatment group at the genus level.

[0028] Figure 15 This is a heatmap of the genus-level distribution of the gut microbiota in rats from each treatment group.

[0029] Figure 16 The results are PLS-DA analysis of the gut microbiota of rats in each treatment group.

[0030] Figure 17 This is a LEFSe multilevel species chromatographic tree of the intestinal flora of rats in each treatment group.

[0031] Figure 18 This is a table showing the LDA discrimination results of LEFSe multilevel species in the gut microbiota of rats in each treatment group (threshold is 3.5). Detailed Implementation

[0032] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0033] The first aspect of this invention provides a strain of Lacticaseibacillus zeae, the preservation number of which is CGMCC NO.26771.

[0034] This invention provides *Lactobacillus zei* isolated and screened from milk, water, soil, and feed samples collected from a dairy factory in Ningxia. The 16S rDNA sequence of *Lactobacillus zei* of this invention is shown in SEQ ID NO:1.

[0035] SEQ ID NO:1:

[0036]

[0037] The *Lactobacillus zei* provided by this invention can produce a large number of live *Lactobacillus zei* cells through liquid culture. The culture method does not have any special requirements, as long as it can enable the *Lactobacillus zei* to proliferate. For example, the live *Lactobacillus zei* cells can be inoculated into the culture medium at an inoculation rate of 1-5 vol.% to obtain a culture solution.

[0038] According to the present invention, the culture medium can be any culture medium suitable for the culture of Lactobacillus maize known in the art, such as MRS medium.

[0039] According to the present invention, the culture conditions may include: a temperature of 25-40°C and a time of 6-48 hours. Preferably, the culture conditions include: a temperature of 35-40°C and a time of 10-15 hours.

[0040] This invention can further isolate live cells of *Lactobacillus cornis* from the above-mentioned culture medium. The method of isolation is not particularly limited, as long as it can enrich the cells from the culture medium. For example, it can be achieved by centrifugation and / or filtration. The conditions for centrifugation and filtration can be known conditions, which will not be elaborated here.

[0041] A second aspect of the present invention provides a microbial agent containing *Lactobacillus cerevisiae* and excipients as described above.

[0042] In this invention, the excipients may be selected from at least one of trehalose, maltodextrin, and glycerol.

[0043] A third aspect of the present invention provides the use of *Lactobacillus cornii* or the bacterial agent described above or its metabolites in the preparation of medicaments for the prevention and / or treatment of digestive tract diseases.

[0044] According to the present invention, the method for preparing the metabolite can be conventionally selected in the art, for example, it can be: inoculating the strain or bacterial agent into a lactic acid bacteria culture medium for culture to obtain a fermentation broth, centrifuging and separating the fermentation broth, and then filtering it with a filter membrane to obtain a culture supernatant, wherein the diameter of the filter membrane can be 0.2-0.5 μm, preferably 0.22 μm.

[0045] In this invention, the digestive tract disease is periodontitis.

[0046] In this invention, the digestive tract disease is periodontitis caused by trauma, wherein the trauma can be abrasion, contusion, laceration, puncture, cut, tear, bite, or burn.

[0047] In this invention, the digestive tract disease is caused by Fusobacterium nucleatum and / or Porphyromonas gingivalis.

[0048] According to the present invention, the metabolites of Lactobacillus zedoaria with accession number CGMCC No. 26771 have the ability to inhibit or hinder the normal growth of pathogenic bacteria in the digestive tract. However, in preferred cases, the antibacterial activity against Fusobacterium nucleatum and Porphyromonas gingivalis is more prominent, thereby achieving the purpose of preventing and treating digestive tract diseases caused by these two bacteria to a certain extent.

[0049] According to the present invention, the Lactobacillus zeaxanthin with accession number CGMCC No.26771 has a high ability to co-aggregate with Fusobacterium nucleatum and Porphyromonas gingivalis. It can effectively co-aggregate and encapsulate pathogens, reducing the probability of pathogens adhering to tissue cells, thereby reducing the probability of infection.

[0050] The fourth aspect of the present invention provides the use of *Lactobacillus zedoaria* or the bacterial agent or metabolites of *Lactobacillus zedoaria* as described above in the preparation of a medicament for reducing inflammatory factors.

[0051] In this invention, the inflammatory factors are interleukin-6 (IL-6) and / or tumor necrosis factor-α (TNF-α).

[0052] Oral lichen planus has a high incidence rate among oral mucosal diseases, but its etiology remains unclear. IL-6 interacts with its specific IL-6 receptor and may be involved in the occurrence and development of oral lichen planus. TNF-α can induce epithelial-mesenchymal transition in oral cancer cells by regulating the expression of the transcription factor Snail, upregulating the expression of MMP-2, MMP-9, MMP-13, and MMP-14, thereby collectively promoting the invasive and metastatic abilities of oral cancer cells SCC4, SCC9, and SCC25. Lowering the levels of both TNF-α and SCC25 can, to some extent, reduce the probability of oral lichen planus and oral cancer.

[0053] The fifth aspect of the present invention provides the use of *Lactobacillus zei* as described above, or the bacterial agent described above, or the metabolites of *Lactobacillus zei* in the preparation of a medicament for inhibiting alveolar bone resorption.

[0054] Major periodontal pathogens in the oral cavity, such as *Porphyromonas gingivalis* and *Aggregatibacter actinomycetemcomitans*, can directly damage periodontal tissues by producing toxins and enzymes such as lipopolysaccharides, proteases, and collagenases. Furthermore, periodontal pathogens can promote osteoclast formation and activity by regulating intracellular signaling pathways, leading to alveolar bone resorption when osteoclast activity is enhanced. Simultaneously, periodontal pathogens can inhibit osteoclast apoptosis, further increasing bone resorption. Secondly, inflammatory factors associated with periodontitis also affect osteoclast expression, with increased TNF-α expression being closely related to osteoclast formation. Therefore, the *Lactobacillus zeylanus* of this invention can inhibit alveolar bone resorption through different mechanisms.

[0055] The sixth aspect of the present invention provides the use of *Lactobacillus zei* or the bacterial agent or metabolites of *Lactobacillus zei* as described above in the preparation of a medicament for regulating the gut microbiota.

[0056] When the oral cavity is infected or injured by bacteria, its colonizing flora becomes imbalanced. Changes in its composition or function may favor the overgrowth and dominance of pathogenic bacteria, leading to abnormalities in the flora of a range of digestive tract components, including the oral cavity and intestines, and causing various diseases. The *Lactobacillus zei* of this invention can reduce the probability of related diseases by regulating certain parts of the digestive tract flora to near-normal levels.

[0057] The present invention will be described in detail below through examples. Unless otherwise specified, the methods described in the following examples are conventional methods in the art, and the reagents are all commercially available.

[0058] In the following examples, *Fusobacterium nucleatum* was purchased from the American Type Culture Collection (ATCC) with accession number ATCC 25586, and *Porphyromonas gingivalis* was purchased from the same collection with accession number ATCC 33277. *Streptococcus salivarius* K12 was purchased from the same collection with accession number ATCC BAA-1024. *Lactobacillus casei* 77, *Lactobacillus casei* 111, *Lactobacillus paracasei* 127, and *Lactobacillus paracasei* 128 were isolated and screened from milk, water, soil, and feed samples collected from a dairy factory in Ningxia.

[0059] All the devices and culture media involved in the following examples have been sterilized before use.

[0060] Example 1

[0061] This embodiment illustrates the inhibitory effect of *Lactobacillus zeaxanthin* on the growth of *Fusobacterium nucleatum* and *Porphyromonas gingivalis*, as described in this invention.

[0062] At 37℃, *Lactobacillus casei* 77, *Lactobacillus casei* 111, *Lactobacillus paracasei* 127, *Lactobacillus paracasei* 128, and *Lactobacillus zeyeris* N165 strains were added to MRS liquid medium and activated for 12 h. The activated strains were then inoculated into MRS medium at 2 vol.% and cultured at 37℃ for 24 h. After the culture, the supernatant was obtained by centrifugation at 10,000 rpm for 10 min at 4℃. The supernatant was then filtered through a 0.22 μm filter membrane to remove bacteria, yielding the natural fermentation supernatant.

[0063] Heat the MRS medium until completely melted, then pour it onto plates, 20 mL per plate, and allow it to solidify. Add 0.1 mL of the medium to a 10⁻⁶ live bacteria concentration. 6 CFU / mL of *Fusobacterium nucleatum* and *Porphyromonas gingivalis* liquid were inoculated onto the prepared plates to obtain indicator plates. 0.1 mL of the naturally fermented supernatant was transferred to an Oxford cup, which was then placed vertically on the indicator plate. Simultaneously, an Oxford cup containing only 0.1 mL of MRS liquid medium was placed in the same position on another identical indicator plate as a negative control. The indicator plates with the Oxford cups were incubated at 4°C for 2 hours, and then cultured at 37°C. After incubation, the size of the inhibition zone was measured using calipers.

[0064] Table 1

[0065]

[0066]

[0067] No inhibition zones were observed in the negative control group, while inhibition zones were observed in all five experimental groups. The diameters of the inhibition zones for *Fusobacterium nucleatum* and *Porphyromonas gingivalis* were 18-21 mm and 15-18 mm, respectively. These results indicate that *Lactobacillus casei* 77, *Lactobacillus casei* 111, *Lactobacillus paracasei* 127, *Lactobacillus paracasei* 128, and *Lactobacillus zeatus* N165 have significant inhibitory effects on *Fusobacterium nucleatum* and *Porphyromonas gingivalis*.

[0068] Example 2

[0069] This embodiment is used to illustrate the extracellular polysaccharide production of Lactobacillus maize involved in the present invention.

[0070] The extracellular polysaccharide yields of Lactobacillus casei 77, Lactobacillus casei 111, Lactobacillus paracasei 127, Lactobacillus paracasei 128, and Lactobacillus zeylindrica N165 were 0.342, 0.370, 0.319, 0.284, and 0.340 g / L, respectively.

[0071] Example 3

[0072] This embodiment is used to illustrate the self-aggregation and co-aggregation forces of *Lactobacillus zei* involved in this invention.

[0073] Five types of Lactobacillus, along with Fusobacterium nucleatum and Porphyromonas gingivalis, were cultured on MRS medium at 37°C for 24 h. The bacterial cells were then collected by centrifugation at 10,000 rpm for 10 min at 4°C. The collected cells were washed twice with PBS and then resuspended in PBS to obtain a Lactobacillus suspension. The Lactobacillus suspension was mixed with Fusobacterium nucleatum and Porphyromonas gingivalis in equal proportions to obtain mixed bacterial suspensions. The absorbance of the Lactobacillus suspension was adjusted to 0.6 ± 0.02 at 600 nm, and the initial absorbance values ​​of the Lactobacillus, Fusobacterium nucleatum, and Porphyromonas gingivalis suspensions were measured. Four mL of each of the Lactobacillus and mixed bacterial suspensions were vortexed for 10 s and incubated anaerobically at 37°C. The absorbance at OD600 was measured at 1 h and 24 h after anaerobic incubation. The autoaggregation and coagulation rates of different lactobacilli are obtained by the following formula:

[0074] Self-aggregation rate (%) = (A0 - A) t ) / A0×100%;

[0075] Coagulation rate (%) = (A0 + B0 - 2C) t ) / (B0+A0)×100%;

[0076] A0 – Initial absorbance of Lactobacillus suspension;

[0077] A t —Absorbance of Lactobacillus suspension after static incubation for t=1 or 24;

[0078] B0—Initial absorbance of the pathogenic bacterial suspension;

[0079] C t —The absorbance of the mixed bacterial suspension after static incubation for t=1 or 24.

[0080] The results of the self-aggregation rate and co-aggregation rate calculated from the absorbance value are as follows: Figure 1 As shown, the self-aggregation rate and co-aggregation rate of Lactobacillus zeatus N165 were significantly different from the other four strains (p<0.05), and were better than the other four lactobacilli. At 24h, the self-aggregation rate and the co-aggregation rate with Fusobacterium nucleatum and Porphyromonas gingivalis were as high as 87.75%, 96.78% and 88.94%, respectively.

[0081] A high self-aggregation rate indicates that *Lactobacillus zei* N165 can effectively and stably adhere to tissue cells in vivo, thereby reducing the probability of pathogen colonization. A high co-aggregation rate indicates that *Lactobacillus zei* can reduce the probability of pathogen adhesion to tissue cells by co-aggregating with pathogens.

[0082] Examples 1-3 collectively illustrate that the Lactobacillus maize N165 of the present invention can reduce the harm of pathogens to organisms by inhibiting pathogen activity and preventing pathogens from adhering to and colonizing tissue cells.

[0083] Example 4

[0084] This embodiment is used to illustrate the lysozyme tolerance of Lactobacillus maize involved in the present invention.

[0085] Lactobacillus casei 77, Lactobacillus casei 111, Lactobacillus paracasei 127, Lactobacillus paracasei 128, and Lactobacillus zeylinum N165 were inoculated separately into MRS broth medium containing 0.1 g / L lysozyme (purchased from Tiangen Biotech Co., Ltd., catalog number RT401) and MRS broth medium without lysozyme, respectively, so that the viable count in the culture medium after inoculation was 10-1. 6 CFU / mL was incubated at 37℃ for 24 h, and the absorbance at OD630 was measured at different time points. The absorbance results are as follows: Figure 2 As shown in (A).

[0086] The results showed that the absorbance of the five strains increased with increasing incubation, indicating that all five strains could grow in an environment with 0.1 g / L lysozyme.

[0087] Example 5

[0088] This embodiment is used to illustrate the salt tolerance of *Lactobacillus cornis* involved in this invention.

[0089] Lactobacillus casei 77, Lactobacillus casei 111, Lactobacillus paracasei 127, Lactobacillus paracasei 128, and Lactobacillus zeylindrica N165 were inoculated into MRS broth media containing 1 wt%, 2 wt%, and 4 wt% NaCl, respectively, as well as into MRS broth media without NaCl, so that the viable count in the media after inoculation was 10-1. 6 CFU / mL. After incubation at 37℃ for 24 h, the absorbance at OD630 was measured at different time points. Results... Figure 2 As shown in (B), (C) and (D).

[0090] The results showed that the absorbance of the five lactobacilli species increased with the increase of incubation time, indicating that all five lactobacilli species could grow stably in 1wt% NaCl and 2wt% NaCl environments. Although the growth in 4wt% NaCl environment was not as good as in 1wt% NaCl and 2wt% NaCl environments, none of them died, indicating that all five lactobacilli species had strong salt tolerance.

[0091] Example 6

[0092] This embodiment illustrates the preventive and therapeutic effects of the Lactobacillus cornis strain of the present invention on periodontitis and related diseases in rats.

[0093] (1) Treatment of the test substance

[0094] The lyophilized bacterial powders of Lactobacillus casei 77, Lactobacillus zei N165, and Streptococcus salivarius K12 were dissolved in sterile 0.85 wt% physiological saline, resuspended, and the viable bacterial concentration was adjusted to 10. 9 CFU / 0.3mL.

[0095] (2) Establishment of a rat periodontitis model and strain intervention regimen

[0096] Forty-eight male SPF-grade Wistar rats aged 6-8 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The rats were randomly divided into four groups based on the principle that there was no significant difference in average body weight among the groups: blank control (CK) group (8 rats), model group (10 rats), Lactobacillus casei 77 group (10 rats), Lactobacillus zeatus N165 group (10 rats), and positive control (Streptococcus salivarius K12) group (10 rats).

[0097] During the first week, the rats were given an adaptive feeding program, during which they had free access to food and water.

[0098] Weeks 2-3 were the pre-lactic acid bacteria intervention phase: rats in the Lactobacillus casei 77 group (hereinafter referred to as group 77), Lactobacillus zei N165 group (hereinafter referred to as group N165), and Streptococcus salivarius K12 group (hereinafter referred to as group K12) were given 0.3 mL / rat / day of the corresponding bacterial suspensions. The CK group and the model group were given 0.3 mL / rat / day of sterile 0.85wt% physiological saline. The intervention method was to use a disposable sterile syringe to slowly spray the bacterial suspension or physiological saline into the entire oral cavity of the rats and swallow it into the stomach, followed by a 30-minute water restriction.

[0099] Weeks 4-6 were the periodontal inflammation modeling and lactic acid bacteria intervention phase. Except for the control group, rats in other groups were anesthetized and their bilateral second molars were ligated with sterile 3-0 non-absorbable sutures, with knots tied on the buccal side. The ligatures were observed every 3 days, and any ligate sutures were promptly re-ligated. Simultaneously, lactic acid bacteria were administered for maintenance intervention, using the same method and frequency as in weeks 2-3.

[0100] Weeks 7-8 were the periodontal disease modeling phase, involving both suture ligation and periodontal pathogen infection, as well as lactic acid bacteria intervention. After three weeks of natural healing of the ligation wounds, infection with *Fusobacterium nucleatum* and *Porphyromonas gingivalis* was initiated. Except for the control group (CK), other rat groups received 0.3 mL / rat / time of a mixed bacterial suspension of *Fusobacterium nucleatum* and *Porphyromonas gingivalis*, once every 48 hours. In the CK group, the mixed bacterial suspension was replaced with 0.85 wt% saline. The infection method was the same as the lactic acid bacteria intervention method, and lactic acid bacteria were used concurrently with the infection, following the same method and frequency as in weeks 2-3.

[0101] (3) Organizational processing

[0102] At the end of week 8, after anesthetizing the rats, oral sampling swabs were used to collect samples from the maxilla, gingival tissue, and rectal contents. The rat maxilla was fixed in 4 wt% neutral formaldehyde solution and stored at room temperature; the oral swabs and other tissues were rapidly frozen in liquid nitrogen.

[0103] (4) Detection method

[0104] a. Alveolar bone resorption in the maxilla of rats

[0105] This invention uses Micro-CT to perform X-ray scanning imaging on rat maxillary bone samples to determine alveolar bone level (ABL): the distance from the cementoenamel junction of the second molar to the alveolar ridge crest. Four sites are measured for each tooth: the proximal and distal ends on the buccal and lingual sides. The average value of the measurements at each site is the alveolar bone resorption amount for that tooth.

[0106] b. Rat gingival inflammatory factors

[0107] This invention uses enzyme-linked immunosorbent assay (ELISA) to detect the levels of inflammatory factors IL-6 and TNF-α in rat gingival tissue.

[0108] c. Oral and intestinal flora of rats

[0109] This invention uses the Meiji Cloud platform to detect microbial diversity and perform bioinformatics analysis on rat oral swabs and intestinal contents.

[0110] (5) Results:

[0111] a. Alveolar bone resorption in the maxilla of rats

[0112] Depend on Figure 3 (A) indicates that the alveolar bone loss in the model group was more severe than that in the CK group, with obvious exposure and resorption of the tooth roots, and the surface was not smooth. In contrast, the alveolar bone loss in groups 77, N165, and K12 was less severe, with no obvious resorption of the tooth roots and a smoother surface.

[0113] The results of alveolar bone resorption measurement are as follows: Figure 3 As shown in (B), the alveolar bone resorption in the model group was significantly higher than that in the CK group. Compared with the model group and the K12 group, the alveolar bone resorption in the N165 group was significantly lower.

[0114] b. Rat gingival inflammatory factors

[0115] Concentrations of inflammatory factors in rat gingival tissue, such as Figure 4 As shown, there was a significant difference in TNF-α levels between the CK and model groups, and the intake of *Lactobacillus casei* N165 and *Lactobacillus casei* 77 significantly reduced TNF-α concentration. At IL-6 levels, the model group showed an increasing trend compared to the CK group, but the difference was not significant. Compared to *Streptococcus salivarius* K12, *Lactobacillus casei* N165 and *Lactobacillus casei* 77 reduced its concentration.

[0116] c. Oral and intestinal flora of rats

[0117] Oral community bar graph at the phylum level as follows Figure 5 As shown in the model group data, periodontitis leads to a downregulation of Proteobacteria abundance and an upregulation of Firmicutes abundance in the oral community. N165 significantly modulates both levels to a level similar to the control (CK) group, and its regulatory effect is similar to that of the K12 group, and significantly better than that of the 77 group. The abundance changes at the genus level are shown in the figure below. Figure 6 As shown, N165 significantly modulates the downregulation of Rothia and Pasteurella abundance and the upregulation of Veillonella abundance caused by periodontitis. The community structure heatmap at the genus level is shown below. Figure 7 As shown, the oral community structure of group N165 was significantly different from that of the model group. Compared with group 77, group N165 showed a stronger similarity to the CK group. The results of the PCoA analysis of oral microbiota at the genus level are as follows: Figure 8 As shown, the data indicates that there are significant differences in oral microbiota structure between the model group and the CK group in terms of habitat diversity (β-diversity). Compared with group 77, group N165 showed greater similarity to group CK, which is consistent with... Figure 7 The phenomenon corresponds to the community structure diagram at the genus level.

[0118] The results of the oral dominant species analysis are as follows: Figure 9 and Figure 10As shown, the dominant species in group N165, *Abiotrophia* and *Chryseobacterium*, had LDA values ​​of 4.05 and 3.85, respectively, indicating a positive correlation with periodontal health. *Ktedonobacteria* has the potential to produce antibacterial bioactive compounds, with an LDA value of 3.51. Groups 77 did not show any bacterial communities associated with periodontal health. Correlation analysis at the genus level is as follows... Figure 11 and Figure 12 As shown. At the genus level, ABL values ​​were negatively correlated with *Rodentibacter pneumophila* and *Rothia*, and... Figure 7 The results also showed that the abundance of both was enriched in group N165. TNF-α and IL-6 were negatively correlated with Rhodanobacter and Pseudonocardia. Among them, Rhodanobacter and unclassified Pasteurella were the dominant species in group N165.

[0119] Lactobacillus zedoaria N165 can regulate ABL, TNF-α, and IL-6 levels by modulating bacterial abundance and dominant species. This phenomenon corresponds to the pathological finding that N165 can significantly reduce alveolar bone resorption. With increasing ABL values, the PC1 axis value in the oral microbiota PCoA decreases, showing a negative correlation with a significant difference (p<0.05). Intake of Lactobacillus zedoaria N165 prevents oral microbiota imbalance.

[0120] Distribution of gut microbiota species, such as Figure 13 , Figure 14 and Figure 15 As shown, at the phylum level, the abundance of Bacteroidota, Actinobacteriota, and Proteobacteria in the model group was lower than that in the CK group, while the abundance of these phyla in the N165 group was higher than that in the model group. At the genus level, the intake of *Lactobacillus zei* N165 regulated the following bacterial groups: *Unclassified f. lachnospiracea*, *Romboutsia*, *Ruminococcus gauvreauii* group, *Ruminococcus*, *Turicibacter*, and *Blautia*. At the genus level, the community structure of the N165 group differed significantly from the model group and showed similarities to the CK group.

[0121] The results of PLS-DA analysis of gut microbiota are as follows: Figure 16 As shown, there were significant differences between the model group and the CK group, and the difference between the N165 group and the CK group was smaller than the difference between the N165 group and the model group. Analysis of dominant species in the gut microbiota is as follows: Figure 17 and Figure 18 As shown, the dominant species in group N165, g_Butyricicoccus, is positively correlated with periodontal health.

[0122] N165 showed better performance than other strains in all of the above aspects, so N165 can be used to reduce the abnormalities in the oral and intestinal flora caused by periodontitis.

[0123] The preferred embodiments of the present invention have been described in detail above with reference to the accompanying drawings; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various specific technical features in any suitable manner. To avoid unnecessary repetition, the present invention will not describe the various possible combinations separately. However, these simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A strain of *Lactaseibacillus zeae*, characterized in that, The preservation number of the Lactobacillus zeatus is CGMCC NO.26771.

2. A microbial agent, characterized in that, The microbial agent contains the *Lactobacillus cornis* as described in claim 1 and excipients.

3. The microbial agent according to claim 2, wherein, The excipients are selected from at least one of trehalose, maltodextrin, and glycerol.

4. The use of the *Lactobacillus zedoaria* of claim 1, or the microbial agent of claim 2 or 3, or the metabolites of *Lactobacillus zedoaria* in the preparation of a medicament for the prevention and / or treatment of digestive tract diseases.

5. The application according to claim 4, wherein, The digestive tract disease mentioned is periodontitis.

6. The application according to claim 4, wherein, The digestive tract disease mentioned is periodontitis caused by trauma.

7. The application according to claim 4, wherein, The digestive tract diseases mentioned are those caused by Fusobacterium nucleatum and / or Porphyromonas gingivalis.

8. The use of the Lactobacillus zedoariae according to claim 1, or the microbial agent according to claim 2 or 3, or the metabolites of Lactobacillus zedoariae in the preparation of a medicament for reducing inflammatory factors; Preferably, the inflammatory factor is interleukin-6 (IL-6) and / or tumor necrosis factor-α (TNF-α).

9. The use of the *Lactobacillus zei* of claim 1, or the bacterial agent of claim 2 or 3, or the metabolites of *Lactobacillus zei* in the preparation of a medicament for inhibiting alveolar bone resorption.

10. The use of the *Lactobacillus zei* of claim 1, or the microbial agent of claim 2 or 3, or the metabolites of *Lactobacillus zei* in the preparation of a medicament for regulating the gut microbiota.

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