Application of saliva combined with lactobacillus in the preparation of medicine for treating obesity

Abstract

The present invention relates to the application of saliva combined with lactobacillus in the preparation of a drug for treating obesity, and belongs to the field of biomedicine technology. The present invention provides the application of saliva combined with lactobacillus XA-1416 in the preparation of a drug for preventing and / or treating obesity, or preventing and / or treating obesity-related diseases. This strain can inhibit the weight gain of mice with obesity model induced by high-fat diet, reduce the blood lipid level of mice with obesity model induced by high-fat diet, improve the liver damage index of mice with obesity model induced by high-fat diet, reduce the area of ​​white fat cells in mice with obesity model induced by high-fat diet, promote the conversion of intestinal conjugated bile acid to free bile acid in mice with obesity model induced by high-fat diet, increase the level of intestinal FXR antagonist in mice with obesity model induced by high-fat diet, improve the intestinal flora imbalance of mice with obesity model induced by high-fat diet, inhibit fat accumulation in Hep G2 model, and inhibit differentiation into adipocytes in 3T3-L1 model.

Description

Technical Field

[0001] The invention relates to application of saliva combined with lactobacillus in preparing medicine for treating obesity, belonging to the technical field of biomedicine. Background Art

[0002] There are currently some weight loss drugs available on the market for obesity, however, these weight loss drugs have obvious adverse reactions and cannot be taken for a long time. For example, orlistat, which targets pancreatic lipase, often has mild to moderate gastrointestinal adverse reactions after taking it, such as fatty stools, diarrhea, and abdominal pain, and occasionally severe liver adverse reactions, such as cholelithiasis, cholestatic hepatitis, and subacute liver failure; and the side effects of GLP-1 receptor agonists targeting GLP-1 receptors represented by semaglutide include nausea, diarrhea, vomiting, constipation, abdominal pain, headache, fatigue, indigestion, dizziness, gastroenteritis, gastroesophageal reflux disease, and hypoglycemia in patients with type 2 diabetes. Therefore, it is necessary to find weight loss drugs that are effective and safe and harmless for long-term use.

[0003] Saliva lactobacillus (Ligilactobacillus salivarius) is a kind of probiotic present in the oral cavity and digestive tract of human body, has multiple health effects such as improving intestinal health, strengthening intestinal barrier, reducing inflammation and strengthening body immune function. Saliva lactobacillus was formally approved as edible strain by the Ministry of Health in 2003, entered in " List of Bacteria that Can Be Used for Food ", and has industrial application in probiotic food and health products. Based on the long-term safe and harmless characteristics of probiotics, existing related research attempts to use saliva lactobacillus for the prevention and treatment of obesity, but the saliva lactobacillus provided by these studies is not ideal in the effect of suppressing obesity and obesity-related diseases. For example, in the patent application text of publication number CN113337440A, although saliva lactobacillus MG-587 can lose weight, its effect in weight loss is not remarkable (when the experimental end point, the body weight of the model group is compared with that of the administration group, and the p value is 0.0678, no statistical difference). Therefore, it is urgent to find salivary Lactobacillus with better weight loss effects in order to develop weight loss drugs that are effective and safe for long-term use. Summary of the invention

[0004] In order to solve the above problems, the present invention provides the use of salivary lactobacillus (Ligilactobacillus Salivarius) XA-1416 in the preparation of medicines, wherein the medicines have any of the following functions:

[0005] (a) prevention and / or treatment of obesity;

[0006] (b) preventing and / or treating obesity-related diseases;

[0007] (c) regulating intestinal flora;

[0008] The saliva-combined Lactobacillus XA-1416 is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit number CGMCC No.40307 and the deposit date September 5, 2022.

[0009] In one embodiment of the present invention, the obesity-related diseases include obesity-induced metabolic syndrome, obesity-induced cardiovascular disease and / or obesity-induced intestinal flora imbalance.

[0010] In one embodiment of the present invention, the obesity-induced metabolic syndrome includes non-alcoholic fatty liver disease; the obesity-induced cardiovascular disease includes hyperlipidemia (high blood lipids) and / or hyperglycemia.

[0011] In one embodiment of the present invention, the prevention and / or treatment of obesity includes inhibiting weight gain, reducing the area of ​​white fat cells, inhibiting the differentiation of preadipocytes into adipocytes, reducing lipid accumulation in liver cells, improving blood lipid levels, improving liver damage indicators, improving bile acid metabolism, increasing intestinal farnesoid X receptor (FXR) antagonist levels and / or improving intestinal flora.

[0012] In one embodiment of the present invention, the prevention and / or treatment of obesity-related diseases includes inhibiting weight gain, reducing the area of ​​white fat cells, inhibiting the differentiation of preadipocytes into adipocytes, reducing lipid accumulation in liver cells, improving blood lipid levels, improving liver damage indicators, improving bile acid metabolism, increasing intestinal farnesoid X receptor (FXR) antagonist levels and / or improving intestinal flora.

[0013] In one embodiment of the present invention, the improvement of blood lipid levels includes lowering serum total cholesterol (TC) and / or low-density lipoprotein cholesterol (LDL-C).

[0014] In one embodiment of the present invention, the improvement of liver damage indicators includes reducing serum alanine aminotransferase (ALT) levels.

[0015] In one embodiment of the present invention, the improving bile acid metabolism includes promoting the conversion of intestinal conjugated bile acid into free bile acid.

[0016] In one embodiment of the present invention, the method for increasing the level of intestinal farnesoid X receptor antagonist comprises increasing the level of ursodeoxycholic acid (UDCA), glycoursodeoxycholic acid (GUDCA) and / or tauroursodeoxycholic acid (TUDCA) in the intestine.

[0017] In one embodiment of the present invention, the improvement of intestinal flora includes reducing the abundance of intestinal obesity-related bacteria, increasing the abundance of intestinal beneficial bacteria, improving the ratio of intestinal obesity-related bacteria and / or improving the functional pathways of intestinal microbiota; the obesity-related bacteria refer to bacteria enriched in the intestines of obese individuals.

[0018] In one embodiment of the present invention, the obesity-associated bacteria include Lachnospiraceae, Acetatifactor and / or Lachnoclostridium.

[0019] In one embodiment of the present invention, the beneficial bacteria include Lactobacillus and / or Bifidobacterium.

[0020] In one embodiment of the present invention, the ratio of obesity-related bacteria includes Firmicutes / Bacteroidetes.

[0021] In one embodiment of the present invention, the improvement of the intestinal microbiota functional pathway includes promoting the functions of pathways related to intestinal energy metabolism and lipid metabolism.

[0022] In one embodiment of the present invention, the energy metabolism and lipid metabolism related pathways include the pentose phosphate pathway, the 4-deoxy-L-threo-hex-4-enopyranuronate degradation pathway and / or the mevalonate pathway.

[0023] In one embodiment of the present invention, the obesity-related disease is hyperlipidemia; the prevention and / or treatment of hyperlipidemia includes improving blood lipid levels, improving bile acid metabolism and / or increasing intestinal farnesoid X receptor (FXR) antagonist levels.

[0024] In one embodiment of the present invention, the obesity-related disease is non-alcoholic fatty liver disease; the prevention and / or treatment of non-alcoholic fatty liver disease includes reducing lipid accumulation in liver cells, improving blood lipid levels, improving liver damage indicators, improving bile acid metabolism and / or increasing intestinal farnesoid X receptor (FXR) antagonist levels.

[0025] In one embodiment of the present invention, the obesity-related disease is obesity-induced intestinal flora imbalance; the prevention and / or treatment of obesity-induced intestinal flora imbalance includes reducing the abundance of intestinal and obesity-related bacteria, increasing the abundance of intestinal beneficial bacteria, improving the ratio of intestinal and obesity-related bacteria and / or improving the functional pathways of intestinal microbiota.

[0026] In one embodiment of the present invention, regulating the intestinal flora includes increasing the abundance of intestinal probiotics and / or reducing the abundance of intestinal harmful bacteria.

[0027] In one embodiment of the present invention, the intestinal probiotics include bifidobacteria and / or lactobacilli; the intestinal harmful bacteria include Clostridium perfringens, enterococci and / or enterobacteriaceae.

[0028] In one embodiment of the present invention, the obesity includes obesity induced by a high-fat diet, obesity induced by chemical drugs and / or obesity caused by genetic modification.

[0029] In one embodiment of the present invention, the obesity-related diseases include obesity-related diseases induced by a high-fat diet, obesity-related diseases induced by chemical drugs and / or obesity caused by genetic modification.

[0030] In one embodiment of the present invention, the drug further contains a drug carrier and / or a pharmaceutical excipient.

[0031] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0032] In one embodiment of the present invention, the pharmaceutical excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids and / or release retardants.

[0033] In one embodiment of the present invention, the dosage form of the drug is powder, granules, capsules, tablets, pills or oral liquid.

[0034] In one embodiment of the present invention, the viable count of saliva-combined Lactobacillus XA-1416 in the drug is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0035] The technical solution of the present invention has the following advantages:

[0036] The present invention provides the use of Ligilactobacillus Salivarius XA-1416 in the preparation of a drug for preventing and / or treating obesity, preventing and / or treating obesity-related diseases, or regulating intestinal flora. Studies have shown that:

[0037] (1) Saliva combined with Lactobacillus XA-1416 can effectively inhibit the weight gain caused by high-fat diet in mice with obesity;

[0038] (2) Saliva combined with Lactobacillus XA-1416 can significantly reduce the levels of TC (total cholesterol) and LDL-C (low-density lipoprotein cholesterol) in the serum of mice with high-fat diet-induced obesity model;

[0039] (3) Saliva combined with Lactobacillus XA-1416 can significantly reduce the level of ALT (alanine aminotransferase) in the serum of mice with high-fat diet-induced obesity model;

[0040] (4) Saliva combined with Lactobacillus XA-1416 can significantly reduce the average area of ​​white adipocytes in tissues (testicular adipose tissue and liver tissue) of mice with high-fat diet-induced obesity model;

[0041] (5) Saliva combined with Lactobacillus XA-1416 can significantly increase the ratio of free bile acid to conjugated bile acid in the ileum of mice with high-fat diet-induced obesity model, and promote the conversion of intestinal conjugated bile acid to free bile acid in mice with high-fat diet-induced obesity model;

[0042] (6) Saliva combined with Lactobacillus XA-1416 can significantly increase the levels of intestinal FXR antagonists (UDCA, GUDCA, and TUDCA) in mice with high-fat diet-induced obesity model;

[0043] (7) Saliva combined with Lactobacillus XA-1416 can significantly improve the intestinal flora imbalance caused by high-fat diet in mice with high-fat diet-induced obesity (reducing the abundance of intestinal bacteria related to obesity, increasing the abundance of intestinal beneficial bacteria, improving the ratio of intestinal bacteria related to obesity, and improving the functional pathways of intestinal microbiota);

[0044] (8) Saliva combined with Lactobacillus XA-1416 can significantly inhibit fat accumulation in the Hep G2 cell model;

[0045] (9) Saliva combined with Lactobacillus XA-1416 can significantly inhibit the differentiation of preadipocytes into adipocytes in the 3T3-L1 cell model;

[0046] (10) Saliva combined with Lactobacillus XA-1416 can regulate intestinal flora (increasing the abundance of intestinal probiotics and reducing the abundance of intestinal harmful bacteria).

[0047] Therefore, saliva combined with Lactobacillus XA-1416 has great application prospects in the preparation of drugs for preventing and / or treating obesity, preventing and / or treating obesity-related diseases (including obesity-induced hyperlipidemia, obesity-induced non-alcoholic fatty liver and / or obesity-induced intestinal flora imbalance, etc.), or regulating intestinal flora.

[0048] In addition, Lactobacillus salivais XA-1416 is an intestinal probiotic and is included in the "List of Bacteria that Can Be Used in Food", which has the advantage of being safe and harmless for long-term use. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 : Utilization of carbon sources salicin (a) and rhamnose (b) by saliva-associated Lactobacillus XA-1416.

[0050] Figure 2 : Phylogenetic analysis of Lactobacillus salivarius XA-1416 and the reference genome (a), and ANI value distribution of genomes with 100% similarity to the 16S rRNA gene sequence of XA-1416 (b).

[0051] Figure 3 : Flowchart of experimental design for high-fat diet-induced obesity model.

[0052] Figure 4 : HE staining of epididymal adipose tissue of mice in each group (×200, scale bar=50 μm).

[0053] Figure 5 : Average area of ​​epididymal white adipocytes in each group of mice.

[0054] Figure 6 : HE staining of liver tissues of mice in each group (×200, scale bar=50 μm).

[0055] Figure 7 : The ratio of free bile acid to conjugated bile acid in the ileum contents of the two groups of mice.

[0056] Figure 8 : Levels of FXR antagonists UDCA, GUDCA and TUDCA in the ileum contents of the two groups of mice. Figure 8 In the figure, A is the UDCA level; B is the GUDCA level; C is the TUDCA level.

[0057] Fig. 9 : Beta diversity of intestinal microbiota in each group of mice at the ASVs level.

[0058] Fig.10 : Beta diversity of intestinal microbiota at the genus level in each group of mice.

[0059] Fig.11 : Beta diversity of intestinal microbiota in the model group and XA-1416 group mice at the ASVs level.

[0060] Fig.12: Beta diversity of intestinal microbiota at the genus level in mice in the model group and XA-1416 group.

[0061] Fig.13 :Differences in the abundance of obesity-related bacteria in the intestinal microbiota of three groups of mice.

[0062] Fig.14 :Differences in the abundance of probiotics Lactobacillus and Bifidobacterium in intestinal microorganisms among three groups of mice.

[0063] Fig.15 : Differences in the abundance of intestinal microorganisms in specific functions among three groups of mice.

[0064] Fig.16 :Differences in the ratio of Firmicutes / Bacteroidetes among intestinal microorganisms of three groups of mice.

[0065] Fig.17 :Effects of saliva combined with Lactobacillus XA-1416 on lipid accumulation in Hep G2 cells.

[0066] Fig.18 :Effects of saliva combined with Lactobacillus XA-1416 on adipogenic differentiation of 3T3-L1 cells (preadipocytes).

[0067] Figures 1 to 18 In, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. DETAILED DESCRIPTION

[0068] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0069] The following experimental examples are not indicated with specific experimental steps or conditions, and the operation or conditions of the conventional experimental steps described in the document in this area can be carried out. The reagents used or the instruments are not indicated with manufacturers, and are the conventional reagent products that can be obtained commercially. The saliva combined lactobacillus XA-1416 involved in the following examples is recorded in the patent application text that publication number is CN115969888A. The animal bifidobacterium XA-768 involved in the following examples is recorded in the patent application text that publication number is CN115778987A.

[0070] The culture medium involved in the following examples is as follows:

[0071] MRS solid culture medium: peptone 10.0 g / L, beef extract powder 5.0 g / L, glucose 20.0 g / L, sodium acetate 5.0 g / L, yeast extract powder 4.0 g / L, triammonium hydrogen citrate 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, magnesium sulfate 0.2 g / L, Tween 80.01 mL / L, agar 15.0 g / L, manganese sulfate 0.05 g / L, pH 6.2.

[0072] MRS liquid culture medium: peptone 10.0 g / L, beef extract powder 5.0 g / L, glucose 20.0 g / L, sodium acetate 5.0 g / L, yeast extract powder 4.0 g / L, triammonium hydrogen citrate 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, magnesium sulfate 0.2 g / L, Tween 80.01 mL / L, manganese sulfate 0.05 g / L, pH 6.2.

[0073] YCFA solid medium: tryptone 10.0 g / L, yeast extract 2.5 g / L, sodium bicarbonate 4.0 g / L, glucose 2.0 g / L, maltose 2.0 g / L, cellobiose 2.0 g / L, cysteine ​​hydrochloride 1.0 g / L, dipotassium hydrogen phosphate 0.25 g / L, potassium dihydrogen phosphate 0.45 g / L, ammonium sulfate 0.9 g / L, sodium chloride 0.9 g / L, magnesium sulfate heptahydrate 0.09 g / L, calcium chloride dihydrate 0.09 g / L, resazurin 1.0 mL / L, hemin 0.01 g / L, VFA mix 6.2 mL / L (VFA mix: acetic acid 17.0 mL, propionic acid 6.0 mL, n-valeric acid 1.0 mL, isovaleric acid 1.0 mL, isobutyric acid 1.0 mL), vitamin solution I 1.0 mL / L (vitamin solution I: Biotin 5.0 mg, Vitamin B12 5.0 mg, p-aminobenzoic acid 15.0 mg, folic acid VB2 5.0 mg, vitamin B6 hydrochloride 75 mg), agar 15.0 g / L, distilled water to 1 L, pH 7.0.

[0074] BHI liquid culture medium: Tryptone 10.0g / L, Sodium Chloride 5.0g / L, Disodium Hydrogen Phosphate 2.5g / L, Dextrose 2.0g / L, Heart Extract Powder 9.8g / L, Brains Extract Powder 7.7g / L, pH 7.4.

[0075] Modified Gifu Anaerobic Medium (mGAM): peptone 5.0 g / L, Peptone 5.0g / L, soy peptone 3.0g / L, yeast extract powder 2.5g / L, beef powder 2.2g / L, digested serum powder 10.0g / L, beef liver extract powder 1.2g / L, glucose 0.5g / L, potassium dihydrogen phosphate 2.5g / L, sodium chloride 3.0g / L, soluble starch 5.0g / L, L-cysteine ​​0.3g / L, L-arginine 1.0g / L, L-tryptophan 0.2g / L, sodium thioglycolate 0.3g / L, pH is 7.3.

[0076] API medium (oligo culture medium): Trypticase 5.0g / L, Yeast extract 5.0g / L, SodiumChloride 2.5g / L, L-tryptophane 0.2g / L, L-cystine 0.4g / L, Hemin 0.005g / L, VitaminK1 0.01g / L, Sodium sulfite, pH 7.3.

[0077] The digestive juices involved in the following embodiments are as follows:

[0078] 0.25% pancreatic digestion solution: first weigh 2.5g porcine trypsin (purchased from Gibco) and 0.2g EDTA and dissolve them in PBS buffer (purchased from Solarbio, product number P1020), then adjust the pH to 7.4 with HCl, and finally add PBS buffer to make the volume to 1L to obtain 0.25% pancreatic digestion solution.

[0079] The preparation methods of the bacterial agents and culture supernatants involved in the following examples are as follows:

[0080] The saliva-associated Lactobacillus bacterial liquid is inoculated into an MRS liquid culture medium at an inoculum amount of 4% (v / v), and is statically cultured in a 37°C constant temperature incubator for 3 days to obtain a culture solution; the culture solution is centrifuged at 8000g for 10 minutes to obtain a saliva-associated Lactobacillus culture supernatant and saliva-associated Lactobacillus bacterial bodies; the saliva-associated Lactobacillus bacterial bodies are washed with physiological saline and then resuspended in a drug solvent (purchased from Solarbio, item number P1020) to obtain a saliva-associated Lactobacillus bacterial agent, and the saliva-associated Lactobacillus bacterial agent is stored at -80°C for use.

[0081] Experimental Example 1: Subspecies determination of saliva-associated Lactobacillus XA-1416

[0082] This experimental example conducted a subspecies determination experiment on saliva-associated Lactobacillus XA-1416. The specific process is as follows:

[0083] Phenotype Microarray 1 and 2A (purchased from BIOLOG, catalog numbers 12111 and 12112) were used to detect the extent to which Lactobacillus salivarius XA-1416 utilized carbon sources salicin and rhamnose. All monoclonal colonies of Lactobacillus salivarius XA-1416 on anaerobic blood agar medium (purchased from Beekman Bio) were collected using a throat swab collection cotton swab, and the cotton swab was inserted into a deoxygenated sterile saline test tube, and the cotton swab was stirred and rubbed against the test tube wall to evenly dissolve all bacteria on the cotton swab in sterile saline to prepare bacterial suspension A. After centrifuging bacterial suspension A (20°C, 7000g, 5min), the supernatant was discarded, and the suspension was resuspended with an equal volume of sterile saline. After centrifugation and discarding the supernatant, API medium (oligo medium) was added to prepare bacterial suspension B. The bacterial suspension B was added to the Phenotype Microarray 1 and Phenotype Microarray 2A plates, the 96-well plates were sealed with a sealing film, and the plates were placed on a microplate reader to detect the growth curve.

[0084] The results are as follows Figure 1 As shown, Lactobacillus salivarius XA-1416 can utilize L-Rhamnose as a carbon source, but does not utilize salicin as a carbon source for growth. According to the classification standard mentioned in the document "Morrison Rogosa et al., Species differentiation of oral lactobacilli from man including descriptionof Lactobacillus salivarius nov spec and Lactobacillus cellobiosus nov spec, J. Bacteriol. 65: 681-699, 1953.", Lactobacillus salivarius XA-1416 can be clearly classified as a salivarius subspecies of Lactobacillus salivarius (Lactobacillus salivarius subsp. salivarius).

[0085] Experimental Example 2: Genome Analysis of Saliva-associated Lactobacillus XA-1416

[0086] In this experimental example, the genome of salivary Lactobacillus XA-1416 was sequenced to obtain the genome sequence, and the difference between XA-1416 strain and the strain reported in the literature was analyzed from a genomic perspective to show its uniqueness, and the BSH activity potential of XA-1416 strain was inferred by predicting the BSH gene in the genome sequence.

[0087] The specific process is as follows:

[0088] The DNA of saliva combined with Lactobacillus XA-1416 strain was extracted by SDS method, and the quality of the extracted DNA was tested by agarose gel electrophoresis. 2.0 was used to quantify the concentration of extracted DNA. The sequencing of the bacterial genome was carried out at Novogene Co., Ltd., using the Nanopore platform to construct a 1D library to complete the third-generation sequencing of the bacterial genome; and using the Illumina platform to construct a 350bp small fragment library to complete the second-generation sequencing of the bacterial genome. After quality control, the sequences obtained by the third-generation and second-generation sequencing were spliced ​​and assembled using Unicycler software, the chromosome and plasmid sequences were screened, and the chromosome sequences were assembled into a circular genome (if it is a linear genome, it is a linear genome sequence), that is, the final 0gap completed map sequence.

[0089] The reference genome of Lactobacillus salivarius was downloaded for phylogenetic analysis based on the core genome to determine the species information of strain XA-1416, and the ANI similarity between the strain and the reference genome was calculated to analyze the similarity between strain XA-1416 and the reference genome. The comparative genome analysis and core genome construction were performed using orthorfinder software, and the phylogenetic analysis was performed using the best calculation model evaluated by iqtree software for phylogenetic calculation. The average nucleotide similarity (ANI) analysis was calculated using fastANI software.

[0090] Prodigal software was used to predict the coding genes of the assembled genome of Lactobacillus salivarius XA-1416. The database was constructed using the Bile salt hydrolase (BSH) gene sequence downloaded from the UniProt database, and the potential BSH genes in the genome were compared to infer the BSH activity potential of the strain.

[0091] The genome of Lactobacillus salivarius XA-1416 consists of 1 circular genome and 1 plasmid, with a total genome length of 1,942,197bp and a GC content of 33.04%. The genome encodes 1,831 genes, with a total base length of 1.71Mbp in the gene coding region, an average base number of 932bp, and a total length of the coding region accounting for 87.87% of the whole genome. A phylogenetic analysis of the core genome consisting of 1401 common homologous genes of Lactobacillus salivarius was performed, and the results are as follows Figure 2 The results showed that strain XA-1416 was clustered into a branch with the reference genome of all Lactobacillus salivarius, so XA-1416 belonged to Lactobacillus salivarius.

[0092] Since the 16S rRNA gene sequences of various species under Lactobacillus are highly similar, the use of 16S rRNA genes as standard molecular markers for differential analysis has great limitations (see reference: Kim E, Yang SM, Lim B, Park SH, Rackerby B, Kim HY. Design of PCR assays to specifically detect and identify 37 Lactobacillus species in a single 96wellplate. BMC Microbiol. 2020Apr 15; 20(1): 96. doi: 10.1186 / s12866-020-01781-z). This includes salivary Lactobacillus. In this experimental example, the 16S rRNA gene sequence of salivary Lactobacillus XA-1416 was compared with the NCBI Refseq genome database, and 37 reference genomes with 100% similarity to the 16S rRNA gene sequence of XA-1416 were found. ANI analysis of these 38 genomes revealed that the ANI similarities between these reference genomes and strain XA-1416 ranged from 96.84% to 98.02%. Figure 2 These results indicate that even though XA-1416 has high similarity with some reference strains in 16S rRNA gene sequence, at the genomic level, strain XA-1416 is highly unique compared to the reference genomes reported so far.

[0093] The results of the BSH gene comparison of Lactobacillus salivae XA-1416 are shown in Table 1. The results show that the genome of Lactobacillus salivae XA-1416 has two genes that are highly homologous to the reference BSH gene sequence, one of which is located on the circular chromosome of the strain and has a similarity of 53.846% to the reference gene; and the other is located on the plasmid of the strain and has a similarity of 97.531% to the reference gene. Therefore, it is inferred that Lactobacillus salivae XA-1416 has BSH activity.

[0094] Table 1 BSH alignment results of saliva-associated Lactobacillus XA-1416 genome

[0095] Gene ID Hit ID Identity(%) Align length Chr1_530 AEZ06356.1 53.846 325 Plas1_91 525364.3.peg.126 97.531 324

[0096] Experimental Example 3: Effects of saliva combined with Lactobacillus XA-1416 on high-fat diet-induced obesity model mice

[0097] This experimental example provides an experiment on the effect of saliva combined with Lactobacillus XA-1416 on high-fat diet-induced obesity model mice. The experimental process is as follows:

[0098] C57BL6 mice (purchased from Guangdong Weitong Lihua Experimental Animal Technology Co., Ltd.), male, 5 weeks (mouse age at the start of the experiment), 50 mice, after quarantine and adaptive feeding, the feed of the high-fat diet group was switched to a high-fat diet. After switching to a high-fat diet for 1 week, the mice except the normal control group were sorted by weight gain, the last 20% of mice were eliminated, and randomly divided into groups of 8 mice each. A total of 5 groups were designed for this experiment, of which Group 1 was a normal control group (Normal diet group), which was provided with feed AIN93M during the experiment, and Groups 2 to 5 were high-fat diet groups, which were fed with a high-fat diet (Research Diets, D12492) during the 12 weeks of the experiment to create an obesity model. Group 2 is a high-fat diet-induced obese mouse model group (Model group), Group 3 is a positive control group (Olistat group), which is gavaged with positive drug Orlistat, Group 4 is a probiotic treatment group (XA-1416 group), which is gavaged with saliva combined with Lactobacillus XA-1416 bacteria, and Group 5 is a probiotic treatment group (XA-768 group), which is gavaged with animal Bifidobacterium XA-768 bacteria. The administration period is 11 weeks, and the test drugs are orally gavaged. The positive drug dose is 10 mg / kg and the bacterial dose is 2×10 8 CFU / mouse, the drug volume was 200 μL, the drug solvent was PBS buffer containing 0.1% (w / v, g / 100 mL) L-cysteine ​​hydrochloride, and the specific experimental design is shown in Figure 3 and Table 2. During the experiment, the mice were observed weekly and their weight and food intake were recorded. At the end of the experiment, fresh feces of the mice were collected into sterile EP tubes, frozen in liquid nitrogen and stored at -80°C, and the mice were dissected and their tissues were collected.

[0099] Testicular adipose tissue and liver tissue of mice were fixed with 4% (w / v, g / 100mL) paraformaldehyde. The fixed tissue was then embedded, and then sliced ​​using a paraffin slicer with a thickness of 3 microns. After the slices were completed, hematoxylin and eosin (Eosin) staining (H&E staining for short) was performed, and the stained slices needed to be sealed. After the sealing was completed, an optical microscope was used for microscopic examination to observe the morphological structure of the tissue and possible pathological changes.

[0100] Weigh 25 mg of mouse ileum contents and add them to a 2 mL thickened centrifuge tube. Add 2 small steel beads and 400 μL of precooled (precooled to 4 °C) precipitant (acetonitrile: methanol = 7:3, v / v) to each tube, and put it into a tissue grinder for grinding (grinding conditions are set to power 50 Hz, time 300 s) to obtain a grinding liquid sample. Remove the steel beads, let the grinding liquid sample stand in a -20 °C refrigerator for 120 min, centrifuge at 25000g, 4 °C for 15 min, take the supernatant to a 1.5 mL EP tube, and obtain supernatant sample A. Add supernatant sample A, standard sample and Blank to the corresponding wells, 50 μL each, and then add 150 μL of internal standard precipitant. After sealing with aluminum film, shake for 2 min, and then centrifuge at 4000 rpm, 4 °C for 30 min to obtain supernatant sample B. 80 μL of pure water was added to a new V-shaped 96 shallow well plate, and 80 μL of supernatant sample B was taken. After sealing with aluminum film, the sample was shaken for 2 minutes and centrifuged at 4000 rpm and 4°C for 2 minutes to obtain the pretreated sample. The 15 bile acids (cholic acid, lithocholic acid, glycocholic acid, glycochenodeoxycholic acid, glycodeoxycholic acid, glycolithocholic acid, glycoursodeoxycholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, taurolithocholic acid, tauroursodeoxycholic acid, ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid and taurocholic acid) in the pretreated sample were quantitatively detected by high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0101] Mouse feces were collected and DNA was extracted using a DNA extraction kit (purchased from QIAGEN, catalog number 47016). PCR amplified the 16S rRNA gene in the V4 region; the PCR amplification reaction was carried out in a volume of 50 μL, containing 25 μL 2×Premix Taq, 1 μL of each primer (10 μM) and 3 μL DNA (20 ng / μL) template; amplification was performed by thermal cycling: 94°C initialization for 5 minutes; 30 cycles of denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 30 seconds; and finally a final extension at 72°C for 10 minutes. The length and concentration of the PCR product were detected by 1% (w / v, g / 100 mL) agarose gel electrophoresis. The PCR product was purified and then used Ultra TM II DNA Library Prep Kit for Generate sequencing library. Use Qubit 2.0 Fluorometer to evaluate library quality. Finally, sequence the library on Illumina Nova6000 platform to generate 250bp double-end read short sequences. For the data downloaded from the machine, use cutadapt (V3.4) to filter sequences such as adapter, primers, poly-A tails, and then use Dada2 (V1.22) and silva138 databases to annotate the data for species. Use Picrust2 (V2.4.1) to perform functional prediction analysis on the samples. Filter out ASVs (Amplicon sequencing variants) with an average abundance of less than 0.1%. Use multiple Packages on R (v3.6.3) to perform species difference analysis (MaAsLin2), Beta diversity analysis (phyloseq), PERMANOVA analysis (vegan v2.6.4), and box plots of strains and functional abundance (ggplot2). For the calculation of inter-group differences, we first performed log transformation on the data, then used analysis of variance (ANOVA) to analyze the differences among the three groups, and finally used Tukey's HSD test (honestly significant difference) to perform multiple comparisons to see the significance between each group.

[0102] As shown in Tables 3 to 7, starting from the 7th week after administration, the body weight of mice in the XA-1416 group was lower than that in the Model group, and significantly lower than that in the Model group and lower than that in the Olistat group at the end of the experiment, while the body weight of mice in the XA-768 group had no significant difference compared with that in the Model group. This indicates that saliva combined with Lactobacillus XA-1416 is more effective than Olistat in inhibiting the weight gain of mice, thereby treating obesity, while animal Bifidobacterium XA-768 has no such effect.

[0103] As shown in Tables 8 to 11, compared with the Normal diet group, the levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and alanine aminotransferase (ALT) in the serum of the Model group mice increased significantly, and the level of high-density lipoprotein cholesterol (HDL-C) decreased significantly. As shown in Table 7, compared with the Model group, the levels of total cholesterol (TC) in the serum of the Olistat group and the XA-1416 group mice decreased, while the level of total cholesterol (TC) in the serum of the XA-1416 group mice was significantly lower than that of the Model group. This shows that saliva combined with Lactobacillus XA-1416 can effectively reduce the level of total cholesterol (TC) in the serum of high-fat diet-induced obese model mice, thereby treating hyperlipidemia (i.e., hyperlipidemia). In recent years, low-density lipoprotein cholesterol (LDL-C) has replaced total cholesterol (TC) in clinical practice as a key lipid measurement indicator for predicting cardiovascular risk. As shown in Tables 9 and 10, compared with the Model group, the serum low-density lipoprotein cholesterol (LDL-C) level of mice in the XA-1416 group was significantly reduced, and this effect significantly exceeded the level of the positive control drug Olistat group. At the same time, the high-density lipoprotein cholesterol (HDL-C) level of mice in the XA-1416 group did not show a significant decrease compared with the model group. This shows that saliva combined with Lactobacillus XA-1416 can effectively reduce the level of low-density lipoprotein cholesterol (LDL-C) in the serum of mice with high-fat diet-induced obesity model, thereby reducing the risk of cardiovascular disease. In addition, alanine aminotransferase (ALT) is a biochemical indicator widely used in clinical practice to assess the health of the liver. As shown in Table 10, compared with the Model group, the serum alanine aminotransferase (ALT) level of mice in the XA-1416 group was significantly reduced, and the degree of reduction was similar to that of the Olistat group. This indicates that saliva combined with Lactobacillus XA-1416 can effectively reduce the level of alanine aminotransferase (ALT) in the serum of mice with obesity induced by a high-fat diet, thereby treating non-alcoholic fatty liver disease.

[0104] Depend on Figure 4-5 It can be seen that compared with the Normal diet group, the area of ​​fat cells in the epididymal adipose tissue (fat tissue attached to the epididymis) of the Model group mice was larger and the cell wall was thinner, while the area of ​​fat cells in the epididymal adipose tissue of the Olistat group and the XA-1416 group was significantly smaller than that of the Model group mice (the average area of ​​epididymal fat cells in the Normal diet group mice was 0.002394mm 2 The average area of ​​epididymal adipocytes in the Model group was 0.006445 mm 2 The average area of ​​epididymal adipocytes in mice in the XA-1416 group was 0.005129 mm 2 ).Depend on Figure 6It can be seen that compared with the Normal diet group, the number of fat cells in the liver tissue of the Model group mice increased significantly, and the white vacuoles became larger, while the number of fat cells in the liver tissue of the Olistat group and the XA-1416 group mice decreased compared with the Model group, and the liver fat infiltration was significantly smaller than that of the Model group. This shows that saliva combined with Lactobacillus XA-1416 can effectively reduce the average area of ​​fat cells in the tissues (testicular fat tissue and liver tissue) of the high-fat diet-induced obese model mice, thereby treating obesity and non-alcoholic fatty liver.

[0105] The intestinal microbiota can have an important impact on the host's metabolic homeostasis by changing bile acid levels. On the one hand, the intestinal microbiota uses bile salt hydrolase (BSH) to deconjugate conjugated bile acids into free bile acids, which increases the excretion of lipids in the feces and reduces the digestion and absorption efficiency of lipids in the intestine, which has a positive effect on lipid metabolism and energy balance (see reference: Yu Wang et al. 2022); on the other hand, the intestinal microbiota plays a key role in the occurrence and development of obesity through the intestinal flora-bile acid-farnesoid X receptor (FXR) signaling pathway. FXR is an important transcription factor mainly present in the liver and ileum. Inhibition of intestinal FXR transcriptional activity can regulate diet-induced lipid and cholesterol metabolic disorders. Studies have shown that functional defects in FXR help reduce the body weight and fat mass of diet-induced obese mice, and the absence of intestinal FXR can effectively prevent weight gain. In addition, FXR antagonists can inhibit lipogenesis by inhibiting the intestinal FXR signaling pathway (see reference: Fei Li et al. 2013). In addition, studies have confirmed that FXR antagonists have the potential to lower blood lipids, and conjugated bile acids in the ileum, such as UDCA (ursodeoxycholic acid), GUDCA (glycoursodeoxycholic acid), and TUDCA (tauroursodeoxycholic acid), as FXR antagonists, can inhibit the intestinal FXR-FGF15 / 19-FGFR4 pathway, activate bile acid synthase in the liver, and promote the conversion of cholesterol to bile acid, thereby lowering cholesterol levels in the blood (see reference: Fengjie Huang et al. 2019), which helps to improve obesity-related metabolic dysfunction in mice (see reference: Lulu Sun et al. 2018). In view of this, strategies that promote the conversion of conjugated bile acids to free bile acids, or target intestinal FXR, may provide safer and more effective treatments for the intervention and treatment of obesity and obesity-related diseases. Figure 7It can be seen that the ratio of free bile acid to conjugated bile acid in the ileum contents of mice in the XA-1416 group was significantly higher than that in the Model group (the ratio of free bile acid to conjugated bile acid in the ileum contents of mice in the Model group was 0.5967, and the ratio of free bile acid to conjugated bile acid in the ileum contents of mice in the XA-1416 group was 1.387), indicating that XA-1416 has a significant regulatory effect on bile acid metabolism, converting conjugated bile acid into free bile acid. Figure 8 It can be seen that compared with the Model group, the levels of UDCA (ursodeoxycholic acid), GUDCA (glycoursodeoxycholic acid) and TUDCA (tauroursodeoxycholic acid) in the XA-1416 group showed an upward trend, especially the levels of UDCA (ursodeoxycholic acid) and GUDCA (glycoursodeoxycholic acid) were significantly upregulated (the average UDCA level in the ileum contents of mice in the Model group was 42.57 ng / mg, and the average UDCA level in the ileum contents of mice in the XA-1416 group was 121.3 ng / mg). / mg; the average GUDCA level in the ileum contents of mice in the Model group was 1.449ng / mg, and the average GUDCA level in the ileum contents of mice in the XA-1416 group was 3.324ng / mg; the average TUDCA level in the ileum contents of mice in the Model group was 373.4ng / mg, and the average TUDCA level in the ileum contents of mice in the XA-1416 group was 495.7ng / mg), indicating that XA-1416 has a promoting effect on the level of intestinal FXR antagonists. This result confirms that saliva combined with Lactobacillus XA-1416 can simultaneously treat obesity and hyperlipidemia (i.e., hyperlipidemia) through the bile acid metabolic pathway and the FXR signaling pathway.

[0106] Based on the composition of intestinal flora, beta diversity analysis was performed, and PCoA graphs were drawn to observe the distribution between groups. Figure 9-10 It can be seen that there are significant differences in species composition among the Normal diet group, Model group and XA-1416 group at the ASV or Genus level (PERMANOVA P = 0.001). Figure 11-12 It can be seen that there are also significant differences between the Model group and the XA-1416 group (PERMANOVA P = 0.016), indicating that a high-fat diet has a significant effect on the intestinal microbial composition of mice, and XA-1416 can significantly change the intestinal flora disorder caused by a high-fat diet. Further differential analysis found 3 related species with significant differences in enrichment in the intestines of obese individuals ( Fig.13 ), 2 significantly changed probiotics ( Fig.14 ) and 4 functional pathways with significant differences ( Fig.15 ).Depend on Figure 13-14As shown, compared with the Normal diet group, the Model group significantly increased the abundance of bacteria under the Lachnospiraceae family of obesity-related intestinal bacteria, as well as Acetatifactor and Lachnoclostridium (see literature: Ruixin Liu et al. 2017, Vanessa Palmas et al. 2021, Li Sun et al. 2021, Guoqiang Yao et al. 2021 and Jiali chen et al. 2021), while the XA-1416 group significantly reduced the abundance of the above bacteria compared with the Model group, making the abundance of these obesity-related bacteria close to the level of the Normal diet group. Fig.15 As shown, in the Model group, the pentose phosphate pathway related to energy metabolism and the 4-deoxy-L-threo-hex-4-enopyranuronate degradation pathway and the Mevalonate metabolic pathway related to lipid metabolism decreased significantly, while in the XA-1416 group, they showed a trend of callback. In the intestinal microbiota of obese people, the increase in the ratio of Firmicutes / Bacteroidetes is an important feature (see the literature: Yong Fan et al. 2020). Fig.16 It can be seen that the ratio of Firmicutes / Bacteroidetes in the Model group increased relative to the Normal diet group, while when the XA-1416 group was compared with the Model group, the ratio fell back and was closer to the normal level. These results show that saliva combined with Lactobacillus XA-1416 can significantly reduce the abundance of bacteria enriched in the intestines in obese individuals (i.e., bacteria associated with obesity), significantly increase the abundance of beneficial bacteria in the intestines, significantly improve the proportion of bacteria enriched in the intestines in obese individuals, and significantly improve the functional pathways of the intestinal microbiota, thereby treating intestinal flora disorders caused by a high-fat diet.

[0107] Table 2 Experimental groups and drug administration design

[0108]

[0109]

[0110] Table 3 Weight of Normal diet group (g)

[0111]

[0112] Table 4 Body weight of Model group (g)

[0113]

[0114]

[0115] Table 5 Body weight of Olistat group (g)

[0116]

[0117] Table 6 Body weight of XA-1416 group (g)

[0118]

[0119]

[0120] Table 7 Body weight of XA-768 group (g)

[0121]

[0122] Table 8 Serum cholesterol level (mmol / L)

[0123]

[0124] Table 9 Serum low-density lipoprotein levels (mmol / L)

[0125]

[0126] Table 10 Serum high-density lipoprotein levels (mmol / L)

[0127]

[0128] Table 11 Serum alanine aminotransferase levels (U / L)

[0129]

[0130] Experimental Example 4: Regulation of intestinal flora by saliva combined with Lactobacillus XA-1416

[0131] According to the criteria for regulating intestinal flora in the "Technical Guidelines for Functional Testing and Evaluation of Health Foods (2023 Edition)", a microbiome analysis was performed on the feces of mice in Experimental Example 3 to evaluate the effects of saliva combined with Lactobacillus XA-1416 on the abundance changes of probiotic Bifidobacterium and Lactobacillus and potentially harmful bacteria Clostridium perfringens, Enterococcus and Enterobacter in the intestine. The experimental methods and analysis methods are detailed in Experimental Example 3.

[0132] Depend on Fig.14It can be seen that compared with the Normal diet group, the abundance of Bifidobacterium and Lactobacillus in the Model group decreased significantly. However, after the treatment of saliva combined with Lactobacillus XA-1416, the abundance of Bifidobacterium and Lactobacillus increased significantly compared with the Model group, while Clostridium perfringens, Enterococcus and Enterobacter were not detected before and after the treatment of saliva combined with Lactobacillus XA-1416. It can be seen that saliva combined with Lactobacillus XA-1416 can regulate the intestinal flora.

[0133] Experimental Example 5: Effects of saliva combined with Lactobacillus XA-1416 on lipid accumulation in Hep G2 hepatocytes

[0134] This experimental example provides an experiment on the effect of saliva combined with Lactobacillus XA-1416 on lipid accumulation in Hep G2 liver cells. The experimental process is as follows:

[0135] Hep G2 cells cultured in T75 flasks (purchased from the Cell Bank of the Chinese Academy of Sciences) were centrifuged at 250×g for 5 minutes, the supernatant was discarded, 5 mL of PBS buffer preheated to 37°C was pipetted into the culture flask, the cells were gently rinsed, and then 3 mL of 0.25% trypsin digestion solution preheated to 37°C was added to the culture flask to cover the cells and the culture flask was transferred to the cell culture incubator for digestion. After 2 minutes of digestion, gaps appeared between the cells under the microscope, and the digestion was terminated when the cells became round. Take 6 mL of complete culture medium (purchased from Gibco) and transfer it to the culture flask. Use a pipette to blow the digested cells evenly and transfer them to a 15 mL centrifuge tube and centrifuge at 250×g for 5 minutes. The supernatant was discarded to obtain a cell pellet. Resuspend the cell pellet with 1 mL of complete culture medium to obtain cell suspension A. Take 10 μL of cell suspension A and mix it with 10 μL of trypan blue (purchased from Biyuntian), and measure the cells twice in parallel for cell counting. According to the counting results, Hep G2 cells were cultured at a cell concentration of 1.5×10 5 The cells were resuspended in fresh complete medium preheated to 37°C to obtain cell suspension B. Cell suspension B was inoculated into a 96-well black multi-well plate at an inoculation volume of 100 μL per well, and the 96-well plate was transferred to a cell culture incubator at 37°C and 5% (v / v) CO2 for culture.

[0136] After 24 h of culture, the 96-well plate was centrifuged at 250 × g for 5 minutes, the supernatant was discarded, and the wells of the 96-well plate were grouped and loaded with samples. 200 μL of complete culture medium was added to the wells of the negative control group, 200 μL of complete culture medium containing 0.4 mM free fatty acids (the final concentration ratio of Palmitic Acid to Oleic Acid was 3:1) was added to the wells of the model group, 50 μM caffeine and 0.4 mM free fatty acids (the final concentration ratio of Palmitic Acid to Oleic Acid was 3:1) were added to the wells of the positive control group, and 5% (5% here refers to 5% of the total volume of the complete culture medium) XA-1416 culture supernatant and 0.4 mM free fatty acids (Palmitic Acid and Oleic Acid) were added to the wells of the XA-1416 intervention group. To the wells of the probiotic medium control group, 200 μL of complete medium containing 5% (5% here means 5% of the total volume of the complete medium) mGAM medium (purchased from Qingdao Haibo Biological) and 0.4 mM free fatty acid (the final concentration ratio of Palmitic Acid to Oleic Acid is 3:1) was added, and 200 μL of complete medium containing 5% (5% here means 5% of the total volume of the complete medium) mGAM medium (purchased from Qingdao Haibo Biological) and 0.4 mM free fatty acid (the final concentration ratio of Palmitic Acid to Oleic Acid is 3:1) was added, see Table 11 for details.

[0137] After the sample addition is completed, the 96-well plate is centrifuged at 250×g for 3 minutes, and then the cells are placed in a 37°C, 5% (v / v) cell culture incubator for 36 hours. After 36 hours of culture, wash once with PBS buffer, discard the supernatant, add 100μL of lipid droplet staining solution (purchased from Biyuntian, product number C2051M) to each well of the 96-well plate, and incubate in the dark at room temperature (25°C) for 20 minutes. After incubation, wash twice with PBS buffer. After Nile red staining, the fluorescence intensity of the cells was detected at Ex / Em=485 / 535nm, and data statistical analysis was performed to compare the effects of different interacting components on cellular lipid accumulation. The test results are shown in Fig.17 .

[0138] like Fig.17 As shown, compared with the negative control group, the fluorescence signal of the model group increased significantly, indicating that after the inducer treatment, the cellular lipid accumulation in the model group increased. The fluorescence intensity of the positive drug 50μM caffeine decreased significantly compared with the model group. When 5% mGAM had no effect on cellular lipid accumulation, 5% XA-1416 culture supernatant showed a significant effect in inhibiting cellular lipid accumulation compared with the model group (the average fluorescence intensity of the negative control group was 0.08903, the average fluorescence intensity of the model group was 0.09247, and the average fluorescence intensity of the XA-1416 group was 0.08822). In summary, saliva combined with Lactobacillus XA-1416 can inhibit lipid accumulation in liver cells.

[0139] Table 11 Grouping and cell treatment methods

[0140]

[0141]

[0142] Experimental Example 5: Effect of saliva combined with Lactobacillus XA-1416 on differentiation of preadipocytes into adipocytes in 3T3-L1 model

[0143] This experimental example provides an experiment on the effect of saliva combined with Lactobacillus XA-1416 on the differentiation of preadipocytes into adipocytes in the 3T3-L1 model. The experimental process is as follows:

[0144] The 3T3-L1 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) cultured in a T75 flask were centrifuged at 250×g for 5 minutes, the supernatant was discarded, 5 mL of PBS buffer preheated to 37°C was pipetted into the culture flask, the cells were gently rinsed, and then 3 mL of 0.25% trypsin digestion solution preheated to 37°C was added to the culture flask to cover the cells and the culture flask was transferred to the cell culture incubator for digestion. After 2 minutes of digestion, gaps appeared between the cells under the microscope, and the digestion was terminated after the cells became round. Take 6 mL of complete culture medium (purchased from Gibco) and transfer it to the culture flask. Use a pipette to blow the digested cells evenly and transfer them to a 15 mL centrifuge tube and centrifuge at 250×g for 5 minutes. Discard the supernatant to obtain a cell pellet. Resuspend the cell pellet with 1 mL of complete culture medium to obtain cell suspension A. Take 10 μL of cell suspension A and mix it with 10 μL of trypan blue (purchased from Biyuntian), and measure the cells twice in parallel for cell counting. According to the counting results, 3T3-L1 cells were plated at a cell concentration of 3×10 4 The cells were resuspended in fresh complete medium preheated to 37°C to obtain cell suspension B. Cell suspension B was inoculated into a 96-well black multi-well plate at an inoculation volume of 100 μL per well, and the 96-well plate was transferred to a cell culture incubator at 37°C and 5% (v / v) CO2 for culture.

[0145] After 24 h of culture, the 96-well plate was centrifuged at 250×g for 5 minutes, the supernatant was discarded, and the wells of the 96-well plate were grouped and loaded with samples. 100 μL of complete medium was added to the wells of the negative control group, 200 μL of complete medium containing 10 μg / mL insulin was added to the wells of the model group, 200 μL of complete medium containing 1 μM retinoic acid (RA) and 10 μg / mL insulin was added to the wells of the positive control group, 5% (here 5% means 5% of the total volume of the complete medium) of XA-1416 culture supernatant and 10 μg / mL insulin were added to the wells of the XA-1416 intervention group, and 5% (here 5% means 5% of the total volume of the complete medium) of mGAM medium (purchased from Qingdao Haibo Biological) and 200 μL of complete medium containing 10 μg / mL insulin were added to the wells of the probiotic medium control group, as shown in Table 12 for details.

[0146] After the sample addition is completed, the 96-well plate is centrifuged at 250×g for 3 minutes, and then the cells are placed in a 37°C, 5% (v / v) cell culture incubator for 36 hours. After 36 hours of culture, wash once with PBS buffer, discard the supernatant, add 100μL of lipid droplet staining solution (purchased from Biyuntian, product number C2051M) to each well of the 96-well plate, and incubate in the dark at room temperature (25°C) for 20 minutes. After incubation, wash twice with PBS buffer. After Nile red staining, the fluorescence intensity of the cells was detected at Ex / Em=485 / 535nm, and data statistical analysis was performed to compare the effects of different interacting components on the differentiation of preadipocytes into adipocytes. The test results are shown in Fig.18 .

[0147] like Fig.18 As shown, compared with the negative control group, after the inducer treatment, the differentiation of preadipocytes into adipocytes in the model group increased significantly. Compared with the model group, the positive control group (1 μM retinoic acid) showed inhibition of the differentiation of preadipocytes into adipocytes, but there was no significant difference. When 5% mGAM had no effect on the differentiation of preadipocytes into adipocytes, 5% XA-1416 culture supernatant significantly inhibited the differentiation of preadipocytes into adipocytes compared with the model group, and the inhibitory effect was better than the positive control drug (the average fluorescence intensity of the negative control group was 0.1506, the average fluorescence intensity of the model group was 0.1746, and the average fluorescence intensity of the XA-1416 group was 0.1313). In summary, saliva combined with Lactobacillus XA-1416 can inhibit the differentiation of preadipocytes into adipocytes.

[0148] Table 12 Grouping and cell treatment methods

[0149] Group Cell treatment methods Negative control group 100 μL cell culture medium Model Group 10 μg / mL insulin Positive control group 1μM retinoic acid (RA) + 10μg / mL insulin XA-1416 intervention group 5% XA-1416 culture supernatant + 10μg / mL insulin Probiotic culture medium control group 5% mGAM + 10μg / mL insulin

[0150] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Saliva combined with Lactobacillus ( Ligilactobacillus Salivarius ) for use in the preparation of a medicament for preventing and / or treating obesity-related diseases, characterized in that: The saliva-combined lactobacillus is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number of CGMCC No.40307; the obesity-related disease is non-alcoholic fatty liver disease.

2. The use according to claim 1, characterized in that Prevention and / or treatment of non-alcoholic fatty liver disease by reducing lipid accumulation in liver cells, lowering serum alanine aminotransferase levels and / or reducing the average area of ​​fat cells in liver tissue.

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