CN115927045B

CN115927045B - Lactobacillus salivarius 069 with cholesterol reducing and liver injury relieving functions caused by hyperlipidemia and application thereof

Info

Publication number: CN115927045B
Application number: CN202210828189.8A
Authority: CN
Inventors: 吴清平; 杨玲双; 代京莎; 李滢; 谢新强; 薛亮; 陈谋通; 王涓; 丁郁; 叶青华; 吴诗; 古其会; 张菊梅; 庞锐
Original assignee: Guangdong Kehuan Biotechnology Co ltd; Institute of Microbiology of Guangdong Academy of Sciences; Guangdong Huankai Biotechnology Co Ltd
Current assignee: Guangdong Kehuan Biotechnology Co ltd; Institute of Microbiology of Guangdong Academy of Sciences; Guangdong Huankai Biotechnology Co Ltd
Priority date: 2022-07-13
Filing date: 2022-07-13
Publication date: 2023-10-20
Anticipated expiration: 2042-07-13
Also published as: CN115927045A

Abstract

The invention discloses lactobacillus salivarius 069 with the functions of reducing cholesterol and relieving liver injury caused by hyperlipidemia and application thereof. Lactobacillus salivarius 069 can reduce 27% cholesterol concentration in vitro, reduce total cholesterol, low density cholesterol and triglyceride in vivo, reduce accumulation of TC in liver, promote discharge of TC and bile acid, and is superior to low dosage lovastatin in relieving liver injury. Lactobacillus salivarius 069 affects disease processes mainly by regulating histidine metabolism, steroid hormone biosynthesis, glycine, serine and threonine metabolism, tryptophan metabolism, primary bile acid biosynthesis, and unsaturated fatty acid biosynthesis, and its material basis is mainly indoleacetic acid and chenodeoxycholic acid. In vitro probiotics and safety characteristic experiments show that the strain has the characteristics of resisting gastrointestinal fluid digestion, antagonizing 6 food-borne pathogenic bacteria, multiple drug-resistant strains, non-hemolysis, antibiotic sensitivity and the like.

Description

Lactobacillus salivarius 069 with cholesterol reducing and liver injury relieving functions caused by hyperlipidemia and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus salivarius 069 with functions of reducing cholesterol and relieving liver injury caused by hyperlipidemia and application thereof.

Background

The American heart society in 2002 pointed out that cardiovascular disease (CVD) is the most important cause of death in developed countries, closely related to hypercholesterolemia, and about 1760 ten thousand people worldwide die from cardiovascular disease in 2016, an increase of 14.5% over 2006. The world health organization predicts that by 2030, cardiovascular disease is still the leading cause of threatening human death, affecting about 2360 tens of thousands of people worldwide. The risk of heart attacks in hypercholesterolemic patients is 3 times higher than in normolipidic patients.

Clinically used statins reduce serum cholesterol levels by inhibiting hydroxymethylglutaryl-CoA (HMG-CoA) reductase activity to reduce endogenous synthesis of cholesterol. The clinical medicine ezetimibe inhibits NPC1L1 to reduce the absorption and transportation of cholesterol; in addition, some traditional Chinese medicines have certain effect on regulating blood fat. Among the drugs commonly used at present, statin drugs have better curative effects in clinic. Statin drugs (statins) are HMG-CoA reductase inhibitors that block intracellular hydroxymethylvalerate metabolic pathways by competitively inhibiting endogenous cholesterol synthesis rate-limiting enzyme (HMG-CoA) reductase, resulting in decreased intracellular cholesterol synthesis, thereby feedback stimulating increased cell membrane surface (primarily hepatocytes) low density lipoprotein (Low Density Lipoprotein, LDL) receptor numbers and activity, and decreased serum cholesterol levels. In 2010, a meta analysis of the data of 129526 participants from 26 trials showed a significant 15% decrease in the incidence of cardiovascular disease events with statin treatment compared to standard treatment. The total mortality was significantly reduced by 10% for each 1mmol/L LDL-C reduction. However, the manufacturing of statin drugs at present mainly depends on chemical synthesis, and the synthesized drugs have adverse reactions, and the whole body is uncomfortable and fever; abdominal discomfort, belching, flatulence, hepatitis, cholestasis; skeletal muscle pain, muscle fatigue, neck pain, joint swelling, and the like.

Functional foods that are safe and effective, such as probiotics that have a healthy function, are increasingly being considered. At present, screening of functional probiotics having cholesterol-lowering effect in human body is a research hotspot.

Lactic acid bacteria are a class of food grade microorganisms with a long recognized safety (GRAS) history of safe consumption. As early as 1963, the first study of sambour tribe in africa on consumption of large amounts of milk fermented by wild lactic acid bacteria and/or bifidobacteria found that serum cholesterol was reduced. To elucidate the specific mechanism of yogurt cholesterol reduction, experiments were designed by parallel, double-blind, placebo-controlled, randomized, etc. The study found that 2x10 was taken twice daily ⁹ The CFU lactobacillus plantarum ECGC 13110402 has significantly reduced Total Cholesterol (TC), low density lipoprotein (LDL-C) and Triglyceride (TG) in the experimental group population. Yet another study showed that probiotic formulations containing lactic acid bacteria and bifidobacteria could reduce the LDL-C content but not increase the TC and HDL-C content in patients with metabolic syndrome. Another study shows that the liver cholesterol concentration of the mixed bacteria and lactobacillus acidophilus is reduced by 23-57%, and the cholesterol concentration of the mixed bacteria is lower than that of the single bacteria. In recent years, more and more researchers have studied probiotic cholesterol lowering. Zhang Fen the separation of enterococcus faecium from infant faeces (Enterococcus faecium) significantly reduces TC, TG and LDL-C in rat serum. So far, a large number of human and animal experiments have fully demonstrated that some lactic acid bacteria have the ability to reduce cholesterol. At present, the mechanism proposed by probiotics for reducing cholesterol at home and abroad mainly comprises the following steps: (1) Probiotics are capable of adsorbing cholesterol into the cell membrane or cytoplasm; (2) the probiotic adsorbs cholesterol to the cell surface; (3) Co-precipitation of cholesterol and free bile salts occurs in an acidic environment; (4) The bile salts are hydrolyzed into free bile salts by the bile salt hydrolase of the probiotics, and the free bile salts have lower solubility and are not easy to be recovered by intestinal tracts; (5) Cholic acid is adhered to the cell surface by the capsular extracellular polysaccharide of the probiotic; (6) Probiotics ferment intestinal indigestible carbohydrates to propionic acid, which is able to inhibit liver cholesterol biosynthesis, resulting in a decrease of serum cholesterol levels; (7) Probiotics reduce the absorption of cholesterol by small intestine cells by down-regulating NPC1L1 protein gene expression; (8) Probiotics reduce LDL-C levels by down-regulating PCSK9 gene; (9) the probiotic inhibits the formation of cholesterol emulsion micelles; (10) Probiotics regulate lipid levels by modulating intestinal microbial composition and producing metabolites. The probiotic bacteria for reducing cholesterol studied at home and abroad are mainly concentrated on several factors: (1) strain specificity and growth status; (2) the viability impact of bile salt hydrolase; (3) type and concentration of bile salts; (4) preparing in vitro screening culture; (5) pH value, etc. For example, wei Yunlu et al studied bifidobacterium animalis LPL-RH, bifidobacterium longum TTF and lactobacillus plantarum LPL-1 with in vitro cholesterol removal rates of 23.80%, 24.50% and 20.90%, respectively. 5 probiotics studied by He Yongyan et al all removed more than 27.00% cholesterol from the culture medium, 35.90% enterococcus faecalis L14-3 and 35.90% lactococcus lactis L14-3. In the study by BELVISO et al, 6 strains of Lactobacillus plantarum and 2 strains of Lactobacillus paracasei were found to have no reduction in cholesterol in the medium at all. The effect of Lin et al on human serum lipoprotein levels was performed in a double-blind experiment with Lactobacillus preparation containing Lactobacillus acidophilus and Lactobacillus bulgaricus, and in a placebo-controlled study. 354 volunteers participated, each taking 4 times per day including 2 kinds of lactobacillus (about 2×10) ⁶ CFU/tablet) and placebo. The results of the experiment after 15 weeks showed that there was no significant difference in the low density lipoprotein levels of the subjects taking the placebo and lactic acid bacteria preparations, although both strains showed cholesterol lowering properties in the in vitro experiments. Many studies have contradictions and are closely related to the influencing factors, screening methods and evaluationThe index is difficult to form a system. Probiotics are widely applied to foods, beverages, medicine and the like in Japan, french, russian, germany and the like, and the probiotics are deeply researched, so that the probiotics are added into fermented milk to play a certain health care role. Probiotics have little side effects compared to drugs, and alleviation of liver damage caused by hyperlipidemia by probiotics is a hotspot of research.

In recent years, studies on cholesterol reduction by lactic acid bacteria have been made, but studies on comprehensive evaluation of probiotic properties, safety evaluation, in vivo function evaluation, and investigation on alleviation of liver injury have been relatively few. At present, few strains are developed and applied to lactobacillus strain resources in China, so that functional lactobacillus is screened, the functional lactobacillus can regulate blood fat of a human body, and the lactobacillus with high safety is further developed.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, and provides the salivary lactobacillus 069 with the functions of reducing cholesterol and relieving liver injury caused by hyperlipidemia and the application thereof.

Lactobacillus salivarius (Lactobacillus salivarius) 069 of the present invention was deposited at the collection of microorganisms and cell cultures (GDMCC) in guangdong province at 1, 6, 2022, accession number: guangzhou city first middle road 100 # college 59 # building 5, post code: 510070 with accession number GDMCC No:62501.

lactobacillus salivarius 069 can reduce 27% cholesterol (TC) concentration in vitro, has effects of reducing total cholesterol, low density cholesterol (LDL-C) and Triglyceride (TG) in vivo, and can reduce accumulation of TC in liver, promote discharge of TC and Bile Acid (TBA), and is superior to low dose (0.1 mg/100 g/day) lovastatin in relieving liver injury effect. Non-targeted metabonomics results indicate that lactobacillus salivarius 069 affects disease processes mainly by modulating histidine metabolism, steroid hormone biosynthesis, glycine, serine and threonine metabolism, tryptophan metabolism, primary bile acid biosynthesis, unsaturated fatty acid biosynthesis, and the substance basis is mainly indoleacetic acid (heteoauxin) and chenodeoxycholic acid (CDCA). In-vitro probiotics and safety characteristic experiments show that the strain has the characteristics of resisting gastrointestinal liquid digestion, antagonizing 6 food-borne pathogenic bacteria, multiple drug-resistant strains, preventing hemolysis, resisting antibiotic sensitivity and the like, and has great application potential and value in preventing hypercholesterolemia, relieving liver injury, fatty liver and prevention and control of pathogenic bacteria.

The second object of the invention is to provide the application of the lactobacillus salivarius 069 in preparing products for reducing cholesterol, relieving liver injury function caused by hyperlipidemia, relieving intestinal metabolite disorder caused by hypercholesterolemia, regulating intestinal flora caused by hyperlipidemia, promoting biosynthesis of primary bile acid and promoting excretion of cholic acid.

The lactobacillus salivarius 069 can be used as a medicament to reduce cholesterol and relieve liver injury caused by hyperlipidemia. For example, lactobacillus salivarius 069 can be made into probiotic powder, tableted fructose, solid beverage or various medicines.

The third object of the invention is to provide the application of the lactobacillus salivarius 069 in the preparation of antibacterial drugs.

Preferably, the composition is an application of anti-escherichia coli, salmonella typhimurium, cronobacter sakazakii, staphylococcus aureus, listeria monocytogenes and bacillus cereus, and the listeria monocytogenes drug-resistant strain, indiana salmonella drug-resistant strain, methicillin-resistant staphylococcus aureus or indiana salmonella drug.

A fourth object of the present invention is to provide a product for lowering cholesterol, alleviating liver injury function caused by hyperlipidemia, alleviating intestinal metabolite disorder caused by hypercholesterolemia, regulating intestinal flora caused by hyperlipidemia, promoting biosynthesis of primary bile acid, promoting bile acid excretion and antibacterial, which contains the above lactobacillus salivarius 069.

The specific sequences SEQ ID NO. 2 and SEQ ID NO. 3 of the lactobacillus salivarius are obtained by a bioinformatics analysis method, and the sequences are subjected to specific verification to determine that the specific sequences are specific molecular targets.

It is therefore a fifth object of the present invention to provide a specific molecular target for detection of lactobacillus salivarius 069, said molecular target being: any one of the nucleotide sequences shown as SEQ ID NO. 2 and SEQ ID NO. 3.

A sixth object of the present invention is to provide a primer set for detecting Lactobacillus salivarius 069, comprising an upstream primer and a downstream primer amplified against the nucleotide sequence shown as SEQ ID NO. 2 and an upstream primer and a downstream primer amplified against the nucleotide sequence shown as SEQ ID NO. 3.

Preferably, the upstream primer and the downstream primer for amplification of the nucleotide sequence shown in SEQ ID NO. 2 are: 5'-GCGTGAGACGAGCACTTAAA-3' and 5'-GCAGTTCGCCAGTAACCCTT-3'.

Preferably, the upstream primer and the downstream primer for amplification of the nucleotide sequence shown in SEQ ID NO. 3 are: 5'-AGAGCGTCATGTTACGGCAA-3' and 5'-TCAGTTTTAGCACCCCAACGA-3'.

A seventh object of the present invention is to provide a method for detecting lactobacillus salivarius 069 for the diagnosis and treatment of non-disease, comprising the steps of:

Extracting genome DNA of the bacteria to be detected, and then amplifying by using the upstream primer and the downstream primer for amplifying the nucleotide sequence shown as SEQ ID NO. 2 and the upstream primer and the downstream primer for amplifying the nucleotide sequence shown as SEQ ID NO. 3, wherein if 292bp and 171bp sequences can be amplified simultaneously, the lactobacillus salivarius 069 is obtained, otherwise, the lactobacillus salivarius 069 is not obtained.

Preferably, the PCR amplification system comprises 2 XPCR Mix, template DNA, primer set and sterilized double distilled water.

Preferably, the PCR amplification system is 2 XPCR Mix 12.5. Mu.L, template DNA, 10. Mu. Mol/L primer 1. Mu.L each, and sterilized double distilled water make up to 25. Mu.L.

Preferably, the PCR amplification is performed by the following amplification procedures: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s; annealing at 68-69 ℃ for 30s; extending at 72 ℃ for 30s; carrying out denaturation, annealing and extension for 30 cycles; finally, the extension is carried out for 10min at 72 ℃.

The invention obtains 1 strain of lactobacillus which has the function of reducing cholesterol and relieving liver injury caused by hyperlipidemia through screening, and is named as lactobacillus salivarius (Lactobacillus salivarius) 069. Experiments prove that the bacteria has the capacity of reducing cholesterol in and out, has better liver injury function than low-dose lovastatin caused by hyperlipidemia in vivo anti-inflammatory and alleviation, has stronger gastrointestinal digestion resistance, has no hemolysis and is sensitive to antibiotics; the strain was identified as lactobacillus salivarius (Lactobacillus salivarius) 069 by 16S rDNA sequence alignment analysis.

The invention provides a culture or processed product thereof comprising any one of the lactobacillus salivarius 069.

The invention provides a pharmaceutical composition comprising the lactobacillus salivarius 069, the culture or the processed product thereof.

The invention has the beneficial effects that:

the lactic acid bacteria provided by the invention are from indigenous strains of longevity village in the world of China. Can reduce cholesterol in vitro; the strain also has better gastrointestinal digestion resistance, inhibits 6 food-borne pathogenic bacteria and multiple drug-resistant strains, has better safety, has the effect of reducing blood fat in vivo, can reduce accumulation of TC in liver, and promotes discharge of TC and TBA; the effect of relieving liver injury is better than the effect of low-dose lovastatin. The probiotic bacteria have good gastric juice tolerance and safety, can regulate intestinal flora and metabolic pathways, and have great application potential and value in the aspects of developing prevention and control of hypercholesterolemia and pathogenic bacteria, regulating intestinal homeostasis, fatty liver and the like.

Drawings

Fig. 1: levels of Total Cholesterol (TC), triglycerides (TG) and Total Bile Acids (TBA) in the feces and liver. (a) liver TC levels, (B) TG levels in the liver, (C) liver TBA levels, (D) TC levels in stool, (E) triglyceride levels in stool, and (F) TBA levels in stool. Data are expressed as the average of three replicatesAnd (5) accuracy difference. P values were determined using one-way ANOVA and the post hoc analysis tukey test, n=6. The differences were statistically significant in the model group compared to the control group or lactobacillus salivarius 069 group ^** P<0.01, ^**** P<0.0001. Control group (Control group), model group (HC group), lactobacillus salivarius 069 group (L.salivarius group), lovastatin group (Lovastatin group).

Fig. 2: lactobacillus salivarius 069 can improve liver injury in rats with hyperlipidemia. (A) serum ALT level, (B) serum AST level, (C) liver TNF-alpha level, (D) liver IL-1 beta level, (E) liver slice H&E, dyeing; (a) control group, (b) HC group, (c) Lactobacillus salivarius 069 group, and (d) lovastatin group. (F) steatosis and hepatocyte inflammation scores. P values were determined using one-way ANOVA and the post hoc analysis tukey test, n=6. The model group has obvious difference from the control group and the lactobacillus salivarius 069 group ^* P<0.05, ^** P<0.01, ^*** P<0.001, ^**** P<0.0001. Control group (Control group), model group (HC group), lactobacillus salivarius 069 group (L.salivarius group), lovastatin group (Lovastatin group).

Fig. 3: lactobacillus salivarius 069 has effects of improving fatty inflammation and fatty hypertrophy of epididymis of hypercholesterolemia rat. (A) epididymal fat MCP-1 level, (B) epididymal fat IL-1 beta level, (C) number of adipocytes in the same field, (D) diameter of adipocytes in the same field, (E) epididymal fat section H&E, dyeing; (a) control group, (b) HC group, (c) Lactobacillus salivarius 069, (d) lovastatin group. P values were determined using one-way ANOVA and the post hoc analysis tukey test, n=6. The model group has obvious difference from the control group and the lactobacillus salivarius 069 group ^* P<0.05, ^** P<0.01, ^*** P<0.001, ^**** P<0.0001. Control group (Control group), model group (HC group), lactobacillus salivarius 069 group (L.salivarius group), lovastatin group (Lovastatin group).

Fig. 4: analysis of differential metabolite enrichment pathways in rat serum and feces. (A) positive and negative ion differential metabolites of faeces of HC group and lactobacillus salivarius 069 group, (B) positive and negative ion differential metabolites enrichment pathway of faeces, (C) positive and negative ion differential metabolites of serum of HC group and lactobacillus salivarius 069 group, and (D) metabolismDifferential serum positive and negative ion pathway, (E) chenodeoxycholic acid (CDCA) level, (F) indoleacetic acid (Heteroauxin) level, (G) N-acetylhistamine (N-acetylhistaminine) level, (H) Histamine (Histamine) level at VIP >1.0 and P<Compounds of different importance in 0.05 are defined as differential metabolites. The HC group was significantly different from the L.salivarius 069 group ^* P<0.05, ^** P<0.01. Control group (Control group), model group (HC group), lactobacillus salivarius 069 group (L.salivarius group), lovastatin group (Lovastatin group).

Fig. 5: each group of rats consists of intestinal microorganisms. (A) an in-group sparse curve based on Shannon index, (B) a Chao1 index to calculate a box plot of alpha diversity, (C) a box plot of Shannon index to calculate alpha diversity, (D) Simpson index to calculate a box plot of alpha diversity, (E) a box plot of bray_curtis distance to calculate Beta diversity, (F) a relative abundance of gate level, (G) a relative abundance of family level, (H) a relative abundance of genus level, (I) a microbiota profile comparing HC and L.salivarius 069 groups using Linear Discriminant Analysis (LDA) and effector (LEfSe). LDA scoring>2, FDR value<0.1.alpha diversity index was tested using Kruskal-Wallis, beta diversity index was tested using permanva, n=6. HC group was different from Control group and L.salivarius 069 group ^* P<0.05， ^** P<0.01. Control group (Control group), model group (HC group), lactobacillus salivarius 069 group (L.salivarius group), lovastatin group (Lovastatin group).

Fig. 6: and (3) enzymatically verifying lactobacillus salivarius 069 BSH. (A) BSH substrate specificity, (B) BSH enzyme reaction kinetics profile, (C) optimum pH of BSH enzyme, (D) optimum temperature of BSH enzyme, and (E) influence of different metal ions on enzyme activity.

Fig. 7: specific target verification electrophoresis pattern of sialyllactose 069. (A) non-sialobacter electrophoresis strips, (B) non-target lactobacillus electrophoresis strips, and (C) non-lactobacillus electrophoresis strips.

Fig. 8: lactic acid bacteria 069SEQ ID NO: and 2, establishing a result schematic diagram of a quantitative detection method of the target qPCR.

Fig. 9: lactic acid bacteria 069SEQ ID NO: and 3, establishing a result schematic diagram of the quantitative detection method of the target qPCR.

Fig. 10: lactobacillus salivarius 069qPCR SEQ ID NO:2 a real-time Ct value schematic diagram of quantitative detection method qPCR.

Fig. 11: lactobacillus salivarius 069qPCR SEQ ID NO:3 a real-time Ct value schematic diagram of quantitative detection method qPCR.

Detailed Description

In order to more clearly describe the technical solution of the present invention, the following description is further given by way of specific examples, but not by way of limitation, only some examples of the present invention.

Example 1 isolation and identification of lactic acid bacteria

Collecting healthy human feces of the longevity rural areas of the world of China as samples, taking about 0.1g of feces samples under a sterile environment, adding 10mL of TPY liquid culture medium, shaking and uniformly mixing, carrying out enrichment culture for 12h under an anaerobic condition at 37 ℃, and absorbing 0.5mL of bacterial liquid for gradient dilution. Adding physiological saline to make into 10 ^-1 To 10 ^-5 Diluting gradient bacterial suspension, selecting 10 ^-3 、10 ^-4 、10 ^-5 Three gradient bacterial suspensions are respectively absorbed from 200 mu L to TPY solid culture medium and MRS solid culture medium, and the coating rod is smeared uniformly and cultured for 48 hours under anaerobic condition at 37 ℃. Representative colonies on the plates were picked, streaked to obtain pure colonies, kept and bacterial DNA extracted, and PCR amplification primer selections were as described in table 1. Reaction conditions: pre-denaturation at 95℃for 5min; for 35 cycles of 95 ℃ 30s,56 ℃ 30s and 72 ℃ 45s, annealing and extending for 10min at 72 ℃. Performing first-generation sequencing on the PCR product, performing sequence comparison on NCBI database, performing homology analysis, selecting 10 strains of lactobacillus salivarius for experiment, and primarily screening 1 strain 069 for subsequent experiment, wherein the 16s amplification sequence is shown as SEQ ID NO: 4.

TABLE 1 primer sequences

Primer sequences
	27F：5’-AGA GTT TGA TCC TGG CTC AG-3’；
1492R：5’-CTAC GGC TAC CTT GTT ACG A-3’

The mass spectrum verification result of the strain 069 selected by the invention through MALDI-TOF MS (BRUKER, germany) shows that the strain is lactobacillus salivarius, which is named as lactobacillus salivarius (Lactobacillus salivarius) 069 and is preserved in the microorganism strain preservation center (GDMCC) in Guangdong province at 1 month 6 of 2022, and the preservation address is: building 5, no. 59, of Guangzhou Md.A. Xian Zhonglu 100, accession number is GDMCC No:62501.

example 2 in vitro cholesterol lowering and probiotic properties

(1) Cholesterol lowering Activity assay

Preparation of cholesterol Medium (CHO) (g/L): cholesterol 0.5g, sucrose ester 0.1g, tween-80 1mL, glacial acetic acid 1mL were thoroughly stirred and sonicated at 60℃for 30min with stirring to add to MRS broth containing 0.2% sodium taurodeoxycholate by mass. Sterilizing at 121deg.C for 20min.

Inoculating 2% of inoculum size into cholesterol medium (CHO), anaerobic culturing at 37deg.C for 72 hr, and inoculating 3 strains each. 1mL of fermentation broth was collected at 4000 Xg for 10min, and the cholesterol content was measured with a cholesterol measurement kit on a Michaelis BS-480 full-automatic biochemical analyzer.

P ₀ (％)＝(1-C/D)×100％

Wherein, C: sample cholesterol content; d: blank cholesterol content.

The result shows that the in vitro cholesterol-reducing capacity of lactobacillus salivarius 069 is 27.0%, which indicates that the lactobacillus salivarius has stronger cholesterol-reducing activity in vitro.

(2) Gastrointestinal fluid simulation

Activating and culturing Lactobacillus salivarius 069 in MRS culture medium, centrifuging (4000 Xg, 10 min) to collect thallus, re-suspending, washing and centrifuging with sterilized normal saline for 2 times, suspending thallus in 5mL sterilized normal saline to obtain bacterial suspension with bacterial concentration of 3×10 ⁷ CFU/ml. Then, 1mL of the bacterial suspension was inoculated into a 9mL artificial gastric juice tube of pH 3.0 subjected to filtration and sterilization treatment, and after thoroughly mixing, anaerobic culture was performed at 37 ℃. And sampling after 0h and 3h of heat preservation at the beginning of the experiment, and measuring the viable count. Then, 1mL of the bacteria-containing artificial gastric juice treated for 3 hours is aseptically sucked, inoculated into 9mL of the filtered and sterilized artificial intestinal juice with pH of 8.0, continuously placed in a 37 ℃ incubator for anaerobic culture, and sampled and measured for viable count after 5 hours.

Artificial gastric juice: the pH value is regulated to 3.0 by 1mol/LHCl, and the mixture is filtered and sterilized for later use, wherein the mixture contains 0.2% of NaCl and 0.35% of Pepsin (sigma) by mass fraction.

Artificial intestinal juice: mixing the solution a and the solution b in a volume ratio of 2:1 to obtain the artificial intestinal juice.

a. Pancreatic juice: contains NaHCO in mass fraction ₃ 1.1%, naCl 0.2% and Trypsin (sigma) 0.1%, and after adjusting the pH to 8.0, filtering and sterilizing for later use.

b. Bile liquid: according to the mass fraction, the Bile Salts are 1.2%, and after the pH is adjusted to 8.0, the mixture is filtered and sterilized for standby.

The calculation formula is as follows:

P(％)＝A/B×100

p: survival rate%

A: number of viable bacteria (CFU) for 3h or 5h

B: number of viable bacteria (CFU) in gastric juice for 0h

The lactobacillus salivarius 069 is subjected to an artificial gastrointestinal fluid simulation experiment, and the viable count can reach 10 after 3 hours of gastric fluid treatment ⁷ CFU enters intestinal juice for 5 hours after passing through gastric juice, and the viable count can reach 10 ⁶ Above CFU, the bacterium has stronger tolerance in gastrointestinal tract.

EXAMPLE 3 inhibition of Lactobacillus salivarius fermentation supernatant on common food-borne pathogenic bacteria and multiple drug-resistant bacteria

The indicator bacteria are Escherichia coli ATCC 8739, salmonella typhimurium ATCC 14028, cronobacter sakazakii ATCC 29544, staphylococcus aureus ATCC 25923, listeria monocytogenes ATCC 19117, bacillus cereus ATCC 14579, and the multi-drug resistant strains separated independently by the team are Listeria monocytogenes 833-1 (drug resistant spectrum: K-CIP-SXT) S-E-OX-DA-TE-C), indonesia 51-38 (resistance profile: NA-CIP-LVX-MXF-AM/CA-CN-SXT-TE-C-FFC), methicillin-resistant Staphylococcus aureus MRSA Sta 177-2 (drug resistance spectrum: AMC-AMP-P-FOX-CAZ) and indiana salmonella 77-5 (drug resistance profile: AMP-AM/CA-CTX-CRO-S-AK-CN-NA-CIP-LVX-MXF-SMZ-TE-C-FFC. Inoculated into MRS broth at a volume ratio of 2%, and cultured anaerobically at 37 ℃ for 48h. The supernatant was collected at 4℃for 4000 Xg for 10min and filtered through a 0.22 μm filter. Regulating living bacteria number of pathogenic indicator bacteria to about 10 ⁷ CFU/mL, spread on LB plate uniformly. Carefully place oxford cups on the plate, draw 200 μl of supernatant into oxford cups and spread for 10 hours in a refrigerator. Carefully place in a 37 ℃ incubator, each experiment was repeated three times. Lactobacillus delbrueckii XJ-51 and MRS broth were used as controls.

TABLE 2 fermentation supernatant bacteriostasis experiments

Note that: ampicillin (AMP); penicillin (P); cefoxitin (FOX); ceftazidime (CAZ); erythromycin (E); amoxicillin (AMC); amoxicillin/clavulanic acid (AM/CA); cefotaxime (CTX); ceftriaxone (CRO); streptomycin (S); amikacin (AK); gentamicin (CN); nalidixic Acid (NA); ciprofloxacin (CIP); levofloxacin (LVX); moxifloxacin (MXF); compound Sulfamethoxazole (SMZ); tetracyclines (TE); chloramphenicol (C); florfenicol (FFC); kanamycin (K); compound neonomine (SXT); oxacillin (OX); clindamycin (DA).

As can be seen from table 2, the MRS broth blank had no bacteriostatic activity. The lactobacillus salivarius 069 has stronger antibacterial activity on 6 food-borne pathogenic bacteria, the diameter of a bacteriostasis circle is more than 20mm, the bacterial supernatant can also inhibit multi-drug-resistant strains which are separated by teams independently, the bacteriostasis circle is more than 17mm, and the activity of the teams independently separated strain lactobacillus delbrueckii XJ-51 is weaker and almost inactive, which indicates that the bacterial inhibition activity of the bacteria is stronger.

EXAMPLE 4 evaluation of lactic acid bacteria safety

(1) Antibiotic susceptibility test

Whether bacteria are sensitive to antibiotics is an important indicator of bacterial safety. The 6 antibiotics, vancomycin (30 mug), ampicillin (10 mug), erythromycin (15 mug), tetracycline (30 mug), ciprofloxacin (5 mug) and chloramphenicol (30 mug) were selected respectively, and a paper sheet diffusion method was used to conduct an antibiotic susceptibility test according to standards established by the Clinical and Laboratory Standards Institute (CLSI).

TABLE 3 antibiotic susceptibility experiments

Note that: s: sensitivity; r: drug resistance; i is uncertain.

From Table 3, it can be seen that Lactobacillus salivarius 069 is naturally resistant to vancomycin, has uncertain resistance to ciprofloxacin, is sensitive to other antibiotics, and has high safety.

(2) Hemolysis experiment

Under aseptic condition, inoculating lactobacillus with inoculating loop in sheep blood plate, culturing at 37deg.C for 48 hr, and observing hemolysis. Hemolysis can form three features on the blood platelets: (1) alpha hemolysis: grass green hemolytic rings appear around colonies, which are generally opportunistic. (2) Beta hemolysis: a broad transparent hemolytic ring appears around the colony, and is generally highly pathogenic. (3) Gamma hemolysis: there were no hemolytic rings around the colonies, and in general the strains were nonpathogenic.

Staphylococcus aureus25923 is positive control, and all colonies have no hemolytic ring, so lactobacillus salivarius 069Is gamma hemolysis, and has no hemolytic activity.

EXAMPLE 5 evaluation of cholesterol reduction in vivo

(1) Grouping of experimental animals and determination of physiological and biochemical indicators

SPF (Specific Pathogen-Free) SD rats at 5 weeks of age, from southern medical university (Guangzhou, china). Rats were placed under controlled environmental conditions (23±3 ℃, 50% -60% relative humidity), light/dark cycles 12/12h, free water and food during the experiment. Rats began to acclimate 1 week prior to the experiment and were randomized into five groups. Control group (Control group) rats were fed Co60 irradiation maintenance diet. Model group (HC group) fed High Cholesterol Diet (HCD), lactobacillus salivarius 069group (L salivarius 069 group) fed at a daily rate of 1X 10 bacteria per 100g rats ⁹ The feeding dose of cfu, lovastatin group (Lovastatin group) was 0.1mg per 100g rats per day. Co60 irradiation maintains the daily ration crude protein not less than 180g/kg, crude fat not less than 40g/kg, crude fiber not less than 50g/kg, crude ash not more than 80g/kg, calcium 10-18 g/kg, total phosphorus 6-12 g/kg, lysine not less than 8.2g/kg, methionine+cystine not less than 5.3g/kg. HCD includes Co60 irradiation maintenance diet, 10% lard by mass, 1% cholesterol by mass, 0.2% bile salts by mass. Body weight was measured once a week over 6 weeks, and food intake and feed utilization were calculated. The experimental design was approved by the institutional animal care and ethics committee of the microbiological study, academy of sciences, guangdong, and rats were maintained according to standard guidelines.

After 42 days of feeding, blood was taken from the heart and sacrificed, and heart, liver, spleen, kidney, epididymal fat, ileum, colon were immediately excised, rinsed, weighed, frozen in dry ice, and stored at-80 ℃ until analysis.

Serum biochemical analysis was performed using heart blood collection in 16h fasted state. Blood was centrifuged at 3500rpm for 10min at 4℃to obtain serum. Serum Total Cholesterol (TC), triglyceride (TG), total Bile Acid (TBA), glutamic-oxaloacetic transaminase (AST), glutamic-pyruvic transaminase (ALT), low Density Lipoprotein (LDL) cholesterol, and High Density Lipoprotein (HDL) cholesterol levels were measured using a Mickey BS-480 full-automatic biochemical analyzer.

Table 4 Biochemical index of blood lipid of rats in each group

p values were analyzed post hoc using one-way ANOVA and Tukeys test, n=6. Significant differences between HC group and control group are shown as ^# P<0.05 ^## P<0.01, ^### P<0.01, ^#### P<0.0001. The significant difference between the lactobacillus salivarius 069group, the lovastatin group and the HC group is that ^* P<0.05, ^** P<0.01 ^*** P<0.001 ^**** P<0.0001

Organ index (%) = (organ weight/body weight) ×100

Control group (Control group), model group (HC group), lactobacillus salivarius group (L.salivarius 069 group), lovastatin group (Lovast atin group)

As can be seen from Table 4, the body weight increase, liver index and epididymal fat index of the model group were higher than those of the control group, indicating successful modeling. After the intervention of lactobacillus salivarius 069 and low-dose lovastatin, compared with a model group, the liver index and epididymal fat index physiological indexes are obviously reduced, and the method has statistical significance. It was demonstrated that lactobacillus salivarius 069 was able to slow down obesity caused by a high fat diet, as well as being effective at low doses of lovastatin.

Table 5 Biochemical index of blood lipid of rats in each group

P values were analyzed post hoc using One-way ANOVA and Tukeys test, n=6. Significant difference between HC and control group

Is that ^# P<0.05 ^## P<0.01, ^### P<0.01, ^#### P<0.0001. Saliency of Lactobacillus salivarius 069group and lovastatin group and HC group

The difference is ^* P<0.05, ^** P<0.01 ^*** P<0.001 ^**** P<0.0001

Control group (Control group), model group (HC group), lactobacillus salivarius 069group (L.salivarius 069 group), lova

Statin group (Lovastatin group)

As can be seen from table 5, the Total Cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL-C), total Bile Acid (TBA) values were significantly different for both the model and control groups (P < 0.05). Both lactobacillus salivarius 069 and lovastatin groups had significantly lower TG and LDL-C levels (P < 0.05) compared to the model group. The TC level of lactobacillus salivarius 069group was in a decreasing trend, but not statistically significant. There was no significant difference in HDL-C, TBA levels in the Lactobacillus salivarius 069group compared to the model group. Through analysis of biochemical indexes of blood fat, the lactobacillus salivarius 069 has good blood fat reducing application in animals, is consistent with the effect of low-dose lovastatin, shows that the lactobacillus salivarius has great potential in reducing blood fat in human bodies, and a specific mechanism is yet to be further explored.

(2) Determination of TC, TG, TBA in feces and liver

5mL chloroform was added per 0.2g of tissue: mixing methanol (2:1, v/v) mixture, shaking, mixing, maintaining at 37deg.C for 30min, centrifuging (8000 r/min,10min, 4deg.C), carefully collecting chloroform layer liquid into a new centrifuge tube, adding 3mL physiological saline, and centrifuging (8000 r/min,10min, 4deg.C). After repeating the above steps once more, collecting the bottom chloroform layer liquid, blow-drying with a nitrogen blower, adding isopropanol into 0.8 mL: re-dissolving TritonX-100 (9:1, v/v) mixture, vortex shaking for 2min, adding 1.2mL distilled water, and vortex shaking for 2min to dissolve thoroughly, wherein the obtained solution is extracted liver tissue total lipid. Total Cholesterol (TC), triglyceride (TG), and Total Bile Acid (TBA) in liver were measured using a Michaelis BS-480 full-automatic biochemical analyzer.

Mixing 0.1g of lyophilized feces sample with 2.5mL of ethanol, extracting at 80deg.C for 1 hr for 2 times, mixing the two extractive solutions, blow drying at 50deg.C under nitrogen protection, and dissolving the residue with 2mL of ethanol. The Total Cholesterol (TC), triglyceride (TG) and Total Bile Acid (TBA) in the feces are measured by a Michaelis-480 full-automatic biochemical analyzer.

The experimental results are shown in FIG. 1. In the liver, the TC, TG values were significantly higher in the model group than in the control group (P < 0.05). The liver TC levels were significantly reduced (P < 0.05) in the lactobacillus salivarius 069 and lovastatin groups compared to the model group (fig. 1A). Liver TG levels tended to decrease compared to the model group, but were not statistically significant (fig. 1B). The liver TBA levels tended to be elevated in lactobacillus salivarius 069 as compared to the model group (fig. 1C). Compared with the model group, the TBA content in the feces of the lactobacillus salivarius 069 group and the lovastatin group is obviously increased (P < 0.05) (figure 1F), the feces TC and TG have a rising trend, and the difference has no statistical significance (figure 1D, E). The lactobacillus salivarius 069 can promote the excretion of bile acid in rats and reduce the accumulation of cholesterol in the liver.

(3) Lactobacillus salivarius 069 can improve liver injury and relieve fat of rat with hyperlipidemia

1) Determination of liver and epididymal fat related inflammatory factor

Sample pretreatment

50.00.+ -. 0.25mg of tissue was weighed into 500. Mu.L of 1 Xprotease inhibitor, 2 stainless steel balls were added to the solution, and the mixture was put into a grinder for grinding (65 HZ,45s, -20 ℃). After completion of the grinding, the mixture was centrifuged at 13000 Xg for 5min at 4℃to obtain a supernatant. The determination of liver and epididymal fat MCP-1, IL-1β, IL-6, TNF- α inflammatory factor content was performed according to the procedure of enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) kit instructions.

Liver and epididymal fat total protein content was performed according to the kit instruction procedure.

The calculation formula is as follows: relative inflammatory factor concentration (pg/mg) =inflammatory factor concentration (pg/mL)/total protein concentration (mg/mL).

2) Liver, epididymal adipose histopathological (H & E staining) analysis

Drawing materials and dehydrating tissues: placing a proper amount of liver and epididymal fat in 10% formalin solution for fixing for 24 hours, cutting a proper tissue by using a surgical knife, placing the tissue in an embedding box, flushing surface fixing liquid, and dehydrating by using a full-automatic dehydrator, wherein the dehydration procedure is as follows: 240min of 75% ethanol, 120min of 85% ethanol, 120min of 90% ethanol, 60min of 90% ethanol, 30min of absolute ethanol I, 30min of absolute ethanol II, 30min of xylene I, 30min of xylene II, 30min of paraffin I, 30min of paraffin II and 30min of paraffin III.

Embedding and slicing: starting the embedding machine, immersing the transparent tissue in wax, adjusting the direction of the tissue block, rapidly transferring to a cooling table to rapidly solidify, and storing at 4 ℃. Starting a semi-automatic tablet spreading machine, and setting slicing parameters when the water temperature in the tablet spreading machine is raised to 42 ℃, wherein the cutting angle is 10 degrees, the thickness of a liver tablet is 3 mu m, and the thickness of a fat tablet is 5 mu m. And (5) placing the slices in a spreading machine for spreading, fishing out the glass slide, airing, and baking in a baking oven at 65 ℃ for later use. Hematoxylin eosin staining and sealing: the slices are baked at 65 ℃ for 1 hour, immediately after wax is melted, the slices are placed into a full-automatic dyeing machine for dyeing, wherein xylene I (dewaxing) is carried out for 5min, xylene II (dewaxing) is carried out for 5min, xylene III (dewaxing) is carried out for 5min, absolute ethanol I (dehydration) is carried out for 1min, absolute ethanol II (dehydration) is carried out for 1min, 95% ethanol 90s, 85% ethanol 1min, 75% ethanol 20s, running water flushing for 1min, hematoxylin (cell nucleus dyeing) is carried out for 10s, running water flushing for 3min, differentiation liquid 9s, running water flushing for 10s, blue returning liquid 1min, running water flushing for 10s, 95% ethanol 1min, eosin (cytoplasm dyeing) is carried out for 4min 40s, 85% ethanol (dehydration) is carried out for 6s, 95% ethanol (dehydration) is carried out for 10s, absolute ethanol I (dehydration) is carried out for 1min, absolute ethanol II (dehydration) is carried out for 3min, xylene I5 min, and xylene II is carried out for 5min.

After dyeing, placing the sheet in a fume hood, volatilizing dimethylbenzene, and sealing the sheet by a sheet sealing machine.

Microscopic examination and photographing: observations were made at 200-fold magnification using the Scomeage 9.0 system.

In liver tissues, the reduction effect of lactobacillus salivarius 069 on liver injury is studied by measuring ALT and AST levels in serum, measuring inflammatory cytokine content in liver, and performing morphological evaluation on liver by HE staining. The serum ALT, AST levels were significantly reduced in lactobacillus salivarius 069 (fig. 2a, b), whereas lovastatin group had a decreasing trend, but no significant difference. Inflammatory factors in the liver are then measured, and increased levels of inflammatory factors in the liver exacerbate liver injury. As can be seen from figures 2c, d, the TNF- α, IL-1β content after lactobacillus salivarius 069 treatment was significantly lower than that of the model group (P < 0.05), and the TNF- α, IL-1β content after lovastatin treatment was also in a decreasing trend. Grading steatosis and hepatocyte inflammation with NAFS score model group rat liver H & E staining showed intracellular vacuolation and significant lipid accumulation compared to control group. The degree of hepatic steatosis and fat deposition was significantly reduced in the lactobacillus salivarius 069 group compared to the model group (fig. 2e, f, p < 0.05) and was superior to the low dose lovastatin group.

In epididymal fat, in order to investigate whether lactobacillus salivarius 069 can improve inflammatory adipocytes induced by high fat diet, we measured inflammatory factors thereof and evaluated pathological sections thereof. The epididymal fat MCP-1 and IL-1β levels were significantly lower in the Lactobacillus salivarius 069 and lovastatin groups than in the model group (FIG. 3A, B, P < 0.05) and then the control group (180.+ -. 5) and Lactobacillus salivarius 069 group (183.+ -. 7) showed a significant increase in the number of adipocytes in the same field, while the HC group (141.+ -. 8; P < 0.05) showed a significant decrease in the adipocyte diameter (FIG. 3C-E). From this, it can be seen that lactobacillus salivarius 069 has the potential to ameliorate fat hypertrophy and inflammation, and the effect is consistent with low doses of lovastatin.

EXAMPLE 6 Lactobacillus salivarius 069 modulation of intestinal and serum metabolites in hyperlipidemic rats

Pretreatment of rat feces: weighing 50+/-5 mg of fecal sample, and adding precooled methanol: acetonitrile: homogenizing (45 HZ,120 s) with a grinder twice with 250 μl of water (4:2:2; v:v:v), performing water bath ultrasonic treatment for 10min, standing in a refrigerator at-20deg.C for 1h, centrifuging at 12000rpm, standing at 4deg.C for 10min, collecting supernatant, and drying at room temperature in a vacuum drying oven; then 200. Mu.L of pre-chilled acetonitrile/water (1:1; v:v) was dissolved, spun for 30s at 12000RPM, centrifuged at 4℃for 15min, and the supernatant was taken as the treated sample, 10. Mu.L of each sample was taken as the QC sample, and 200. Mu.L was taken as the sample size.

The serum pretreatment was carried out by mixing 200. Mu.L of serum with 1mL of methanol/acetonitrile (1:1; v:v), sonicating (ice bath) for 20min, and placing in a refrigerator at-20deg.C for 1h. 1mL of the supernatant was dried at 13000rpm/15min under vacuum at 4 ℃. A 50% acetonitrile mixture was added, vortexed at 30 ℃, centrifuged, and the supernatant was taken. The reconstitution method is similar to fecal pretreatment.

Liquid color using UPLC UltiMate3000 (Thermo Fisher Scientific)The profile was subjected to metabonomic analysis of the processed stool/serum samples. The UPLC system was equipped with an RSLC system, an autosampler, and a DAD-3000 (RS) detector using ESI source positive and negative ion mode scanning (ESI+/ESD-), sheath gas flow rate 40%, assist gas flow rate 10%, scavenge gas flow rate 0%, spray voltage 3.50KV, capillary temperature 320 ℃, S-lens RF level 50.0, assist gas heater temperature 350 ℃. By usingHSS T3.8 μm column (Waters, 2.1X100 mm) was used for separation. The mobile phase in positive ion mode is 1/1000 formic acid water, the mobile phase in negative ion mode is 5mmol acetic acid, the mobile phase in positive ion mode is acetonitrile, the sample injection amount is 3 mu L, and the column temperature is 35 ℃. The liquid phase conditions are shown in Table 6. Statistical analysis: raw data obtained by UPLCQ-TOF-MS using Thermo Compound Discoverer ^TM 3.1 software is used for extracting chromatographic peaks, the software can automatically complete pretreatment such as baseline calibration, peak alignment, chromatographic peak extraction, deconvolution and the like, and the structure of the chromatographic peaks is determined by searching through databases such as HMDB (http:// www.hmdb.ca /), KEGG (http:// www.keggjp /), chemsps (http:// www.chemspider.com /), and the like, and the multi-element statistical analysis mainly adopts PCA and OPLS-DA analysis. In addition, in order to verify whether the variables screened by the multivariate statistical analysis are different, a difference test (t test) and a Fold difference test (Fold Change) and a variable projection importance index (VIP not less than 1) are carried out by using Metaboanalysis (https:// www.metaboanalyst.ca /), VIP not less than 1 and P not more than 0.05 are regulated as differential metabolites, and finally, the obtained differential metabolites are identified to be input into MetaboaAnalyst and KEGG for analysis of metabolic pathways.

TABLE 6 liquid phase elution conditions

The distribution of the stool and serum positive and negative ion scans of the raw data was shown by Principal Component Analysis (PCA) with no outliers in the sample analysis. In addition, the arrangement 200 was used for the fitting test, and all points to the left of R2 and Q2 below the right were found to be greater than 0, demonstrating that the orthogonal partial least squares discriminant analysis (OPLS-DA) did not overfit (FIG. 4). We performed a cluster analysis of these stool and serum differential metabolites and analyzed by KEGG metabolic pathways. A total of 52 differential metabolites were detected in these rat feces, mainly enriched in histidine metabolism, steroid hormone biosynthesis, glycine, serine and threonine metabolism, tryptophan metabolism, primary bile acid biosynthesis, unsaturated fatty acid biosynthesis (fig. 4a, b); the 17 serum differential metabolites were mainly enriched in primary bile acid biosynthesis, unsaturated fatty acid biosynthesis, starch and sucrose metabolism, pentose phosphate pathway (fig. 4c, d). It is worth noting that lactobacillus salivarius 069 treatment affects mainly carbohydrate metabolism, fatty acid metabolism, bile acid metabolism, cholesterol metabolism and amino acid metabolism. Interestingly, we found that lactobacillus salivarius 069 had significantly elevated chenodeoxycholic acid (CDCA) and indoleacetic acid (Heteroauxin) compared to HC group (fig. 4e, f), and significantly reduced levels of N-acetylhistamine (N-acetylhistaminine) and Histamine (histaminine) (fig. 4g, h). The results show that the lactobacillus salivarius 069 can promote the biosynthesis of primary bile acid and the metabolism of tryptophan, inhibit the metabolism of histidine and improve hyperlipidemia.

EXAMPLE 7 Lactobacillus salivarius 069 to ameliorate intestinal micro-ecological disorders

The composition of the intestinal flora is related to hyperlipidemia, for which we explored lactobacillus salivarius 069 to regulate intestinal microbial function. As shown in FIG. 5A, the depth of sequencing between groups is deeper, and the curve is gentle, which indicates that the depth of sequencing is reasonable. Secondly, the alpha diversity (richness and uniformity) index of Chao1, shannon and Simpson was significantly improved for the Lactobacillus salivarius 069 group compared to the HC group (FIGS. 5B-D), indicating that the Lactobacillus salivarius 069 group had higher richness and diversity. Next, it can be seen from the PCoA graph (branch_curtis distance) that the intestinal flora of different groups is well-clustered and distributed in different circles (fig. 5E). The PERMANOVA multidimensional statistical analysis of PCoA showed that the differences between the control group (p=0.002) and lactobacillus salivarius 069 group (p=0.013) compared to the HC group were statistically significant.

At the gate level, the abundance of lactobacillus salivarius group 069, thick-walled, was increased, the relative abundance of bacteroides and actinomycetes was decreased, but the tenericut ratio was significantly increased in the control group compared to the HCD group (fig. 5F). At the family level, the heat map showed a significant decrease in the abundance of the erysipelotorich acid and the Veillonellaceae of lactobacillus salivarius 069 (P < 0.05) and lower abundance of other families such as Porphyromonadaceae, peptostreptococcaceae, oxalobacteraceae, prevotellaceae, coriobacteriaceae, paraprevotellaceae (fig. 5G) compared to HC group. Whereas s24_7, ruminococcaceae, spirochaetaceae, lachnospiraceae and corynebacterium are more abundant in lactobacillus salivarius 069 than in HC group. At the genus level, the proportion of Paraprevatellaceae was significantly increased in the control group CF231 compared to the other groups. Both Succinispira, prevotella, ruminococcus, bacteroides, collinsella, paraprevotella and Eubacterium were reduced in the lactobacillus salivarius 069 group compared to the HC group (fig. 5H). The proportions of Corynebacterium, butyricicoccus, clostridium, lactobacillus and Lactobacillus are lower than those of Lactobacillus salivarius 069. After FDR adjustment, the biomarker was detected using LDA effector, and the abundance of Eubacterium, veillonellaceae, coriobacteriaceae, ruminococcus, erysipelotrichaceae in HC group was significantly higher than in other groups. The ratio of spirochaeteae in lactobacillus salivarius 069 was significantly higher (fig. 5I). The results show that lactobacillus salivarius 069 tends to control the composition of intestinal flora of a rat with hyperlipidemia, and can regulate the unbalance of the intestinal flora caused by hyperlipidemia.

Example 8 enzymatic validation of Lactobacillus salivarius 069 to improve the mechanism of hyperlipidemia

Cloning bile salt hydrolase (EC 3.5.1.24, the nucleotide sequence of which is shown as SEQ ID NO: 1) genes (98 ℃ C. 30s;98 ℃ C. 10 s; 58 ℃ C. 20 s; 72 ℃ C. 40 s; 35 cycles; 72 ℃ C. Extension 10 min) by using the whole genome sequence, and connecting the target genes with pET-28a (+) plasmid by using Nhe and Xho as enzyme cleavage sites. The plasmid was introduced into E.coli DH 5. Alpha. Receptor cells and screened with kanamycin-resistant plates. The correct plasmid was designated pET-28a-BSH. Finally, the correct plasmid was introduced into competent cells of E.coli BL21 and heterogeneous expression was induced using 0.4mmol/L IPTG. The crude enzyme solution was purified by bacterial lysis using a HisTrap Fast Flow nickel column (Cyctiva Manufacturer, danaher Group, USA). Collecting the pureAdding 20% glycerol into enzyme solution, and preserving at-80deg.C. Substrate specificity of BSH the reaction system consisted of 20. Mu.L enzyme solution, 0.5. Mu.L 1mol/L vitamin C solution, 100. Mu.L pH=6, 6mmol/L cholic acid and 80. Mu.L dd H ₂ O composition. After mixing, incubating in a constant-temperature water bath at 37 ℃ for 30min, and inactivating enzymes at high temperature after the reaction is finished. The supernatant was centrifuged at 16000rpm for 10 minutes, and then 60. Mu.L of ninhydrin was added thereto for color development, and the supernatant was rapidly placed in a boiling water bath for 15 minutes and an ice bath for 3 minutes. The reaction mixture was left for 5 minutes, and absorbance was measured at 570nm. Actual absorbance value of sample = absorbance value-controlled absorbance value. The amino acid production was calculated from the glycine standard curve, and the specific enzyme activity was calculated. Protein concentration was determined using the modified Bradford protein concentration determination kit. The highest enzyme activity was 100%, and the relative enzyme activity was calculated. The experiment was repeated three times. Different cholic acids include 6mmol/L sodium Glycocholate (GCA), 6mmol/L sodium Glycodeoxycholate (GDCA), 6mmol/L sodium Glycodeoxycholate (GCDCA), 6mmol/L sodium Taurocholate (TCA), 6mmol/L sodium Taurocholate (TDCA), 6mmol/L sodium Taurocholate (TCDCA). BSH total enzyme activity is defined as the number of micromoles (μmol min) of amino acid produced by hydrolysis of choline-bound enzyme solution per unit time per unit volume ^-1 mL ^-1 ) The method comprises the steps of carrying out a first treatment on the surface of the 1U = μmol/min. BSH specific enzymatic activity is defined as the number of micromoles of amino acid produced by enzymatic hydrolysis of conjugated bile salts per unit time and per unit mass of total protein in. Mu. Mol min ^-1 mg ^-1 。

BSH enzyme reaction kinetics the relative enzyme activities were calculated by adding different concentrations (0.1 mmol/L, 0.2mmol/L, 0.4mmol/L, 0.6mmol/L, 0.8mmol/L, 1mmol/L, 2mmol/L, 4mmol/L, 6mmol/L, 8 mmol/L) of GCDCA to the reaction system for 30 min. The maximum reaction rate (V) of BSH was calculated by fitting the data according to Michaelis-menten equation using GraphPad Prism 8.4.3 software _max ) And Michaelis constant (K) _m ). The effect of BSH optimum pH, temperature and different metal ions on enzyme activity (1) GCDCA is used as substrate, and the reaction system is used for respectively placing the GCDCA and the GCDCA at different pH values (3, 4, 5, 5.50, 6, 7, 8, 9 and 10) to react for 30 minutes to calculate specific enzyme activity; (2) Taking 6mmol/L GCDCA as a substrate, reacting for 30min at different temperatures (4 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃ and 70 ℃), and calculating specific enzyme activity; (3) At 6mmol/LGCDCA is used as a substrate, and different metal ions (NaCl and CaCl) are respectively added into a reaction system with the final concentration of 5mmol/L ₂ 、MgSO ₄ 、MnCl ₂ 4H ₂ O、FeSO ₄ 7H ₂ O、LiCO ₃ 、(Al) ₂ (SO ₄ ) ₃ 8H ₂ O、NiSO ₄ 6H ₂ O、CoSO ₄ 7H ₂ O、ZnSO ₄ ) The specific enzyme activity without metal ions was 100%.

Based on the previous team foundation, we found that lactobacillus salivarius 069 promotion of bile acid excretion may be related to the action of Bile Salt Hydrolase (BSH) in vivo. The BSH gene of lactobacillus salivarius 069 is cloned through the whole genome sequence and introduced into BL21 for exogenous expression. BSH was purified by a nickel column and its enzymatic properties were studied. Interestingly, in the substrate specificity experiments, BSH was found to have a preference for hydrolyzing glycochenodeoxycholic acid (GCDCA) (fig. 6). The results indicate that lactobacillus salivarius 069 may promote cholic acid excretion by hydrolyzing GCDCA. The results show that the enzyme V _max 47.46, K _m The pH optimum was 1.27, the pH optimum was 5.50, and the temperature optimum was 37 ℃. Almost all metal ions affect enzyme activity, al ³⁺ Approximately 90% of the enzymatic activity is lost. These results provide a theoretical basis for further understanding the effect of lactobacillus salivarius 069 on lipid metabolism.

Example 9 validation of specific molecular targets of lactic acid bacteria

(1) Excavating of diverse species-specific new molecular targets

Firstly, carrying out whole gene sequencing on lactobacillus salivarius 069, carrying out bioinformatics analysis by utilizing a GenBank database, and screening to obtain two specific gene fragments of the lactobacillus salivarius 069, wherein the nucleotide sequences of the gene fragments are shown as SEQ ID NO:2 and SEQ ID NO: 3.

(2) PCR detection primer availability

According to the sequence SEQ ID NO: 2-NO: 3 design of specific PCR amplification primer set (including forward primer and reverse primer), primer set sequences are shown in Table 7 below.

TABLE 7 PCR detection primer set for specific sequences

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Step S1, DNA template preparation: bacteria are added and cultured in MRS liquid culture medium respectively, bacterial genome DNA is extracted respectively by using a bacterial genome DNA extraction kit as a template to be detected, non-target lactobacillus and non-lactobacillus DNA are extracted simultaneously, and the concentration of bacterial liquid is 10 ⁸ CFU/mL；

Step S2, PCR amplification: the PCR amplification system was as follows:

wherein: the two sets of primers were added simultaneously for amplification, as shown below in the PCR amplification procedure. Wherein the annealing temperature was 67.5 ℃.

Step S3: and (3) taking PCR amplified products to carry out gel electrophoresis, and observing whether two amplified bands exist at the position of each primer group corresponding to the size of the products. If other strain templates do not have two bands at the same time, the corresponding target is a strain-specific molecular target.

41 other Lactobacillus salivarius strains, 44 non-target Lactobacillus and 85 non-Lactobacillus salivarius strains were used for PCR detection of Lactobacillus salivarius 069 specific targets as described in example 7. Wherein, the step S1DNA template is prepared to extract the genome DNA of each bacterium respectively; in the step S2PCR amplification, the primers used are the primers in the primer set. A blank is set, and the template of the blank is an aqueous solution without genome.

The strains of each bacterium used and the results of the tests are shown in Table 8 below, in which "-" in the column of the results of the tests indicates negative. The result of electrophoresis of PCR products is shown in FIG. 7, "+" indicates the target band, "M" is 2000Maker, "-" is blank control.

TABLE 8 test results of test for the detection specificity of Lactobacillus salivarius 069

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As can be seen from FIG. 8, only the target strain showed two simultaneous amplified bands, and both non-target and non-lactobacillus did not contain two bands, indicating that only the target strain contained specific molecular targets in the present method.

(3) Primer availability for fluorescent quantitative PCR detection

Primer SEQ ID NO: 2-NO: 3 availability and specificity. First, lactobacillus salivarius 069 to 10 is cultured ⁸ CFU/mL, with ddH ₂ O is diluted according to a gradient of 10 times to obtain the concentration of 10 ² ，10 ³ ，10 ⁴ ，10 ⁵ ，10 ⁶ ，10 ⁷ ，10 ⁸ Pure culture of CFU/mL strain, extracting DNA, namely qPCR standard, and performing three times of parallelization on each template and ddH ₂ O served as a blank. Standard curves were drawn with Cq (Ct) in qPCR on the ordinate and the logarithm of the pure culture concentration of the strain of the standard on the abscissa.

qPCR was performed with the strains shown in table 8 to verify SEQ ID NO: 2-NO: 3 availability and specificity, at a concentration of 10 ⁸ CFU/mL. The Cq value is substituted into a standard curve, and the concentration of the lactobacillus salivarius 069 obtained by amplification is obtained.

The qPCR amplification system was as follows:

wherein: the two sets of primers were added separately to the amplification system and the PCR amplification procedure was as follows. Wherein the annealing temperature was 67.5 ℃. The qPCR amplification procedure was as follows:

fig. 8 is SEQ ID NO:2, the primer detection line in the invention is 10 ³ CFU/mL, fitted standard curve y= -3.0955x+40.218, R ² =0.9514, fig. 9 is SEQ ID NO:3, the primer detection line is 10 ³ CFU/mL, fitted standard curve is y= -3.8393x+44.424, r2=0.9961. FIGS. 10 and 11 are schematic diagrams of real-time Ct values of the quantitative detection method qPCR of Lactobacillus salivarius 069qPCR SEQ ID NO:2-NO: 3. The strain was confirmed to be consistent with the results of Table 8, and the detection concentration of more than 10 could not be satisfied at the same time ³ CFU/mL. Therefore, the two target primers have good specificity and can be used for specially detecting the bacteria.

SEQ ID NO.1：

ATGTGTACAGCAATTACTTTAAATGGTAATAATAATTATTTTGGAAGAAACTTAGATTTGG

ATTTTTCATATGGTGAGCAGGTAATCATTACTCCGGCTGAATATGAGTTTAAATTTAGAAA

GGAAAAGGCTATAAAGAATCATAAATCATTAATAGGTGTTGGAATTGTTGCTAACGATTA

CCCATTGTATTTTGATGCTATTAATGAGGATGGACTAGGAATGGCAGGATTGAATTTTCCT

GGAAATGCATATTATAGCGATGCTTTAGAGAATGACAAAGATAATATTACGCCGTTCGAG

TTTATTCCATGGATTCTGGGACAGTGTAGCGATGTTAATGAAGCAAGAAATTTAGTTGAA

AAAATAAATCTTATTAATCTTAGTTTTAGCGAACAGTTACCTTTAGCAGGATTACATTGGT

TAATTGCTGATAGAGAAAAATCCATCGTAGTAGAAGTAACTAAATCTGGAGTACATATTTA

TGATAATCCAATTGGAGTATTGACTAATAATCCAGAATTTAATTATCAGATGTACAATCTG

AATAAATATCGCAATTTATCTATCAGTACACCACAAAATACATTCTCAGATAGCGTGGATT

TAAAGGTAGACGGTACTGGTTTTGGTGGTATTGGCTTGCCAGGCGATGTATCTCCCGAAT

CTCGTTTTGTGAGAGTTGCTTTTAGTAAGTTAAATTCAAGTAAAGGGACGACCGTAGAA

GAAGATATTACTCAGTTTTTCCATATACTAGGGACAGTAGAACAGATAAAGGGTGTTAAT

AAGACAGAATCAGGAAAAGAAGAATACACTGTATATTCGAATTGCTATGATTTGGACAA

CAAGACGCTATATTATACAACCTATGAAAATAGACAAATAGTGGCTGTCACTTTAAATAA

AGATAAGGACGGTAATAGGTTAGTTACATATCCATTTGAAAGGAAACAAATAATAAATAAGTTGAATTAA。

SEQ ID NO.2：

GCGTGAGACGAGCACTTAAAATGACAACCAAGCGAAATAGAGGAATTTCTCTGAACGAT

ATGTTCAAAGAAATATACCAGAAAATGCGTGGTTGGTTACAGTATTACTCAATAGGGAGA

ATGACTACTTTTATCAGACGTCTTGACCAATGGCTAAGGTCAAGAATAAGGCAATATATC

TGGAAACAATGGAAGAAACCTAAAACAAGATTTGTTAACTTGAAGAAGTTAGGGTTGTCACAACAAGATGCATATACATTTGCCATGACCCGTAAGGGTTACTGGCGAACTGC。

SEQ ID NO.3：

AGAGCGTCATGTTACGGCAAATTCTGAATCAGTTTTAAATACACCTAAACCAAAACCACAAGCAGTTACTTATGATTCTTGGACTGACAATCTAGGTGATGTTTGGTACAAAGAAGATGGTACATTTACACTATCTGAAAGTATTAATCTTCGTTGGGGTGCTAAAACTGA。

SEQ ID NO.4：

CTGGCTCCTTGCGGTTACCCCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGACATGCTGATTCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTAAACCTCGCGGTCTTGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCGCCAGAGTGCCCAACTTAATGCTGGCAACTGACAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCACTTTGTCCCCGAAGGGAAAGCCTAATCTCTTAGGTGGTCAAAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTATTGCGTTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTACCCACGCTTTCGAACCTCAGCGTCAGTTACAGACCAGAGAGCCGCTTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGTCTTCCAGTTTCCAATGCACTACTCCGGTTAAGCCGAAGGCTTTCACATCAGACTTAAAAGACCGCCTGCGTTCCCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTGCTGGTTAGATACCGTCATCGAATGAACAGTTACTCTCACTCGTGTTCTTCTCTAACAACAGAGTTTTACGATCCGAAGACCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAACCTCTCAGTTCGGCTACGTATCATCACCTTGGTAGGCCGTTACCCCACCAACTAGTTAATACGCCGCGGGTCCATCTAAAAGCGATAGCAGAACCATCTTTCATCTAAGGATCATGCGATCCTTAGAGATATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTTCTTTTAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCACTCAACTTCTTACGGTGAATGCAAGCATTCGGTGTAAGAAAGTTTCGTTCGA。

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. Lactobacillus salivarius @Lactobacillus salivarius) 069, accession No. GDMCC No:62501.

2. use of lactobacillus salivarius 069 as claimed in claim 1 in the manufacture of a medicament for the treatment of liver injury caused by hyperlipidemia or gut metabolite disorders caused by hypercholesterolemia.

3. The method for preparing the anti-escherichia coli agent from lactobacillus salivarius 069 as claimed in claim 1Escherichia coli) Salmonella typhimurium (Salmonella typhimurium)Salmonella typhimurium) Cronobacter sakazakiiCronobacter sakazakii) Staphylococcus aureus @ sStaphylococcus aureus) Listeria monocytogenesListeria monocytogenes) Bacillus cereusBacillus cereus) Or Indiana salmonellaSalmonlla Indiana) Is used in the medicine.

4. A medicament for treating liver injury caused by hyperlipidemia or intestinal metabolite disorder caused by hypercholesterolemia, which comprises the Lactobacillus salivarius of claim 1Lactobacillus salivarius）069。

5. A specific molecular marker combination for detecting lactobacillus salivarius 069 as claimed in claim 1, wherein the molecular marker combination comprises a molecular marker 1 with a nucleotide sequence shown in SEQ ID NO. 2 and a molecular marker 2 with a nucleotide sequence shown in SEQ ID NO. 3.

6. A primer set for detecting lactobacillus salivarius 069 as claimed in claim 1 comprising an upstream primer and a downstream primer for amplification of the nucleotide sequence shown in SEQ ID No. 2 and an upstream primer and a downstream primer for amplification of the nucleotide sequence shown in SEQ ID No. 3.

7. The primer set of claim 6, wherein the upstream primer and the downstream primer for amplification against the nucleotide sequence shown in SEQ ID NO. 2 are: 5'-GCGTGAGACGAGCACTTAAA-3' and 5'-GCAGTTCGCCAGTAACCCTT-3'; the upstream primer and the downstream primer for amplifying the nucleotide sequence shown in SEQ ID NO. 3 are as follows: 5'-AGAGCGTCATGTTACGGCAA-3' and 5'-TCAGTTTTAGCACCCCAACGA-3'.

8. A method for detecting lactobacillus salivarius 069 as claimed in claim 1 for non-disease diagnostic and therapeutic purposes comprising the steps of:

extracting genome DNA of the bacteria to be detected, and then amplifying by using the upstream primer and the downstream primer for amplifying the nucleotide sequence shown as SEQ ID NO. 2 and the upstream primer and the downstream primer for amplifying the nucleotide sequence shown as SEQ ID NO. 3 as set forth in claim 7, wherein if 292bp and 171bp sequences can be amplified simultaneously, the sequence is lactobacillus salivarius 069, otherwise, the sequence is not.