CN113337440B

CN113337440B - Lactobacillus salivarius MG-587 and application thereof

Info

Publication number: CN113337440B
Application number: CN202110684279.XA
Authority: CN
Inventors: 刘庆军; 沈鹤霄; 李国龙; 张帆
Original assignee: Maintain Biomedical Wuhan Co ltd
Current assignee: Maintain Biomedical Wuhan Co ltd
Priority date: 2021-06-21
Filing date: 2021-06-21
Publication date: 2023-02-21
Anticipated expiration: 2041-06-21
Also published as: CN113337440A

Abstract

The invention discloses Lactobacillus salivarius MG-587 and application thereof, wherein the Lactobacillus salivarius MG-587 is preserved in China general microbiological culture Collection center at 04.01.2021, with the preservation numbers as follows: CGMCC No. 21588. The Lactobacillus salivarius MG-587 is separated and identified from human excrement for the first time, and researches show that the strain can obviously reduce fat cells, reduce the weight of visceral fat, and reduce the contents of cholesterol, triglyceride and low-density lipoprotein, so that the effects of reducing blood fat and weight and improving liver functions are achieved, and the strain also has an excellent blood sugar reducing function; the choline hydrolase activity and the antibiotic resistance are obtained from healthy human bodies, and the choline hydrolase activity and the antibiotic resistance are nontoxic and have no antigen reaction, can be used as probiotics to prepare multifunctional medicines, functional foods or health-care foods, and have wide market prospect and application value.

Description

Lactobacillus salivarius MG-587 and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus salivarius MG-587 and application thereof.

Background

Lactobacillus salivarius belongs to the family of Lactobacillus, the genus Lactobacillus is gram-positive, does not produce spores, does not have catalase, oxidase and motility, can produce lactic acid, can grow in aerobic and anaerobic environments, belongs to facultative heterogeneous acidic strains, and does not produce gas during glucose metabolism. Researches find that the lactobacillus salivarius is a probiotic which can stimulate immune cells to secrete anti-allergy related cell hormones, has certain adsorption and immunoregulation functions, and can be used for improving the immune function and enhancing the immunity of the organism; and the lactobacillus colonized in the intestinal tract can secrete substances such as antibiotics, bacteriocins and the like in the growth and metabolism process, and has various effects of inhibiting the growth of pathogenic bacteria and the like. The lactobacillus salivarius is one of the strains approved by the Ministry of health in China for food production and processing, has good acid resistance and choline resistance, and can survive in intestines and stomach of human bodies and animals. It is widely used as a probiotic lactobacillus with great potential to be prepared into probiotic preparations suitable for human and animals.

Hyperlipidemia (HLP) is a systemic abnormal metabolism disease caused by high blood lipid level, such as atherosclerosis, coronary heart disease, pancreatitis, etc., in which the levels of cholesterol, triglyceride and low-density lipoprotein in blood plasma are increased and the level of high-density lipoprotein is too low. With the rapid development of economy and the continuous improvement of living standard of people in China, cardiovascular and cerebrovascular diseases are increasing day by day, wherein hyperlipidemia is an important risk factor of cardiovascular and cerebrovascular diseases. The main approaches to improving blood lipids include lifestyle changes and hypolipidemic medications. The main cause of the disorder of fat metabolism is mostly due to the imbalance and shift of microbial flora inhabiting the gastrointestinal tract of the human body caused by the preference of high-fat food or unreasonable diet, which results in the continuous passive lipid substance absorption of the human body. The treatment and prevention of hyperlipidemia based on intestinal flora is therefore the current major research direction.

The glucose in blood becomes blood sugar, and most of the energy required for the activities of the cells of each tissue in the body comes from glucose, so that the blood sugar needs to be kept at a certain level to maintain the needs of each organ and tissue in the body, and when the blood sugar value is higher than a normal value, the blood sugar is high. Under normal conditions, a human body can ensure the balance of blood sugar sources and approaches through two regulating systems of hormone regulation and nerve regulation so as to maintain the normal blood sugar, but under the action of genetic factors, unreasonable diet, obesity and the like, the two regulating functions can be disordered to further cause abnormal rise of blood sugar level, and long-term hyperglycemia can cause pathological changes of all tissues and organs of the whole body to cause various acute and chronic complications. However, since there are many side effects in regulating blood sugar level by drugs such as acarbose, metformin, insulin, etc., which are commonly used, it is currently an important research direction to improve blood sugar level by diet therapy and regulation of intestinal probiotics.

Disclosure of Invention

One of the purposes of the invention is to provide a Lactobacillus salivarius MG-587 strain, wherein the Lactobacillus salivarius MG-587 strain is separated from human excrement and has been preserved in China general microbiological culture Collection center (CGMCC) at 04 th 2021, with the preservation number: CGMCC No. 21588.

The invention also aims to provide application of the Lactobacillus salivarius MG-587 in preparation of medicines, functional foods and health-care foods for treating and/or preventing hyperlipidemia.

Further, the Lactobacillus salivarius MG-587 can reduce blood lipid by reducing cholesterol, triglyceride and low density lipoprotein levels.

The invention also aims to provide application of the Lactobacillus salivarius MG-587 in preparation of hypoglycemic drugs, functional foods and health foods.

The fourth purpose of the invention is to provide the application of the Lactobacillus salivarius MG-587 in preparing medicines, functional foods and health foods for treating and/or preventing fatty liver.

The fifth purpose of the invention is to provide the application of the Lactobacillus salivarius MG-587 in preparing weight-reducing medicines, functional foods and health-care foods.

Further, the Lactobacillus salivarius MG-587 achieves the effect of losing weight by reducing the weight of the liver and reducing the contents of gonadal fat and perirenal fat.

Furthermore, the medicine also comprises pharmaceutically acceptable auxiliary materials.

Further, the medicine is a tablet, a capsule, powder, a pill, granules or a solution.

Further, the functional food or health food comprises: biscuit, cake, beverage, oral liquid, granule, beverage, liquid milk, and milk powder.

The sixth purpose of the invention is to provide a probiotic preparation, which comprises: a bacterial liquid containing the Lactobacillus salivarius MG-587, or a dry powder microbial inoculum containing the Lactobacillus salivarius MG-587.

Compared with the prior art, the invention has the beneficial effects that: the Lactobacillus salivarius MG-587 is separated and identified from human excrement for the first time, and researches show that after the strain is dried, fat cells of a mouse are obviously reduced, the weight of visceral fat is obviously reduced, and the contents of cholesterol, triglyceride and low-density lipoprotein are all obviously reduced, namely the strain has the effects of reducing blood fat, improving liver function, losing weight and the like; meanwhile, the strain also has an excellent blood sugar reducing function; meanwhile, the Lactobacillus salivarius MG-587 is derived from a healthy human body, has the ability of producing small molecular acid, choline hydrolase activity and antibiotic resistance, is non-toxic and free of antigen reaction, can be used as probiotics to prepare multifunctional medicines, functional foods or health foods with the functions of reducing blood fat, losing weight and reducing blood sugar, and has wide market prospect and application value.

Drawings

FIG. 1 is an observation of adipocytes before and after probiotic intervention in example 2 of the present invention;

FIG. 2 shows the results of the bile salt hydrolase activity assay of Lactobacillus salivarius MG-587 according to example 2 of the present invention;

FIG. 3 is a graph showing the results of observing liver tissues of mice in ND group, HFD group and probiotic-treated group in example 3 of the present invention;

FIG. 4 is an observation result of adipose tissues of mice in ND group, HFD group and probiotic-treated group in example 3 of the present invention;

FIG. 5 is a genome map of Lactobacillus salivarius MG-587 in example 4 of the present invention;

FIG. 6 is an alignment result of the whole gene sequence of Lactobacillus salivarius MG-587 in the PAIDB database in example 4 of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 isolation, identification and Performance determination of the strains

1. 16S rDNA identification

Adding 5g of a feces sample from a healthy human body into 50mL of sterile water, shaking, uniformly mixing, performing gradient dilution, respectively performing plate coating on MRS + bromocresol purple and MRS + mupirocin lithium salt selective culture media, placing at 37 ℃, performing anaerobic overnight culture, selecting a single colony for purification, and then performing 16S rDNA strain identification.

16S rDNA identification: extracting genome DNA by adopting a universal primer:

27F：AGAGTTTGATCCTGGCTCAG；

1492R：TACGGCTACCTTGTTACGACTT

PCR amplification of 16S rDNA fragment is carried out, the amplified fragment is submitted to the department of Oncology company for sequencing, and the sequencing result is shown in the sequence table SEQ ID NO. 1. Through comparison, a strain of Lactobacillus salivarius is obtained, named as Lactobacillus salivarius MG-587 and preserved in the China general microbiological culture Collection center (address: china, beijing area of the open world, postal code: 100101) at 04 th 2021, with the preservation numbers: CGMCC No. 21588.

2. Cholic acid and choline resistance detection

MRS culture media with pH 2.0 (cholic acid) and MRS culture media containing 0.3% choline salt are prepared respectively, strain mother liquor is inoculated in the MRS culture media, the pH 2.0 (cholic acid) and the MRS culture media containing 0.3% choline salt respectively in an inoculation amount of 5%, and plates are counted after 2 hours of treatment, and the results are shown in the following table:

the results show that the Lactobacillus salivarius MG-587 cannot survive under the condition of pH 2.0 cholic acid, but can survive under the condition of 0.3% choline salt, namely the strain has the effect of resisting choline salt, and is more beneficial to the application of the strain in human bodies.

3. Detection of ability to produce small molecule acids

(1) Sample treatment: taking 1mL of strain mother liquor, carrying out centrifugation at 5000r/min for 10min, and reserving supernatant.

(2) And (3) chromatographic column: 7890A-5975C GC MS/MS, VF-WAXms (30m0.25mm0.25 μm) capillary chromatography column.

(3) And (3) sample introduction mode: and (3) headspace sample injection, constant temperature furnace temperature of 70 ℃, sample flow path temperature of 90 ℃, transmission line temperature of 110 ℃, sample bottle pressurization pressure of 160.0kPa, sample bottle constant temperature time of 10min, sample bottle pressurization time of 1min and GC cycle time of 22min.

(4) GC operation: temperature programming is carried out, the initial temperature is 100 ℃, and after the temperature is increased to 150 ℃ at the speed of 5 ℃/min, the temperature is increased to 240 ℃ and kept for 3min; the sample inlet temperature is 230 ℃, the auxiliary temperature is 240 ℃, the quadrupole temperature is 150 ℃, the ion source temperature is 230 ℃, the sample injection amount is 1 mu L, and the sample injection mode is as follows: non-shunt sample introduction and carrier gas: high purity nitrogen (> 99.999%), carrier gas flow rate: 2mL/min.

The results are shown in the following table:

the results show that the Lactobacillus salivarius MG-587 can produce small molecule acid, such as acetic acid, butyric acid, etc.

4. Detection of antibiotic resistance in bacterial strains

And detecting the resistance of the candidate strain to the antibiotic by a double-layer plate method. The lower layer of the double-layer flat plate is 1.5 percent of agar nutrient medium, and the upper layer of the double-layer flat plate is prepared by mixing 0.5 percent of soft agar nutrient medium with a strain mother liquor of 100:1 and mixing uniformly. After the plate was solidified and the surface dried, commercial antibiotic susceptibility tablets (Tet, cm, fz, amp, ery, NA, rif, gen and Neo) were placed and cultured overnight, and the resistance of the probiotic to the antibiotic was observed according to the size of the zone of inhibition, as shown in the following table, wherein the zone of inhibition indicates that the bacteria are susceptible to the antibiotic:

the results show that said Lactobacillus salivarius MG-587 is insensitive to, i.e. resistant to, some antibiotics, including Cm (chloramphenicol), neo (neomycin), gen (gentamicin) and NA (nafcillin).

5. Coagulation test assay for bacterial strains

(1) KRT buffer solution (NaCl 7.5g, KCl 0.383g, mgSO 7.5g ₄ ·7H ₂ O 0.318g，CaCl ₂ 0.305g, pH 7.4 adjusted with hydrochloric acid);

(2) Washing fresh sheep blood red blood cells once by using PBS buffer solution, centrifuging at 3000rpm for 5min, and resuspending in KRT buffer solution at a final concentration of 10%;

(3) The overnight culture of the strain is diluted in KRT buffer solution by 2 times gradient;

(4) After 25. Mu.L of sheep blood erythrocytes were added to the bacterial suspension (25. Mu.L) diluted in a gradient, the aggregation was detected after 2 hours at room temperature, and the results are shown in the following table:

in general, the Escherichia coli is diluted 8 times, and the Lactobacillus salivarius MG-587 is diluted 6 times according to the above results. The bacterial strain does not have the coagulation phenomenon, so that the bacterial strain is not easy to cause the antigen reaction of the organism, namely the side effect is small, and the bacterial strain can be applied to the human body as probiotics.

6. Evaluation of pathogenicity of Strain

The bacterial strain to be evaluated is infected with the animal through two ways of intraperitoneal injection and oral gavage, and the pathogenicity of the bacterial strain to the animal is evaluated.

(1) Experimental animals: c57 mice and males with the weight of 18.0 g-22.0 g are selected.

(2) And (3) intraperitoneal injection:

strain activation and strain suspension preparation: inoculating strain into MRS broth, culturing at 37 deg.C under anaerobic condition for 24hr, transferring to MRS agar plate, culturing for 24hr, scraping colony on the plate, and suspending inMixing with sterile normal saline, adjusting the concentration of the bacterial suspension with appropriate amount of sterile normal saline, and turbidimetry to obtain final concentration of thallus cells of 1 × 10 ⁸ CFU/mL (requirement at least 10) ⁷ CFU/only).

Mice were grouped, including: a mouse bacterial suspension inactivation control group and a mouse bacterial suspension group. Each group of 10 mice was injected with 0.2mL of a 2X 10 bacterial dose per mouse ⁷ CFU/only.

Animals were observed 1 time per day after i.p. injection for at least 21 days. The animals were observed for the following (but not limited to) abnormalities: (1) skin and hair; (2) eyes and mucous membranes; (3) a respiratory system; (4) limb movement; (5) a behavioral pattern; (6) Specially pay attention to the observation of phenomena such as tremor, convulsion, diarrhea, lethargy, salivation, coma and the like; (7) body weight: all mice were weighed before and weekly after injection and the body weight of dead, eventually sacrificed mice during the experiment was measured. If the survival time of the mouse exceeds 1d, recording the weight change of the mouse; (8) the death time of the mice was recorded as accurately as possible. The results of observation of abnormal characteristics after intraperitoneal injection are shown in table 1, and the changes in body weight of mice before and after injection are shown in table 2.

TABLE 1 Observation of abnormal characteristics after intraperitoneal injection

TABLE 2 weight changes of mice before and after intraperitoneal injection

(3) Via oral gavage

Strain activation and bacterial suspension preparation: except that the concentration of the prepared bacterial suspension is 5.0 multiplied by 10 ⁸ CFU/mL (not less than 2.5X 10 is required) ⁸ CFU/mL), othersThe operation steps are the same as above.

Mice were grouped, including: and (3) respectively performing intragastric administration on the mouse by using the mouse bacterial suspension inactivation control group and the male mouse bacterial suspension group at the dose of 2.0mL/100 g.BW, and feeding the mice after the intragastric administration for 3-4 h after the one-night fasting.

After oral gavage, the observation is carried out for 1 time every day for at least 21 days, and the observation method is the same as that of the observation. The observation results of abnormal characteristics after oral gavage are shown in table 3, and the body weight changes of mice before and after gavage are shown in table 4.

TABLE 3 Observation of abnormal characteristics after oral gavage

TABLE 4 weight changes of mice before and after gastric lavage

By combining the observation results in tables 1-4, after the Lactobacillus salivarius MG-587 bacterial suspension is subjected to intraperitoneal injection or intragastric perfusion treatment for 3 weeks, the mice do not have abnormal bodies, grow normally and maintain normal body weight, which indicates that the strain is high in safety, nontoxic and nonpathogenic.

Example 2 lipid-lowering efficacy of Lactobacillus salivarius MG-587

1. Fat cell assay

Cell differentiation induction: when the cell confluence rate is 70-80%, according to

CL-173 ^TM The cells were harvested by trypsinization as provided in the product instructions in connection with subculture. Inoculating cells into preadipocyte expansion medium(90% DMEM,10% calf serum), for 48 hours, or until the culture reaches 100% confluence. After another 48 hours of incubation, preadipocyte expansion medium was removed from each well and the same volume of differentiation medium was added (90% DMEM,10% fetal bovine serum FBS, 1.0. Mu.M dexamethasone, 0.5mM methyl isobutyl xanthine (IBMX), 10. Mu.g/mL insulin). Simultaneously, according to the following steps of 1:1000, adding inactivated bacteria solution.

After 48 hours of incubation in differentiation medium, the differentiation medium was replaced with adipocyte maintenance medium (90% DMEM,10% fetal bovine serum, 10. Mu.g/mL insulin), and the adipocyte maintenance medium was replaced every 48 hours. Between 6 and 10 days after induction, lipid droplet formation was observed by oil red staining. The content of triglyceride in the cells is detected by the kit.

(1) Oil red dyeing

Preparing oil red staining solution: dissolving 1g of oil red in 200mL of 60% isopropanol, mixing uniformly, stirring and dissolving to prepare a storage solution. Storing at 4 ℃ in the dark. Temporary use 3:2 preparing stock solution with distilled water, standing for a while, and filtering.

Dyeing: the fat cells are washed by PBS for 2 times, fixed in 4% formaldehyde for 20min, discarded, washed by PBS for 1-2 times, washed by oil red O staining solution for 0.5-1h, quickly rinsed by 60% isopropanol for 3s, washed by PBS for 2-3 times, and observed by a microscope, wherein the more red, the more oil drops.

The observation results are shown in FIG. 1, in which FIGS. 1-A and 1-B are blank control groups without probiotic bacteria, and FIGS. 1-C and 1-D are the results of adipocytes for the prognosis of Lactobacillus salivarius MG-587 stem. The results show that the number of fat cells is remarkably reduced after the Lactobacillus salivarius MG-587 is intervened in vitro compared with a blank control group, namely the Lactobacillus salivarius MG-587 has certain lipid-lowering effect.

(2) Measurement of triglyceride content in adipocytes

Performed according to the Triglyceride (TG) test kit instructions (Nanjing Pan-built A110-1-1).

1) Cell collection: taking out the prepared cell suspension, centrifuging for 10 minutes at 1000 rpm, removing supernatant, and leaving cell precipitate; washing with phosphate buffer solution for 1-2 times at the same speed of 1000 rpm, centrifuging for 10min, removing supernatant, and collecting cell precipitate;

2) Cell disruption: adding 0.2-0.3mL buffer solution for homogenizing, performing ultrasonic disruption in ice water bath (300W, 3-5 s/time, 30 s interval, 3-5 times repetition), and directly measuring the prepared homogenate without centrifugation.

3) Calculating the formula: enzyme-linked immunosorbent assay: triglyceride content (mmol/gprot) = (sample OD value-blank OD value)/(calibration OD value-blank OD value) × standard concentration (mmol/L) ÷ protein concentration of the sample to be measured (gprot/L, which can be directly calculated from the sample concentration) as shown in the following table:

strain of bacillus	Triglyceride content (mmol/L)
		Control	0.728342
Lactobacillus salivarius MG-587	0.624147

The results show that the content of triglyceride in adipocytes is remarkably reduced after the Lactobacillus salivarius MG-587 is dried, and further confirm that the Lactobacillus salivarius MG-587 has excellent lipid-lowering efficacy.

2. Bile Salt Hydrolase (BSH) activity assay

0.5. Mu.L of fresh culture broth of Lactobacillus salivarius MG-587 was inoculated dropwise into MRS medium and MRS +0.5% TDCA (choline salt), respectively, and cultured overnight at 37 ℃ to observe the formation of a colony-surrounding zone, as shown in FIG. 2. The results show that, compared with the MRS medium, a larger precipitation ring appears on the TDCA-added medium, which indicates that the strain has a very excellent bile salt hydrolase activity, i.e., that the strain can tolerate the bile salt pressure in the intestinal tract, so that the colonization effect of the strain in the intestinal tract can be enhanced, the survival rate and stability in the intestinal tract can be improved, and the effect of the probiotic can be exerted to the maximum extent, and the strain can also participate in the bile acid regulation to achieve the effect of reducing cholesterol.

Example 3 animal experiments with Lactobacillus salivarius MG-587

1. Material

Preparing a bacterial suspension: after the strain is activated and inoculated twice, the strain is cultured to a stationary phase under a suitable culture condition at 6000rpm/min, and the strain is centrifuged for 10min to collect the strain and washed by PBS for heavy suspension.

Grouping: a conventional diet control group, a high fat diet control group (one rat and two biotechnology limited, yozhou rat), and a probiotic-treated group, each of which contains 5 mice.

2. Establishment of high-fat mouse model

1) Experimental animals: male C57BL/6J mice (SPF grade) of 8 weeks old were kept in an environment of constant temperature 25. + -. 2 ℃ and relative humidity 50. + -. 5 with a 12/12h day-night cycle.

2) After one week of adaptation to the Normal Diet (ND), the diets were randomized into two groups according to body weight, one group was given a normal maintenance diet as a blank control, and the remaining experimental mice were fed a High Fat Diet (HFD) for 8 weeks to induce obesity. During which food intake was recorded weekly and body weight was weighed. Before formal experiments, the weight change condition is observed, mice without obvious weight change are eliminated, and the mice are regrouped according to the average value no difference among groups.

3) Wherein the conventional feed control group (ND group) is on normal diet; high fat diet control group (HFD group) high fat diet; the probiotic treatment groups were: the high-fat mouse model was continuously fed with high-fat diet and gavage probiotic (1 × 10) ⁸ CFU/mL), the gavage amount is 200 mu L; the ND group and HFD group were gavaged with PBS buffer solution of the same volume as the former, gavaged at the same time every day, and fed during gavage feeding period, and the body weight (g) and food intake of the mice were measured periodicallyAmount (v).

The results of the body weight statistics of the conventional diet control group (ND group) are shown in table 5, the results of the body weight statistics of the high fat diet control group (HFD group) are shown in table 6, and the results of the body weight statistics of the probiotic-treated group are shown in table 7.

Table 5 statistical results for body weight in ND group

TABLE 6 statistical body weight in HFD group

Table 7 weight statistics of probiotic treated groups

By combining the statistical results in tables 5-7, compared with the high fat diet control group (HFD group), the weight of the mice is significantly reduced after the Lactobacillus salivarius MG-587 dry prognosis, which indicates that the Lactobacillus salivarius MG-587 dry prognosis has a certain fat-reducing effect, and the bacteria can be prepared into weight-reducing drugs, functional foods or health-care foods.

3. Index detection

After the completion of the gavage experiment, the mouse feces were collected. Animals were fasted overnight. Mice were sacrificed and blood, fat, liver were rapidly removed. The blood samples were centrifuged at 1000-2500rpm/min for 15min and serum was collected for the following analysis. All samples were stored at-80 ℃ prior to use. Each mouse was dissected accurately, weighed and collected for further analysis (3 whole samples were taken for weighing). The results of measuring the liver weight (g) are shown in table 8, and the results of measuring the fat weight are shown in table 9.

Table 8 liver weight measurement results

TABLE 9 fat weight measurement results

By combining the detection results in tables 8 and 9, compared with the HFD group, the weight of the liver and the weight of fat in the mice are reduced to different degrees after the intervention of Lactobacillus salivarius MG-587, and the lipid-lowering effect of the strain is further illustrated.

4. Biochemical analysis

(1) Biochemical parameters of serum

According to the kit for four items of established blood fat in Nanjing, the blood fat levels including cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL-C) and high-density lipoprotein (HDL-C) are measured. According to the requirements of ALT and AST kits, the content changes of alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) in serum are measured to reflect the liver function status. The results are shown in tables 10 to 11.

TABLE 10 results of four tests for blood fat

TABLE 11 ALT and AST content determination

According to the detection results in tables 10 and 11, compared with the HFD group, after intervention of Lactobacillus salivarius MG-587, the contents of cholesterol, triglyceride and low-density lipoprotein in the mice are all remarkably reduced, and the content of high-density lipoprotein is in a relatively balanced state, so that the probiotic has excellent blood fat reducing effect; meanwhile, the content of alanine aminotransferase and aspartate aminotransferase is also obviously reduced, which shows that the liver function state of the mice is improved compared with that of the HFD group mice after the intervention of Lactobacillus salivarius MG-587.

(2) Blood glucose determination

Blood glucose levels of mouse tail vein whole blood were measured using a glucometer (LifeScan). Blood glucose levels were measured at 0min,30min,75min and 120min, respectively, 1d before sacrifice, after fasting for 12h, i.e., intraperitoneal injection of glucose at 5g/kg, after tail trimming.

TABLE 12 blood glucose measurement results

The result shows that compared with the HFD group, after the intervention of Lactobacillus salivarius MG-587 for 120 minutes, the blood sugar level of the mice is obviously reduced, and reaches the level equivalent to that of the ND group, which indicates that the strain has very excellent blood sugar reducing effect, and can be prepared into blood sugar reducing medicines, functional foods or health foods for regulating blood sugar of hyperglycemia patients.

(3) Histopathological analysis

The same part of liver, 40mg (0.5 cm. Times.0.5 cm), and about 50mg of adipose tissue were fixed in 4% paraformaldehyde for 24 hours, and HE stained. The stained slides were observed by inverted microscope and image acquisition was performed, white circles representing fat vacuoles, more circles representing more severe fatty liver.

The results of observation of liver tissue are shown in fig. 3, and the results of observation of adipose tissue are shown in fig. 4, in which a is ND group, B is HFD group, and C and D are probiotic-treated groups. Compared with the ND group, the HFD group has more fat vacuoles in liver tissues, large fat cells and disordered arrangement, which indicates that the fatty liver is serious, and compared with the HFD group, after probiotic bacteria are dried by Lactobacillus salivarius MG-587, the fat vacuoles in the liver tissues of mice are obviously reduced, and the fat cells are reduced and arranged orderly, which indicates that the strain has a certain relieving and treating effect on the fatty liver, so that the strain can be prepared into a medicament, a functional food or a health-care food for treating and/or preventing the fatty liver.

(4) Inflammatory factor detection

Serum of each group of mice is taken, and six different inflammatory factor contents of IL-6, IL-10, MCP-1, IFN-gamma, TNF and IL-12 are detected by using a CBA mouse inflammatory multifactorial factor kit. The specific steps were performed according to the kit instructions (CBA # 560485). Firstly, fully mixing six different microspheres in a vortex mode according to actual using amount, adding 30ul of mixed microspheres into each experimental tube, adding 25ul of PE-labeled cytokine detection antibody, and incubating for 3 hours at room temperature in a dark place. Before loading, each tube was washed with wash solution, centrifuged at 200g for 5min, the supernatant was decanted, and 300ul of wash solution was added to resuspend the cells. After the samples were prepared, they were measured by flow cytometry, and the results were analyzed, and the average values of the results of the measurements of different inflammatory factors are shown in Table 13.

TABLE 13 inflammatory factor test results

The results show that the content of the inflammatory factors of the mice is not obviously abnormal after the intervention of Lactobacillus salivarius MG-587.

In conclusion, the Lactobacillus salivarius MG-587 is separated and screened from human excrement for the first time, has excellent effects of reducing blood fat, alleviating fatty liver and reducing blood sugar, is prepared into medicines or health-care foods for treating and preventing diseases such as hyperlipidemia and hyperglycemia, and has wide application value.

Example 4 genomic analysis of Lactobacillus salivarius MG-587

Purifying the Lactobacillus salivarius MG-587 again, extracting DNA, and sending to An Nuo Youda Gene technology (Beijing) Limited company for whole genome re-sequencing; after the DNA sample is received, the sample is tested, and the DNA sample qualified by electrophoresis is randomly broken into fragments with the length of about 350bp by a Covaris ultrasonic disruptor. After the treatment, the DNA fragment is subjected to end repair, A tail addition, sequencing joint addition, purification, PCR amplification and other steps to complete the preparation of the whole library; after the library is constructed, firstly, carrying out primary quantification by using Qubit2.0, diluting the library to 2ng/ul, then, detecting an insert fragment (insert size) of the library by using Agilent 2100, and accurately quantifying the effective concentration of the library by using a Q-PCR method after the insert size meets the expectation so as to ensure the quality of the library; and after the library is qualified, carrying out Illumina Novaseq sequencing on different libraries according to the effective concentration and the requirement of target offline data volume.

The genome map of Lactobacillus salivarius MG-587 is shown in FIG. 5, and the whole genome sequence of the strain is compared with the existing database, wherein the comparison result in virulence/pathogenic island database (PAIDB) is shown in FIG. 6, and the comparison result in virulence gene database (VFDB) is shown in Table 14.

TABLE 14 VFDB database alignment results

http://www.mgc.ac.cn/cgi-bin/VFs/v5/main.cgiJobID＝Dec_2-435516720

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.

Sequence listing

<110> Meiyi Adoration biomedical (Wuhan) Co., ltd

<120> lactobacillus salivarius MG-587 strain and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

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<211> 899

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<213> Lactobacillus salivarius MG-587

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tggcgtccgc tatgatgcaa gtcgaacgaa actttcttac accgaatgct tgcattcacc 60

gtaagaagtt gagtggcgga cgggtgagta acacgtgggt aacctgccta aaagaagggg 120

ataacacttg gaaacaggtg ctaataccgt atatctctaa ggatcgcatg atccttagat 180

gaaagatggt tctgctatcg cttttagatg gacccgcggc gtattaacta gttggtgggg 240

taacggccta ccaaggtgat gatacgtagc cgaactgaga ggttgatcgg ccacattggg 300

actgagacac ggcccaaact cctacgggag gcagcagtag ggaatcttcc acaatggacg 360

caagtctgat ggagcaacgc cgcgtgagtg aagaaggtct tcggatcgta aaactctgtt 420

gttagagaag aacacgagtg agagtaactg ttcattcgat gacggtatct aaccagcaag 480

tcacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttgtccggat 540

ttattgggcg taaagggaac gcaggcggtc ttttaagtct gatgtgaaag ccttcggctt 600

aaccggagta gtgcattgga aactggaaga cttgagtgca gaagaggaga gtggaactcc 660

atgtgtagcg gtgaaatgcg tagatatatg gaagaacacc agtggcgaaa gcggctctct 720

ggtctgtaac tgacgctgag gttcgaaagc gtgggtagca aacaggatta gataccctgg 780

tagtccacgc cgtaaacgat gaatgctagg tgttggaggg tttccgccct tcagtgccgc 840

agctaacgca ataagcattc cgcctgggga gtacgaccgc aaggttgaaa ctcaaagga 899

Claims

1. Lactobacillus salivariusLactobacillus salivariusMG-587, wherein said Lactobacillus salivarius isLactobacillus salivariusThe MG-587 has a deposit number of: CGMCC No. 21588.

2. Lactobacillus salivarius according to claim 1Lactobacillus salivariusApplication of MG-587 in preparing medicine for treating and/or preventing hyperlipemia.

3. Use according to claim 2, wherein the Lactobacillus salivarius is present in a composition comprising a lipid fraction derived from Lactobacillus salivarius and a lipid fraction derived from Lactobacillus salivariusLactobacillus salivariusMG-587 lowers blood lipids by lowering cholesterol, triglycerides and low density lipoproteins.

4. Lactobacillus salivarius according to claim 1Lactobacillus salivariusMG-587 in preparing hypoglycemic medicine.

5. Lactobacillus salivarius according to claim 1Lactobacillus salivariusApplication of MG-587 in preparing medicine for treating and/or preventing fatty liver is provided.

6. Lactobacillus salivarius according to claim 1Lactobacillus salivariusApplication of MG-587 in preparing weight reducing medicine is provided.

7. The use according to claim 2 or claim 4 or claim 5 or claim 6, wherein the medicament further comprises a pharmaceutically acceptable excipient.

8. Use according to claim 7, wherein the medicament is a tablet, capsule, powder, pill, granule or solution.

9. A probiotic inoculum, comprising: comprising the Lactobacillus salivarius according to claim 1Lactobacillus salivariusMG-587, or a bacterium solution containing the sameLactobacillus salivariusDry powder microbial inoculum of MG-587.