阅读说明：本技术 一株可干预代谢综合征的罗伊氏乳杆菌ccfm1178及应用 (Lactobacillus reuteri CCFM1178 capable of intervening metabolic syndrome and application thereof ) 是由 杨波 陈卫 郑富莉 崔树茂 王刚 翟齐啸 赵建新 张灏 于 2021-07-08 设计创作，主要内容包括：本发明公开了一株可干预代谢综合征的罗伊氏乳杆菌CCFM1178及应用,属于微生物技术领域。本发明筛选的罗伊氏乳杆菌CCFM1178能显著改善高脂饮食导致的代谢综合征小鼠血清总胆固醇含量和低密度脂蛋白胆固醇的升高,缓解代谢综合征个体体重上升,降低每日饮食摄入和能量转化效率；改善由高脂导致的小鼠肠道菌群失衡；改善代谢综合征小鼠肝脏中促炎因子IL-6和TNF-α的水平,提高抗炎因子IL-10水平来预防代谢综合征,抑制血清脂多糖内毒素和维持结肠屏障结构。本发明的罗伊氏乳杆菌CCFM1178可用于制备缓解代谢综合征、非酒精性脂肪肝、糖尿病等疾病发生的药物或功能性食品,具有非常广泛的应用前景。(The invention discloses lactobacillus reuteri CCFM1178 capable of intervening in metabolic syndrome and application thereof, and belongs to the technical field of microorganisms. The lactobacillus reuteri CCFM1178 screened by the method can obviously improve the serum total cholesterol content and the low-density lipoprotein cholesterol increase of a mouse with metabolic syndrome caused by high-fat diet, relieve the weight rise of an individual with the metabolic syndrome, and reduce daily diet intake and energy conversion efficiency; improving the imbalance of intestinal flora of mice caused by high fat; improving the level of proinflammatory factors IL-6 and TNF-alpha in the liver of a mouse with metabolic syndrome, improving the level of an anti-inflammatory factor IL-10 to prevent the metabolic syndrome, inhibiting serum lipopolysaccharide endotoxin and maintaining the colon barrier structure. The lactobacillus reuteri CCFM1178 can be used for preparing medicines or functional foods for relieving the diseases such as metabolic syndrome, non-alcoholic fatty liver, diabetes and the like, and has very wide application prospect.)

1. Lactobacillus reuteri (Lactobacillus reuteri) CCFM1178, which was deposited with the Guangdong province collection of microorganisms at 19.3.2021 with the deposit number GDMCC No. 61574.

2. Use of lactobacillus reuteri CCFM1178 according to claim 1 for the preparation of a medicament.

3. The use according to claim 2, wherein the medicament has at least one of the following functions:

(a) relief of metabolic syndrome;

(b) regulating intestinal flora;

(c) relieving irritable bowel syndrome;

(d) relieving inflammation and/or fibrosis of liver tissue.

4. A composition comprising lactobacillus reuteri CCFM1178 of claim 1.

5. The composition of claim 4, wherein the composition is a microbial agent; the microbial preparation comprises wet cells or lyophilized cells of Lactobacillus reuteri CCFM1178 of claim 1.

6. The composition of claim 4, wherein the composition is a functional food or health product.

7. The composition of claim 4, wherein the composition is a fermented food product; the fermented food is prepared by fermenting Lactobacillus reuteri CCFM1178, and comprises solid food, liquid food or semisolid food.

8. The medicament containing the Lactobacillus reuteri CCFM1178 of claim 1, wherein the content of the Lactobacillus reuteri CCFM1178 in the medicament is more than or equal to 1 x 10⁸CFU/g or 1X 10⁸CFU/mL。

9. The medicament of claim 8, further comprising a pharmaceutically acceptable excipient.

10. Use of lactobacillus reuteri CCFM1178 according to claim 1 for the preparation of a fermented food product.

Technical Field

The invention relates to lactobacillus reuteri CCFM1178 capable of intervening in metabolic syndrome and application thereof, belonging to the technical field of microorganisms.

Background

Metabolic Syndrome (MS) is a group of clinical syndromes that are characterized by the presence of central obesity, hyperglycemia (diabetes or impaired glucose regulation), dyslipidemia (including high TG and/or low HDL-C blood disease), hypertension, etc., and the accumulation of various risk factors based on pathological changes of glucose metabolism and lipid metabolism, and the promotion of development of various cardiovascular and cerebrovascular diseases such as type II diabetes and atherosclerosis. In recent years, with the continuous development of the economic and social level of China, the living standard of the public is continuously improved, the specific gravity of carbohydrates and grease in the dietary structure is increased day by day, and the people have a habit of sedentary sitting due to the limitation of various working conditions. The external environmental factors are combined with the genetic factors of the external environmental factors to greatly improve the prevalence rate of the metabolic syndrome of the population in China. It has been shown that about 7700 million patients with metabolic syndrome exist in the country, i.e. 1 in 8 adults have metabolic syndrome. The prevalence rate of metabolic syndrome of old people over 60 years old in China is 25%, and the prevalence rate is gradually increased with the age. Therefore, it is necessary to find a suitable means for alleviating metabolic syndrome.

The treatment measures adopted aiming at the metabolic syndrome at present mainly comprise strengthening exercise and improving diet and combining drug therapy, and the aims are all various risk factors for controlling and improving the metabolic syndrome, and mainly comprise weight-reducing drugs such as orlistat and lorcaserin; insulin resistance reducing agents such as metformin, thiazolidinediones (rosiglitazone, pioglitazone, etc.); fibrates that lower blood lipids such as fenofibrate, and statins that lower cholesterol such as simvastatin, etc.; angiotensin converting enzyme inhibitor for lowering blood pressure such as captopril. These drugs have good curative effects on single risk factors of metabolic syndrome, but have certain side effects after long-term administration, such as stimulation of gastrointestinal tract of some patients caused by long-term administration of metformin and possible influence on absorption of vitamin B12 by the patients, and rosiglitazone caused liver function damage and edema.

There are many studies reporting that the intestinal flora plays an important role in the physiological metabolism of the human body, and the dysbacteriosis of the intestinal flora is related to various diseases, including gastrointestinal diseases (irritable bowel syndrome, inflammatory bowel disease and the like), metabolic diseases (obesity, hyperlipidemia, diabetes and the like). The occurrence of metabolic syndrome is closely related to the imbalance of intestinal flora, and common intestinal flora regulating preparations comprise probiotics, prebiotics and the like. The probiotics is edible microorganisms beneficial to human health, and has the potential functions of relieving blood sugar and dyslipidemia, and regulating intestinal flora structure and brain and intestinal axis. For example, CN107699517A discloses a bifidobacterium adolescentis strain and application thereof, which can obviously improve pathological damage of liver and duodenum of a mouse with metabolic syndrome caused by high fat diet, and increase of triglyceride and total cholesterol content and oral glucose tolerance in serum; CN107523526A discloses a Lactobacillus reuteri and its use, which can reduce the serum lipid and blood sugar level of mice with metabolic syndrome; CN105567586A discloses a Lactobacillus plantarum NCU116 with an anti-diabetic function, which can achieve the anti-diabetic effect by regulating the blood sugar, blood fat and hormone levels of the organism and regulating the metabolic pathways in the organism. However, the above patents all use probiotics to relieve metabolic syndrome, and few studies are currently made on how probiotics inhibit endotoxin and protect colon intestinal barrier.

Disclosure of Invention

The invention aims to provide Lactobacillus reuteri (Lactobacillus reuteri) CCFM1178 which is preserved in Guangdong province microorganism culture preservation center at 19.03.2021, with the preservation number being GDMCC No.61574 and the preservation address being No. 59 building 5 of Miyaolu 100, Guangzhou city.

The lactobacillus reuteri CCFM1178 has the following biological characteristics:

(1) the characteristics of the thallus are as follows: the product is gram-positive, slender rod-shaped, has no flagellum and no spore;

(2) colony characteristics: culturing for 36 hours to form obvious convex colonies, wherein the edges are irregular, milky white and opaque, the surface is moist and smooth, and no pigment is generated;

(3) growth characteristics: culturing in MRS culture medium at 37 deg.C for about 18 hr to reach stationary phase;

(4) the medicinal safety is as follows: has no toxic and side effects on C57BL/6J mice.

The Lactobacillus reuteri (Lactobacillus reuteri) CCFM1178 can relieve metabolic syndrome, regulate intestinal flora and relieve irritable bowel syndrome, and is specifically embodied in that:

(1) reducing the body weight of individuals with metabolic syndrome, and improving high fat diet to cause excessive weight gain;

(2) reducing fasting blood glucose level of individuals with metabolic syndrome, and improving insulin resistance index of individuals with metabolic syndrome;

(3) reducing serum total cholesterol in individuals with high fat diet-induced metabolic syndrome;

(4) restoring abnormal intestinal flora of individuals with metabolic syndrome caused by high fat;

(5) serum low density lipoprotein of individual with metabolic syndrome;

(6) reducing body fat and epididymis fat index of individual with metabolic syndrome;

(7) has positive effect on bacterial lipopolysaccharide endotoxin induced inflammation;

(8) helping to maintain the colon barrier structure.

The second purpose of the invention is to provide the application of the lactobacillus reuteri CCFM1178 in preparing medicines.

In one embodiment, the medicament has at least one of the following functions:

(a) relief of metabolic syndrome;

(b) regulating intestinal flora;

(c) relieving irritable bowel syndrome;

(d) relieving inflammation and fibrosis of liver tissue.

In one embodiment, the symptoms exhibited by the metabolic syndrome include hyperglycemia, hyperlipidemia, and/or hepatic oxidative stress.

In one embodiment, the content of the Lactobacillus reuteri CCFM1178 in the medicament is more than or equal to 1 x 10⁸CFU/g or 1X 10⁸CFU/mL。

In one embodiment, the medicament further comprises a pharmaceutically acceptable excipient.

In one embodiment, the pharmaceutically acceptable excipients include one or more of fillers, binders, wetting agents, disintegrants, lubricants, flavoring agents.

In one embodiment, the dosage form of the medicament is granules, capsules, tablets, pills or oral liquid.

The third purpose of the invention is to provide a composition containing the lactobacillus reuteri CCFM 1178.

In one embodiment, the composition is a microbial preparation comprising wet cells or lyophilized cells of said lactobacillus reuteri CCFM 1178.

In one embodiment, the viable count of Lactobacillus reuteri CCFM1178 in the microbial preparation is greater than or equal to 1 × 10⁶CFU/g。

In one embodiment, the microbial preparation is prepared by: inoculating Lactobacillus reuteri CCFM1178 into MRS culture medium, culturing at 37 deg.C under anaerobic condition for 18-20 hr, collecting thallus, and resuspending thallus cells with protective agent to make the concentration of thallus reach 1 × 10¹⁰CFU/ml, culturing the suspension at 37 deg.C under anaerobic condition for 50-70min, and drying.

In one embodiment, the MRS medium contains per liter: 10g of tryptone, 10g of beef extract, 5g of yeast powder, 20g of glucose, 5g of sodium acetate, 2g of diammonium hydrogen citrate, 2g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate heptahydrate, 801ml of tween and 0.25g of manganese sulfate monohydrate.

In one embodiment, the protective agent comprises skimmed milk powder, maltodextrin, trehalose.

In one embodiment, the protectant comprises: 100g/L-150g/L skimmed milk powder, 100g/L-150g/L maltodextrin and 140g/L-160g/L trehalose.

In one embodiment, the collected bacterial cells are washed 2-4 times with a phosphate buffer having a pH of 6.8-7.2.

In one embodiment, the drying may be performed by any one of a number of methods including, but not limited to, vacuum freeze drying.

In one embodiment, the vacuum freeze-drying is performed after pre-freezing at-15 to-20 ℃ for 8 to 14 hours.

In one embodiment, the composition is a functional food or nutraceutical.

In one embodiment, the composition is a fermented food product produced by fermentation using lactobacillus reuteri CCFM1178, including solid food products, liquid food products, semi-solid food products.

In one embodiment, the fermented food product comprises a dairy product, a soy product, or a fruit and vegetable product; the dairy product comprises milk, sour cream or cheese; the raw materials of the fruit and vegetable product comprise cucumber, carrot, beet, celery or cabbage product.

The invention also claims the application of the lactobacillus reuteri CCFM1178 in preparing food or health care products.

The invention has the following beneficial effects and advantages:

the lactobacillus reuteri CCFM1178 has the effect of improving metabolic syndrome; specifically, the amelioration of metabolic syndrome includes: reducing the body weight of a subject with metabolic syndrome, reducing the fasting blood glucose level of a subject with metabolic syndrome, reducing the glucose tolerance of a subject with metabolic syndrome, reducing serum total cholesterol in a mouse with high fat diet-induced metabolic syndrome, reducing serum low density lipoprotein in a subject with metabolic syndrome, reducing the body fat of a subject with metabolic syndrome, and/or reducing the epididymal fat index and increasing the brown fat index of a subject with metabolic syndrome; restoring the imbalance of the intestinal flora of the mice caused by high fat; can restore the abnormal abundance of flora in the excrement of mice with the high fat diet induced metabolic syndrome, has positive effect on the induction of inflammation by bacterial lipopolysaccharide endotoxin, and has better effect on maintaining the colon barrier structure. The lactobacillus reuteri CCFM1178 can be used for preparing health-care food or medicine for relieving metabolic syndrome and regulating intestinal flora to relieve irritable bowel syndrome, and has very wide application prospect.

Biological material preservation

Lactobacillus reuteri (Lactobacillus reuteri) is classified and named as Lactobacillus reuteri, and is preserved in Guangdong province microorganism culture collection center in 2021, 3 and 19 days, and the preservation address is No. 59 building 5 of Michelia Tokyo No. 100, Michelia Tokyo, Guangzhou city, and the preservation number is GDMCC No. 61574.

Drawings

FIG. 1 shows the effect of Lactobacillus reuteri CCFM1178 on the intestinal flora of mice with metabolic syndrome;

FIG. 2 shows the effect of Lactobacillus reuteri CCFM1178 on the percent weight gain, epididymal fat index and brown fat index in mice with metabolic syndrome;

FIG. 3 shows the effect of Lactobacillus reuteri CCFM1178 on the daily dietary intake and energy conversion efficiency of the mice with metabolic syndrome;

FIG. 4 shows the effect of Lactobacillus reuteri CCFM1178 on serum Total Cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) in mice with metabolic syndrome;

FIG. 5 shows the effect of Lactobacillus reuteri CCFM1178 on the hepatic proinflammatory factors IL-6 and TNF- α in mice with metabolic syndrome;

FIG. 6 shows the effect of Lactobacillus reuteri CCFM1178 on the hepatic anti-inflammatory factor IL-10 in mice with metabolic syndrome;

FIG. 7 shows the effect of Lactobacillus reuteri CCFM1178 on serum Lipopolysaccharide (LPS) endotoxin in mice with metabolic syndrome;

FIG. 8 shows the effect of Lactobacillus reuteri CCFM1178 on the expression level of the zon-1, Occludin and Claudin-1 genes in colon tissue of the metabolic syndrome mouse;

(Note: p <0.05, p <0.01)

Detailed Description

The lactobacillus reuteri CCFM1178 has the following biological characteristics:

(1) the characteristics of the thallus are as follows: the product is gram-positive, slender rod-shaped, has no flagellum and no spore;

(2) colony characteristics: culturing for 36 hours to form obvious convex colonies, wherein the edges are irregular, milky white and opaque, the surface is moist and smooth, and no pigment is generated;

(3) growth characteristics: culturing in MRS culture medium at 37 deg.C for about 18 hr to reach stationary phase;

(4) the composition has no toxic or side effect on C57BL/6J mice;

(5) can remarkably improve the excessive weight gain caused by high fat diet;

(6) can remarkably regulate the inflammation level of the liver of a mouse with high fat diet;

(7) can remarkably recover the imbalance of the flora in the intestinal tract caused by high fat diet;

(8) reduce appetite and efficiency of dietary intake;

(9) can regulate the total cholesterol and low density lipoprotein cholesterol content in serum, and reduce the total cholesterol and low density lipoprotein cholesterol content to normal level;

(10) has positive effect on bacterial lipopolysaccharide endotoxin induced inflammation;

(11) maintaining the colon barrier structure has a good effect.

Example 1: screening and identification of Lactobacillus reuteri CCFM1178

Separation and screening of lactic acid bacteria

(1) 1g of pig manure from Lijiang City of Yunnan province is taken. After gradient dilution, the suspension was spread on a solid culture medium (each 1L of MRS medium contains 0.02g of vancomycin and 20g of agar, and the pH value was 5.0. + -. 0.1) for lactobacillus high selection, and cultured at 37 ℃ for 48 to 72 hours.

(2) Observing and recording colony morphology, picking colonies and streaking for purification.

(3) The colonies were gram-stained in MRS liquid medium at 37 ℃ for 24 hours, and the morphology of the colonies was recorded.

(4) Removing gram-negative bacteria strains and gram-positive cocci from the colonies, and selecting to obtain gram-positive bacilli.

(5) After catalase analysis, catalase-positive strains were discarded, and catalase-negative strains were retained.

Molecular biological identification of lactobacillus reuteri

(1) Extraction of a Single bacterial genome (following the TIANAmp Bacteria DNA kit protocol)

A. Culturing the lactic acid bacteria obtained by screening in the step (II) overnight, taking 1mL of the bacterial suspension into a 1.5mL centrifuge tube, centrifuging for 1min at 10,000 rpm (11,500 Xg), and sucking the supernatant as far as possible;

B. to the pellet was added 180. mu.L of a buffer (20mM Tris, pH 8.0; 2mM Na)₂-EDTA; 1.2% Triton; lysozyme with a final concentration of 20mg/mL (the lysozyme must be prepared by dissolving the lysozyme dry powder in a buffer solution, otherwise the lysozyme would cause dissolutionNo activity of bacteria and enzyme), treating at 37 deg.C for more than 30 min;

C. adding 20 mu L of protease K solution into the tube, and uniformly mixing;

D. adding 220 μ L buffer solution GB, oscillating for 15sec, standing at 70 deg.C for 10min, cleaning the solution, and centrifuging briefly to remove water droplets on the inner wall of the tube cover;

E. adding 220 μ L of anhydrous ethanol, shaking thoroughly, mixing for 15sec, wherein flocculent precipitate may appear, and centrifuging briefly to remove water drop on the inner wall of the tube cover;

F. adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (the adsorption column is placed into a collecting pipe), centrifuging at 12,000 rpm (-13,400 Xg) for 30sec, pouring the waste liquid, and placing an adsorption column CB3 into the collecting pipe;

G. adding 500 μ L buffer GD (checking whether absolute ethanol is added before use) into adsorption column CB3, centrifuging at 12,000 rpm (-13,400 Xg) for 30sec, pouring off waste liquid, and placing adsorption column CB3 into a collection tube;

H. adding 600 μ L of rinsing liquid PW (checking whether absolute ethanol is added before use) into adsorption column CB3, centrifuging at 12,000 rpm (-13,400 Xg) for 30sec, discarding the waste liquid, and placing adsorption column CB3 into the collection tube; repeating the operation once;

I. putting the adsorption column CB3 back into the collecting pipe, centrifuging at 12,000 rpm (13,400 Xg) for 2min, pouring off waste liquid, and placing the adsorption column CB3 at room temperature for a plurality of minutes to thoroughly dry the residual rinsing liquid in the adsorption material;

J. transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50-200 mu L of elution buffer TE into the middle part of the adsorption membrane, standing for 2-5min at room temperature, centrifuging for 2min at 12,000 rpm (13,400 Xg), and collecting the solution into the centrifuge tube.

35 strains are obtained by co-screening, and the strain with the serial number of CCFM1178 is identified as Lactobacillus reuteri (Lactobacillus reuteri) through 16S rDNA amplification and sequence comparison.

Example 2: tolerance of lactobacillus reuteri CCFM1178 to simulated gastrointestinal fluids

Inoculating the frozen lactobacillus reuteri CCFM1178 into an MRS solid culture medium (MRS culture medium + 0.05% cysteine hydrochloride) in a streak manner, carrying out anaerobic static culture at 37 ℃ for 48h, carrying out subculture for 2-3 times by using the MRS culture medium, mixing 1mL of the culture medium of the lactobacillus reuteri CCFM1178 with 9.0mL of artificial simulated gastric juice (MRS culture medium containing 1% pepsin and pH 2.5), carrying out anaerobic culture at 37 ℃, sampling at 0h, 0.5h, 1h and 2h respectively, carrying out pouring culture by using an agar MRS culture medium, carrying out plate colony counting, and calculating the survival rate according to the viable count. The survival rate is the ratio of the number of viable bacteria at the time of sampling the culture medium to the number of viable bacteria at 0h, and is expressed as%.

Adding 1mL culture solution of Lactobacillus reuteri CCFM1178 into 9.0mL artificial simulated intestinal fluid (MRS culture medium containing 0.3% of bovine bile salt, 1% of trypsin and pH 8.0), anaerobically culturing at 37 deg.C, sampling at 0h, 0.5h, 1h and 2h, respectively, pouring and culturing with MRS agar culture medium, counting plate colony, and calculating survival rate according to viable count. The survival rate is the ratio of the number of viable bacteria at the time of sampling the culture medium to the number of viable bacteria at 0h, and is expressed as%. The results of the experiment are shown in tables 1 and 2. The result shows that the Lactobacillus reuteri CCFM1178 has better tolerance to the artificial gastrointestinal fluid.

TABLE 1 tolerance of Lactobacillus reuteri CCFM1178 in simulated gastric fluid

TABLE 2 tolerance of Lactobacillus reuteri CCFM1178 in artificially simulated intestinal fluid

Example 3: safety verification of C57BL/6J mice by Lactobacillus reuteri CCFM1178

Suspending cells of Lactobacillus reuteri CCFM1178 in physiological saline to obtain a concentration of 1 × 10¹⁰CFU/mL of bacterial suspension. Taking 8 healthy male C57BL/6J mice with the weight of about 20g, after adapting to the environment for one week, giving the bacterial suspension with the concentration every dayThe solution was gavaged once at 0.2ml for one week and death and body weight were recorded.

The results are shown in Table 3. Feeding concentration of 1 × 10¹⁰The CFU/mL lactobacillus reuteri CCFM1178 does not cause obvious influence on the mouse, the weight of the mouse does not have obvious change, and the appearance of the mouse has no obvious pathological symptoms and no death phenomenon.

TABLE 3 weight change and mortality in mice

Note-no death of the mice.

Example 4: lactobacillus reuteri CCFM1178 has recovery effect on intestinal dysbacteriosis caused by high-fat feed

48 healthy male C57BL/6J mice, weighing 20-21g, were acclimated for 1 week and randomized into 6 groups: blank control group (NC), high fat model control group (HFD), Simvastatin control group (Simvastatin), Metformin control group (Metformin), Lactobacillus reuteri CCFM1178 dry control group (CCFM1178), Lactobacillus reuteri NCIMB 30242 dry control group (NCIMB 30242), each group contains 8 mice; wherein, the lactobacillus reuteri NCIMB 30242 is a commonly used commercial health product strain, the simvastatin is a commonly used medicament for reducing blood fat, and the metformin is a commonly used medicament for controlling type 2 diabetes.

Respectively culturing Lactobacillus reuteri CCFM1178 and NCIMB 30242, mixing the bacterial sludge with protective agent (100g/L-150g/L skimmed milk powder, 100g/L-150g/L maltodextrin, 140g/L-160g/L trehalose) at a ratio of 1:2(w/v, 1g bacterial sludge: 2mL protective agent solution), pre-freezing at-15-20 deg.C for 8-14h, vacuum freeze-drying to obtain lyophilized bacterial powder, and suspending the lyophilized bacterial powder in physiological saline to make the concentration of Lactobacillus reuteri in physiological saline suspension be 1 × 10¹⁰CFU/mL, used for gavage of mice, and the grouping and treatment method of experimental animals are shown in Table 4.

TABLE 4 groups of experimental animals

The body weight of the mice was monitored periodically during the experiment.

Fresh excrement of the mice is collected at the final stage of the test and is frozen and stored at the temperature of minus 80 ℃, genome DNA in the excrement is extracted for 16s rDNA sequencing, and the structure of intestinal flora is analyzed by a second-generation sequencer. At the end of the test, the mice are fasted and are not forbidden to be watered for 12 hours, after the mice are anesthetized by injecting a 10% chloral hydrate solution into the abdominal cavity, the heart is sampled and killed by means of cervical dislocation. Centrifuging blood sample at 4 deg.C for 15min at 3000 Xg, collecting supernatant, and freezing at-80 deg.C for measuring related serum index. Collecting part of liver, rapidly placing in pre-cooled normal saline for rinsing and removing blood, placing in paraformaldehyde for fixation, freezing the rest part of liver in liquid nitrogen at a medium speed, transferring to-80 ℃ for freezing storage, and subsequently preparing into liver homogenate for measuring related indexes, wherein the specific preparation method comprises the following steps: weighing a certain amount of liver tissue, and carrying out the following steps: adding physiological saline at a ratio of 9, grinding tissue, centrifuging at 3000r for 10min, and freezing the supernatant at-80 deg.C.

The results of the flora analysis experiments are shown in FIG. 1. Compared with the HFD group, the intervention of lactobacillus reuteri CCFM1178 (CCFM1178 group) can alleviate the abnormal abundance of intestinal microorganisms of bacillus, acetti factor, ruminococcus, Eubacterium _ coprostanogens _ group, Clostridium _ sense _ stricoto _1, and rombouuthsia in mouse intestinal flora caused by high fat diet.

Example 5: lactobacillus reuteri CCFM1178 reduces the weight gain percentage, epididymal fat index and brown fat index of mice with metabolic syndrome

The grouping, modeling and handling methods of the C57BL/6J mice were the same as in example 4. The weight gain percentage, epididymal fat index and brown fat index were calculated by weighing.

The results of the experiment are shown in FIG. 2. The body weight of the mice in the model group is obviously increased, and the lactobacillus gasseri CCFM1178 obviously reduces the weight gain percentage and the white fat index (white fat/body weight) of the mice, reduces 19.38 percent and 36.7 percent relative to the model group, increases the brown fat index (brown fat/body weight), is 1.65 times of that of the model group, and is also superior to the strain CCFM1145 (brown fat index 0.0045) disclosed in CN 112322527A. Its ability to reduce body weight levels in mice was significantly higher than both drug groups and lactobacillus reuteri NCIMB 30242.

Example 6: lactobacillus reuteri CCFM1178 for reducing daily dietary intake and energy efficiency of mice with metabolic syndrome

The grouping, modeling and handling methods of the C57BL/6J mice were the same as in example 4. The dietary intake and energy efficiency were calculated by weighing.

The results of the experiment are shown in FIG. 3. The daily dietary intake and energy efficiency of the mice in the model group are obviously improved, and the daily dietary intake and energy efficiency of the mice are obviously reduced by lactobacillus reuteri CCFM 1178. The ability to reduce the daily dietary intake level of the mice is slightly higher than that of simvastatin, metformin and lactobacillus reuteri NCIMB 30242, which are reduced by 4.93%, 5.01% and 3.87% respectively.

Example 7: lactobacillus reuteri CCFM1178 reduces serum Total Cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) levels in mice with metabolic syndrome

Grouping, modeling and treatment methods of C57BL/6J mice are the same as example 4, and at the end of the test, the mice are fasted for 12 hours without water prohibition, and blood is collected from the heart after the mice are anesthetized by intraperitoneal injection of 10% chloral hydrate solution. The blood sample was centrifuged at 3000 Xg at 4 ℃ for 15min, the supernatant was collected, and the Total Cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) contents in the blood were measured by the detection method of the kit.

The results of the experiment are shown in FIG. 4. As can be seen from FIG. 4, the serum total cholesterol level of the mice in the high-fat diet group is obviously increased to 5.05mmol/L, the Lactobacillus reuteri CCFM1178 for intragastric administration can reduce the serum total protein cholesterol and low-density lipoprotein cholesterol levels to 3.68mmol/L and 0.71mmol/L, the recovery capability of the Lactobacillus reuteri CCFM1178 on the serum total cholesterol level is obviously stronger than that of a control medicament and the Lactobacillus reuteri NCIMB 30242, and is also better than that of the strain CCFM1145 disclosed in CN112322527A (TC 3.78mmol/L, LDL-C0.72 mmol/L), and the Lactobacillus reuteri CCFM1178 has a positive effect on blood lipid metabolism is suggested.

Example 8: lactobacillus reuteri CCFM1178 reduces the level of proinflammatory factors IL-6 and TNF-alpha in liver of mice with metabolic syndrome

The grouping, modeling and treatment method of the C57BL/6J mice are the same as example 4, and at the end of the test, the mice are fasted for 12 hours without water prohibition, and after the mice are anesthetized by injecting a 10% chloral hydrate solution into the abdominal cavity, the heart is sampled, and the cervical vertebra is dislocated and killed. Freezing and storing liver at-80 deg.C, weighing a certain amount of liver tissue during measurement, and performing the following steps: adding physiological saline at a ratio of 9 for tissue grinding, centrifuging at 3000r for 10min, taking supernatant, and determining the content of proinflammatory factors IL-6 and TNF-alpha in the liver according to the detection method of the kit. Since the drug ameliorated metabolic syndrome by a different route from microorganisms, the drug group was not tested in this example.

The results of the experiment are shown in FIG. 5. The experimental result shows that compared with a normal control group, the contents of proinflammatory factors IL-6 and TNF-alpha in the liver of a mouse in a high-fat diet group are obviously increased, the Lactobacillus reuteri CCFM1178 in gavage can reduce the contents of the proinflammatory factors IL-6 and TNF-alpha in the liver to 1173.36pg/mg protein and 4862.37pg/mg protein, and the levels are recovered to be equivalent to that of a blank group, and the intervention capability of the Lactobacillus reuteri CCFM1178 on the proinflammatory factors in the liver is obviously stronger than that of the control group and the Lactobacillus reuteri NCIMB 30242.

Example 9: lactobacillus reuteri CCFM1178 reduces the level of anti-inflammatory factor IL-10 in the liver of mice with metabolic syndrome

The grouping, modeling and treatment method of the C57BL/6J mice are the same as example 4, and at the end of the test, the mice are fasted for 12 hours without water prohibition, and after the mice are anesthetized by injecting a 10% chloral hydrate solution into the abdominal cavity, the heart is sampled, and the cervical vertebra is dislocated and killed. Freezing and storing liver at-80 deg.C, weighing a certain amount of liver tissue during measurement, and performing the following steps: adding physiological saline at a ratio of 9 for tissue grinding, centrifuging at 3000r for 10min, collecting supernatant, and determining the content of anti-inflammatory factor IL-10 in liver according to the detection method of the kit. Since the drug ameliorated metabolic syndrome by a different route from microorganisms, the drug group was not tested in this example.

The results of the experiment are shown in FIG. 6. As can be seen from the experimental results, compared with the normal control group, the content of the liver anti-inflammatory factor IL-10 of the mice in the high-fat diet group is obviously increased to 1169.76pg/mg protein, the content of the liver anti-inflammatory factor IL-10 of the lactobacillus reuteri CCFM1178 in the gavage can be increased to 1169.72U/mg protein, and the level of the lactobacillus reuteri CCFM1178 in preventing the metabolic syndrome is obviously higher than that of the control drug and the lactobacillus reuteri NCIMB 30242.

Example 10 Lactobacillus reuteri CCFM1178 reduction of serum Lipopolysaccharide (LPS) endotoxin levels in mice with Metabolic syndrome

Grouping, modeling and treatment methods of C57BL/6J mice are the same as example 4, and at the end of the test, the mice are fasted for 12 hours without water prohibition, and blood is collected from the heart after the mice are anesthetized by intraperitoneal injection of 10% chloral hydrate solution. Centrifuging blood sample at 3000 Xg and 4 deg.C for 15min, collecting supernatant, and determining Lipopolysaccharide (LPS) endotoxin content in blood according to the detection method of the kit.

The results of the experiment are shown in FIG. 7. As can be seen from FIG. 7, the serum LPS content of the mice in the high-fat diet group is obviously increased to 348.7EU/L, and the Lactobacillus reuteri CCFM1178 for intragastric administration can reduce the serum LPS content to 123.2EU/L, and the recovery capability of the Lactobacillus reuteri CCFM1178 for the serum LPS content is obviously stronger than that of the control drug and the Lactobacillus reuteri NCIMB 30242, which indicates that the Lactobacillus reuteri CCFM1178 has a positive effect on the bacterial Lipopolysaccharide (LPS) endotoxin induced inflammation.

Example 11 Lactobacillus reuteri CCFM1178 increase the expression level of claudin in colonic tissue of mice with metabolic syndrome

The grouping, modeling and treatment method of the C57BL/6J mice are the same as example 4, and at the end of the test, the mice are fasted for 12 hours without water prohibition, and after the mice are anesthetized by injecting a 10% chloral hydrate solution into the abdominal cavity, the heart is sampled, and the cervical vertebra is dislocated and killed. Freezing and storing the colon of the mouse at the temperature of-80 ℃, weighing a certain amount of colon tissues during measurement, and carrying out the following steps: adding physiological saline at a ratio of 9 for tissue grinding, centrifuging at 3000r for 10min, taking supernatant, and determining expression levels of zon-1, Occludin and Claudin-1 genes in colon tissue according to the detection method of the kit.

The results of the experiment are shown in FIG. 8. The expression levels of ZO-1, Occludin and Claudin-1 genes of the lactobacillus reuteri CCFM1178 stem group are obviously higher than those of the model group, and the expression levels of the ZO-1, Occludin and Claudin-1 genes are respectively raised back to 0.912, 0.976 and 1.121, which are close to the blank group; and the expression levels of ZO-1, Occludin and Claudin-1 genes of the lactobacillus reuteri NCIMB 30242 stem group have no significant difference with the expression levels of the model group, which shows that the lactobacillus reuteri CCFM1178 has better effect on maintaining the colon barrier structure.

Example 12: preparation of fermentation agent containing Lactobacillus reuteri CCFM1178

MRS culture medium: 10g of tryptone, 10g of beef extract, 5g of yeast powder, 20g of glucose, 5g of sodium acetate, 2g of diammonium hydrogen citrate, 2g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate heptahydrate, 801ml of tween and 0.25g of manganese sulfate monohydrate, wherein the volume of water is fixed to 1000ml, the pH value is adjusted to 6.5, and the sterilization is carried out for 15-25min at the temperature of 119-.

A protective agent: 100g/L-150g/L skimmed milk powder, 100g/L-150g/L maltodextrin and 140g/L-160g/L trehalose.

Inoculating Lactobacillus reuteri CCFM1178 into MRS culture medium, culturing at 37 deg.C under anaerobic condition for 18-20 hr, collecting thallus, and resuspending thallus cells with protective agent to make the concentration of thallus reach 10¹⁰CFU/ml, culturing the suspension at 37 deg.C under anaerobic condition for 50-70min, and drying.

Optionally, the drying is vacuum freeze drying after pre-freezing for 8-14h at-15 to-20 ℃.

Example 13: application of lactobacillus reuteri CCFM1178

(1) Lactobacillus milk beverage prepared from Lactobacillus reuteri CCFM1178

The raw material skim milk is sterilized by heat at 95 ℃ for 20min, then cooled to 4 ℃, and then added with the lactobacillus reuteri CCFM1178 screened in example 1 or the leaven prepared in example 12 to ensure that the thallus concentration reaches 10⁶And (4) preserving at the temperature of more than 4 ℃ by refrigeration to obtain the milk beverage containing the viable bacteria of the lactobacillus reuteri CCFM 1178.

(2) Soybean milk prepared from Lactobacillus reuteri CCFM1178

Soaking soybeans in soft water, wherein the volume of the water is three times that of the original soybeans, soaking the soybeans for 1-2 hours at the temperature of 80 ℃, and then removing soybean hulls. And then, draining the soaking water, adding boiling water, grinding into slurry, and keeping the temperature for 10-15 min under the condition that the temperature is higher than 80 ℃. Filtering the slurry with 150 mesh filter membrane, centrifuging to obtain centrifugate as coarse soybean milk, heating to 140-150 deg.C, and rapidly introducing hot coarse soybean milk into a vacuum containerAnd vacuumizing the air cooling chamber, wherein the peculiar smell substances in the coarse soybean milk are quickly discharged along with the water vapor. After vacuum degassing, the temperature is reduced to about 37 ℃, and then the lactobacillus reuteri CCFM1178 screened in the embodiment 1 or the leaven prepared in the embodiment 12 is inoculated to ensure that the concentration of the lactobacillus reuteri CCFM1178 reaches 10⁶And (3) preserving the soybean milk at the temperature of more than 4 ℃ by refrigeration to obtain the soybean milk containing the viable bacteria of the lactobacillus reuteri CCFM 1178.

(3) Fruit and vegetable beverage prepared from lactobacillus reuteri CCFM1178

Selecting fresh vegetables (such as one or more of cucumber, carrot, beet, celery or cabbage), cleaning, juicing, instantly sterilizing at 140 deg.C for 2s, immediately cooling to about 37 deg.C, inoculating the Lactobacillus reuteri CCFM1178 starter to make its concentration reach 10⁶And (3) storing the fruit and vegetable beverage containing the viable bacteria of the lactobacillus reuteri CCFM1178 at the temperature of more than 4 ℃ by refrigeration to obtain the fruit and vegetable beverage containing the viable bacteria of the lactobacillus reuteri CCFM 1178.

(4) Capsule product prepared from lactobacillus reuteri CCFM1178

Culturing lactobacillus reuteri CCFM1178 on an MRS culture medium for 24 hours, centrifuging for 20 minutes at the temperature of 4 ℃ and under the condition of 4000r/min, washing twice by PBS, adding skim milk powder 4 percent and lactose 6 percent based on the weight of the powder finally obtained containing the lactobacillus reuteri CCFM1178, mixing for 10 minutes, adding sterile 2 percent calcium chloride and sodium alginate 3 percent, stirring for 10 minutes at the speed of 150r/min, standing and solidifying for 30 minutes, finally cleaning and filtering, freezing and drying the obtained filtrate for 20 hours to obtain powder containing the lactobacillus reuteri CCFM1178, and filling the powder into commercial medicinal microcapsules to obtain the capsule product.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.