Lactobacillus rhamnosus GYX-9 and application thereof

Abstract

The invention discloses a lactobacillus rhamnosus GYX-9 strain and an application of the lactobacillus rhamnosus GYX-9 strain. The lactobacillus rhamnosus GYX-9 disclosed by the invention can be used for regulating the problems of blood lipid metabolism disorder, liver lipid accumulation or pathological injury and the like caused by high-fat diet. The lactobacillus rhamnosus GYX-9 disclosed by the invention can be used for reducing the level of TNF-alpha, increasing the content of IL-10 and regulating colonic immune disorder; the abundance of intestinal microbiome communities is adjusted; according to the invention, the lactobacillus rhamnosus GYX-9 is combined to regulate and control the metabolic disorder caused by high-fat diet on the basis of'intestinal flora-intestine-liver axis'. The normal-temperature viable bacterium fermented milk with the viable bacterium number not less than 108 cfu / mL is also prepared, the normal-temperature viable bacterium fermented milk has remarkable capabilities of lowering cholesterol, combining taurocholate and inhibiting pancreatic lipase, and the pancreatic lipase inhibition rate can reach 67.06%.

Description

Technical Field

[0001] The invention belongs to the field of food and medicine, and specifically relates to a Lactobacillus rhamnosus GYX-9 and an application thereof. Background Art

[0002] With factors such as sedentary lifestyle, reduced physical labor, and excessive intake of high-energy-density foods, obesity has become one of the main problems that endanger human health. The relationship between obesity caused by a high-fat diet (HFD) and intestinal inflammation has gradually become a hot topic of research. Studies have reported that a high-fat diet can change the structure of the intestinal community, such as containing a higher proportion of Firmicutes and a lower proportion of Bacteroidetes. Studies on animal models and human populations have shown that there are differences in the intestinal flora of organisms with different degrees of fatness and thinness. Therefore, considering changing the intestinal flora can be used as a therapeutic method for treating obesity. The metabolic activity of the flora can improve the host's acquisition of energy from the diet; conventional flora or specific symbiosis can also affect energy balance; regulate the expression of genes in the metabolic pathways of nutrients such as fat. In addition, a high-fat diet can also stimulate intestinal cells to produce inflammatory factors. Lipopolysaccharide (LPS) is one of the components of the cell membrane of Gram-negative bacteria in the intestinal flora. It is released into the intestinal cavity after the death of Gram-negative bacteria and enters the circulatory system through the intestinal capillaries. LPS entering the circulatory system can cause an inflammatory response by activating mononuclear macrophages and releasing some pro-inflammatory factors such as TNF-α, IL-6, IL-8 and IL-1β. A high-fat diet can directly increase the level of LPS in plasma.

[0003] There is increasing evidence that probiotics can improve obesity, inflammation, and related metabolic diseases through some mechanisms, including inhibiting the adhesion of pathogens to the intestinal mucosa, stabilizing the structure of the microbial community, and improving the integrity and barrier function of the mucosa. Some studies have shown that supplementation with bifidobacteria and other lactobacilli can effectively improve fatty degeneration and insulin resistance in mice induced by a high-fat diet. Probiotics, as a supplement to daily diet, have a good effect on obesity caused by a high-fat diet and a high-sugar diet.

[0004] Dietary control therapy has become a basic project in clinical treatment. Nutritional imbalance is the main factor leading to simple obesity. The main treatment strategy should be to adjust the structure and type of diet, increase physical activity, and supplement probiotic dietary supplements that improve obesity, regulate intestinal flora and regulate immunity. Therefore, studying the preventive and improvement effects of probiotics on obesity and the intestinal inflammation caused by obesity is of great significance to human health. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a strain of Lactobacillus rhamnosus GYX-9 that can alleviate obesity caused by a high-fat diet.

[0006] The technical problem that the present invention needs to solve is to provide a microbial preparation, food or medicine.

[0007] The technical problem that the present invention also aims to solve is to provide the use of the Lactobacillus rhamnosus GYX-9 and the microbial preparation in the preparation of drugs for improving blood lipid metabolism disorders, liver fat accumulation, improving intestinal flora imbalance and / or regulating colon immune disorders in obese animals caused by a high-fat diet.

[0008] The final technical problem to be solved by the present invention is to provide an application in preparing a medicine for alleviating obesity or colitis caused by a high-fat diet.

[0009] Technical solution: In order to solve the above technical problems, the present invention provides a strain of Lactobacillus rhamnosus GYX-9, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on June 14, 2024, with a deposit number of CGMCC No. 30946 and a deposit address of Beijing, China.

[0010] The present invention also includes a microbial preparation, food or medicine, which contains the Lacticaseibacillus rhamnosus GYX-9, its fermentation liquid or supernatant.

[0011] Preferably, the microbial preparation, food or medicine further comprises a lyophilization protective agent, and preferably, the lyophilization protective agent comprises whey protein powder rich in milk fat globule membrane; preferably, the microbial preparation further comprises a commercial starter. Preferably, the microbial preparation or medicine comprises a single or compound preparation, and the preparation type comprises a liquid preparation or a solid preparation; preferably, the mass ratio of the whey protein powder rich in milk fat globule membrane to the rhamnosus casei bacterial slurry GYX-9 is 1:1; preferably, the mass ratio of the rhamnosus casei GYX-9 to the commercial starter is 5:1; preferably, the content of the rhamnosus casei GYX-9 is not less than 1×10 8cfu / mL; preferably, the food is room temperature fermented milk, and preferably, the room temperature fermented milk comprises embedded Lactobacillus rhamnosus GYX-9 and inactivated Lactobacillus rhamnosus GYX-9.

[0012] The present invention also includes the use of the Lacticaseibacillus rhamnosus GYX-9, the microbial preparation, the food or the medicine in the preparation of medicines or fermented milk for improving blood lipid metabolism disorders, liver fat accumulation, improving intestinal flora imbalance and / or regulating colon immune disorders of obese animals caused by a high-fat diet.

[0013] The present invention also includes the use of the Lacticaseibacillus rhamnosus GYX-9, the microbial preparation, the food or the medicine in preparing products for alleviating obesity or colitis caused by a high-fat diet.

[0014] The present invention also includes a method for preparing room temperature live bacteria fermented milk, comprising the following steps:

[0015] 1) embedding Lactobacillus rhamnosus GYX-9;

[0016] 2) using raw milk as a raw material, inoculating Lactobacillus rhamnosus GYX-9 live bacteria, fermenting, and then sterilizing to obtain sterilized milk;

[0017] 3) adding the embedded Lactobacillus rhamnosus GYX-9 from step 1) to the sterilized milk from step 2);

[0018] Preferably, the embedding method in step 1) includes freeze-drying or emulsification embedding;

[0019] Preferably, step 2) further comprises inoculating a commercial starter culture.

[0020] The present invention also includes room-temperature live bacteria fermented milk prepared by the preparation method.

[0021] The application includes any one of the following (a) to (e):

[0022] (a) preparing products for regulating indicators of blood lipid metabolism;

[0023] (b) preparing a product for improving fat accumulation and oxidative stress in liver tissue caused by a high-fat diet;

[0024] (c) preparing products that reduce pro-inflammatory factors in the colon and increase anti-inflammatory factors in the colon;

[0025] (d) preparing products that increase the diversity of intestinal flora and regulate the abundance of intestinal microbiome communities;

[0026] (e) preparing products for lowering cholesterol, increasing taurocholate binding rate and / or pancreatic lipase activity inhibition rate.

[0027] Among them, the blood lipid metabolism indicators include TG, TC, HDL-C, LDL-C, leptin, adiponectin, ALT and / or AST; the improvement of liver tissue oxidative stress includes a significant increase in antioxidant enzyme activity; the improvement of liver fat accumulation is manifested as a significant decrease in liver free fatty acid FFA, liver coefficient and epididymal fat coefficient.

[0028] Wherein, the pro-inflammatory factors in the colon include TNF-α, IL-1β and / or IL-6, and the anti-inflammatory factors mainly include IL-10.

[0029] Among them, the improving of intestinal flora diversity and regulating the abundance of intestinal microbiome communities include increasing the relative abundance of Candidatus_Saccharimonas, Romboutsia, Erysipelatoclostridium, and Colidextribacte, and reducing the relative abundance of norank_f__norank_o__RF39, Gordonibacter, and norank_f__Ruminococcaceae.

[0030] The formula of the room temperature live bacteria fermented milk is as follows: the addition amount of whey protein powder (M-WPI) rich in milk fat globule membrane is 5%, the addition amount of rhamnosus casei GYX-9 and commercial starter is 5:1 (m / m), and then after 72°C, 30s pasteurization heat treatment, freeze-dried or emulsified embedded rhamnosus casei GYX-9 is added, wherein the addition amount of freeze-dried bacterial powder is 1%, or the addition amount of emulsion is selected to be 10%.

[0031] Beneficial effects: Compared with the prior art, the present invention has the following advantages: the Lactobacillus rhamnosus GYX-9 and its functional bacterial agents, foods or drugs of the present invention can regulate problems such as dyslipidemia, liver lipid accumulation or pathological damage, colon immune disorders, intestinal flora imbalance caused by a high-fat diet, and can alleviate obesity caused by a high-fat diet and be used for fasting blood glucose tolerance abnormalities caused by a high-fat diet. Specifically, the Lactobacillus rhamnosus GYX-9 of the present invention can reduce the level of TNF-α, increase the content of IL-10 to regulate colon immune disorders; compared with the high-fat model group, the Lactobacillus rhamnosus GYX-9 can increase the relative abundance of Candidatus_Saccharimonas, Romboutsia, Erysipelatoclostridium, and Colidextribacte, and reduce

[0032] The relative abundance of norank_f__Ruminococcaceae, norank_f__norank_o__RF39, and Gordonibacter regulates the intestinal flora. The present invention combines Lactobacillus rhamnosus GYX-9 to regulate the metabolic disorders caused by high-fat diet based on the "intestinal flora-intestine-liver axis". The present invention also prepares a viable bacterial count of 10 8 cfu / mL of room temperature live bacteria fermented milk has significant pancreatic lipase inhibitory activity, which can reach 67.06%. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the developmental tree diagram of Lactobacillus rhamnosus GYX-9;

[0034] Figure 2 This is the weight gain trend of mice within 6 weeks of administration;

[0035] Figure 3 Lactobacillus rhamnosus GYX-9 affects fasting blood glucose in mice (A); OGTT decrease area (B);

[0036] Effect of insulin level (C) on lipid metabolism in serum (n=10);

[0037] Figure 4 The effect of Lactobacillus rhamnosus GYX-9 on lipid metabolism in mouse serum (n=10);

[0038] Figure 5 The effect of Lactobacillus rhamnosus GYX-9 on leptin (A) and adiponectin (B) in mice;

[0039] Figure 6 The effect of Lactobacillus rhamnosus GYX-9 on AST activity (A); ALT activity (B); LPS content (C) in mouse serum;

[0040] Figure 7 Effects of Lactobacillus rhamnosus GYX-9 on the liver weight (A); liver coefficient (B); epididymal fat weight (C); and epididymal fat coefficient (D) of mice

[0041] Figure 8 Effects of Lactobacillus rhamnosus GYX-9 on SOD activity (A), MDA content (B), and CAT activity (C) in mouse liver.

[0042] Fig. 9 The effect of Lactobacillus rhamnosus GYX-9 on the free fatty acid content in mouse liver;

[0043] Fig.10 Effects of Lactobacillus rhamnosus GYX-9 on inflammatory factors IL-1β(A); TNF-α(B); IL-6(C); IL-10(D) in mouse colon tissue. Fig.11 The effect of Lactobacillus rhamnosus GYX-9 on the OTU species of the intestinal flora of mice;

[0044] Fig.12 Alpha diversity analysis of intestinal flora species in the Lactobacillus rhamnosus GYX-9 treated group of mice;

[0045] Fig.13 Beta diversity analysis of intestinal flora in the Lactobacillus rhamnosus GYX-9 treated mice;

[0046] Fig.14 This is the composition analysis of the intestinal flora of mice treated with Lactobacillus rhamnosus GYX-9 at the phylum level;

[0047] Fig.15 This is the composition analysis of the intestinal flora of mice treated with Lactobacillus rhamnosus GYX-9 at the genus level;

[0048] Fig.16 The changes in the relative abundance of the genera at the genus level in the intestinal microorganisms of the mice treated with Lactobacillus rhamnosus GYX-9;

[0049] Fig.17 The changes in the relative abundance of the genera at the genus level in the intestinal microorganisms of the mice treated with Lactobacillus rhamnosus GYX-9 were shown;

[0050] Fig.18 This is the LDA discrimination result of the difference in intestinal microbial expression of mice in the Lactobacillus rhamnosus GYX-9 treatment group;

[0051] Fig.19 Correlation analysis between intestinal bacteria and biochemical indicators;

[0052] Fig. 20 The effect of different embedding methods on the survival rate of Lactobacillus rhamnosus GYX-9;

[0053] Fig.21 It has lipid-lowering properties for room temperature fermented milk.

[0054] Note: a, b, c indicate that the groups represented by different letters have significant differences (P<0.05). DETAILED DESCRIPTION

[0055] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0056] The Lactobacillus rhamnosus GYX-9 used in the present invention was screened from infant feces in Rugao, Jiangsu Province. An appropriate amount of fecal sample was diluted with physiological saline gradient, spread on an MRS plate, and cultured at 37°C for 24 hours. Different single colonies were picked and streaked for purification and cultured. The purified single colonies were picked and inoculated into 5mL MRS culture medium and cultured at 37°C for 12 hours. 1mL of bacterial solution was extracted with the Shanghai Sangon Biological Bacterial Genome Rapid Extraction Kit and sequenced by Shanghai Sangon Biological. The identification result was Lactobacillus rhamnosus, and its sequence development tree was shown in Figure 1 As shown. The strain GYX-9 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on June 14, 2024, with the deposit number: CGMCC No.30946, and the deposit address: Beijing, China. High-fat mice were provided by Nanjing Bioray Pharmaceutical Research Institute Co., Ltd.; various mouse ELISA kits were provided by Shanghai Zhuocai Biotechnology Co., Ltd.; various enzyme activity assay kits were provided by Beijing Solebow Technology Co., Ltd.

[0057] Microscopes, automatic biological tissue dehydrators, and slicers were provided by Leica Microsystems (Shanghai) Trading Co., Ltd.; biological tissue freezing embedding machines, embedding machine freezing tables, biological tissue spreaders, and biological tissue baking machines were provided by Jinhua Yidi Medical Equipment Co., Ltd.

[0058] Example 1 Preparation of Lactobacillus rhamnosus GYX-9

[0059] The activated Lactobacillus rhamnosus GYX-9 (1×10 8 CFU / mL) was inoculated into MRS broth at a ratio of 1% (v / v) and cultured at 37°C for 16h. Centrifuged at 3520×g and 4°C for 10min, the supernatant was discarded, and the bacterial precipitate was collected. The bacterial cells were washed with sterilized 0.9% saline solution, and the concentration of the bacterial solution was adjusted to 10 after three times with 0.9% sterile saline. 8 CFU / mL is reserved.

[0060] Example 2 Establishment of high-fat diet model

[0061] 6-week-old C57BL / 6 male mice, weighing 15-20g. The temperature of the animal laboratory was 22±2℃, the humidity was 40-60%, and the light-dark cycle was 12h (lighting time 8:00A.M~8:00P.M). The mice were free to drink water and eat SPF stick feed, and were pre-fed for one week to adapt to the environment. All mice were divided into two groups, a normal group (15 mice) and a model group (35 mice). The normal group was fed with normal feed, and the model group was fed with high-fat feed. The mice were kept in separate cages, 5 mice per cage, and the weight of the mice was measured once a week. After 9 weeks of feeding and successful modeling, 10 normal mice (Control) and 30 successfully modeled high-fat mice were taken.

[0062] Thirty high-fat mice were randomly divided into three groups (n=10): model group (Model), Lactobacillus rhamnosus GYX-9 group (SLT) and drug positive control group (Orlistat). The blank group continued to be fed with normal feed, and the other groups continued to be fed with high-fat feed. The specific gavage arrangement of each group of mice is shown in Table 1. Every day before treatment, the growth status of the mice was recorded, and the death, mental state, hair gloss and activity were observed. The body weight was weighed once before gavage at a fixed time every week.

[0063] Table 1 Grouping of mice and daily gavage

[0064]

[0065] 1. Determination of mouse weight and body mass index

[0066] The mice were weighed once a week and their weight gain was recorded. The body length of the mice was measured after they were killed (body length is the distance from the nose tip to the anus). The liver and epididymal fat were removed and weighed after the mice were dissected. The organ index was calculated according to the following formula:

[0067]

[0068] The mice were weighed at a fixed time every week from the beginning of the intervention, and the weight gain of the mice was recorded. The intervention lasted for 16 weeks in total. A high-fat diet-induced obesity model was established by feeding the mice with a high-fat diet for 9 weeks, and Lactobacillus rhamnosus GYX-9 was used to intervene in the mice fed a high-fat diet. Figure 2As shown, compared with the mice in the Control group fed with a normal diet for 9 weeks, the weight of the mice in the Model group increased significantly (P < 0.05), with a weight gain of 15%-20%, and the obese mouse model was successfully established. During the 6-week oral gavage intervention, the weight gain of the Control group was 3.18g, and the weight gain of the Model group was 3.03g. The weight loss in the sixth week of drug intervention was more obvious. The possible reason is that oral gavage damaged the esophagus of mice and suppressed appetite. The weight gains of the high-fat diet + SLT group and the Orlistat group were 3.23 ± 2.32g and 2.02 ± 1.42g, respectively, with no significant difference, indicating that the live bacteria of Lactobacillus rhamnosus GYX-9 can inhibit the weight gain of mice caused by a high-fat diet. The groups of mice constructed in this example will be used in the subsequent experiments in the following examples.

[0069] Example 3 Regulatory effect of Lactobacillus rhamnosus GYX-9 on obesity in mice

[0070] 1. Determination of Insulin Sensitivity in Mice

[0071] Glucose tolerance test (OGTT): After fasting for 12 hours, the baseline blood glucose level was measured (0 minutes), and then 2 g / kg glucose was taken orally. Blood glucose levels were measured at 0, 15, 30, 60, 90 and 120 minutes.

[0072] Determination of fasting blood glucose in mice: Blood glucose was measured before killing mice, and mice were fasted for 12 hours before measurement. During the measurement, the tail of the mouse was gently pricked with a blood collection needle, a drop of blood was squeezed out by hand, and then the blood was quickly dripped onto a blood glucose test strip, and then the reading was taken with a rapid blood glucose meter.

[0073] Determination of fasting serum insulin in mice: Collect blood from mice to prepare serum. When measuring insulin, draw 10 μL of serum for ELISA method to measure the insulin level in serum, and refer to the instructions of the kit for determination.

[0074] from Figure 3 A shows that the fasting blood glucose of mice in the Control group was the lowest. Compared with the Model group, the fasting blood glucose of the SLT group showed a downward trend, indicating that Lactobacillus rhamnosus GYX-9 has a certain effect on alleviating the increase of fasting blood glucose in obese mice induced by a high-fat diet. Figure 3B shows that 15 minutes after the injection of glucose, the blood glucose levels of mice in all groups increased, and showed a downward trend after 30 minutes. The fasting blood glucose of mice in the Control group remained at a low level throughout the experimental period. After a small increase, it basically returned to normal levels after 2 hours. The fasting blood glucose of mice in the Model group increased significantly, and the blood glucose level decreased more slowly than other groups, and remained at a high level after 2 hours. Compared with the Model group, the blood glucose levels of each treatment group were significantly lower than those of the Model group within 2 hours. The fasting blood glucose of the SLT group decreased rapidly after 60 minutes, and began to stabilize after 90 minutes, and the blood glucose trend was relatively stable. This shows that mice given Lactobacillus rhamnosus GYX-9 by gavage have better tolerance to glucose injection.

[0075] Similar to blood sugar levels, insulin levels also increase in obesity. Insulin is an important hub of human metabolism, and maintaining low insulin levels may be the key to longevity and health. Insulin levels are also an important indicator for determining insulin sensitivity in mice. Figure 3 As shown in C, the insulin level of mice in the Control group was the lowest. Compared with the Control group, the insulin level of the Model group increased significantly. Compared with the Model group, the serum insulin levels of mice in the SLT group and the Orlistat group decreased significantly, indicating that Lactobacillus rhamnosus GYX-9 has a protective effect on high-fat diet-induced hyperinsulinemia in mice.

[0076] 2. Determination of serum leptin, adiponectin, ALT, AST and blood lipids in mice

[0077] The serum of mice in each intervention group was obtained, and the levels of blood biochemical indices (TG, TC, HDL-C, LDL-C, leptin, adiponectin, ALT, AST) of mice in different groups were determined using the kit.

[0078] The four blood lipids include TC, TG, LDL-C and HDL-C, which are closely related to atherosclerosis, diabetes, coronary heart disease, obesity, etc. Figure 4 Compared with the Control group, the serum TG, TC, and LDL-C levels of the Model group mice were significantly increased, and the HDL-C level was significantly decreased, indicating that the blood lipid metabolism of the Model group mice was abnormal after continuous high-fat diet feeding. Compared with the Model group, the SLT group and the Orlistat group could significantly reduce the serum TC, TG, and LDL-C levels and increase the serum HDL-C level (P<0.05); the results showed that Lactobacillus rhamnosus GYX-9 could improve the lipid metabolism disorder of mice induced by high-fat diet by reducing the serum TC, TG, and LDL-C levels and increasing the HDL-C level.

[0079] like Figure 5High-fat diet significantly increased the level of leptin in serum. Compared with the Model group, the serum leptin levels of mice in the SLT group and Orlistat group decreased significantly (P<0.05), indicating that Lactobacillus rhamnosus GYX-9 can more effectively improve the leptin resistance caused by obesity in mice. Compared with the Control group, the adiponectin level of mice in the Model group decreased significantly. After intervention in each treatment group, the adiponectin content of the SLT group and Orlistat group increased significantly compared with the Model group (P<0.05). The results showed that Lactobacillus rhamnosus GYX-9 and Orlistat had a certain improvement effect on the decrease of adiponectin level induced by high-fat diet.

[0080] like Figure 6 The AST and ALT activities in the serum of mice in the Model group were significantly increased compared with those in the Control group (P<0.05). Compared with the Model group, the AST and ALT activities in the serum of mice in the SLT group and Orlistat group were significantly decreased (P<0.05), indicating that Lactobacillus rhamnosus GYX-9 and Orlistat had an inhibitory effect on the AST and ALT activities.

[0081] The increase of LPS, the active ingredient of endotoxin, will increase the synthesis and release of pro-inflammatory factors in the intestine, causing intestinal inflammation and intestinal mucosal barrier damage. Figure 6 As shown in C, after long-term high-fat diet feeding, the LPS content in the serum of the Model group mice increased significantly (P<0.05). However, the intervention of Lactobacillus rhamnosus GYX-9 significantly reduced the LPS content in the serum of mice with obese liver injury (P<0.05), indicating that Lactobacillus rhamnosus GYX-9 can effectively inhibit the increase of LPS content in the serum caused by high-fat diet.

[0082] 3. Determination of lipid accumulation in mouse liver

[0083] Determination of free fatty acids in mouse liver: Take fresh mouse liver of similar size, homogenize the tissue and take the supernatant for the determination of FFA level. The determination principle is that FFA will combine with copper ions to produce yellow-green color, and the color depth is proportional to the concentration of fatty acids. The level of FFA in the sample is calculated by comparing the absorbance value with the standard and blank.

[0084] Determination of oxidative stress indexes in mouse liver: Weigh an appropriate amount of liver, take 9 times the weight of the tissue block of physiological saline, put them together in a manual homogenizer, grind them in an ice bath, and after forming a homogenate, centrifuge the liver homogenate at 2500×g for 10 minutes at 4°C, and take out the supernatant. Take an appropriate amount of supernatant and detect the oxidation indexes (SOD, CAT and MDA content) in the liver according to the kit.

[0085] Depend on Figure 7It can be seen that compared with the Control group, the liver weight, liver coefficient, epididymal fat weight and epididymal fat coefficient of the Model group mice increased significantly (P<0.05), indicating that a high-fat diet can lead to abnormal liver status and a large amount of fat accumulation in mice. Compared with the Model group, the liver weight, liver coefficient, epididymal fat weight and epididymal fat coefficient of the SLT group decreased significantly (P<0.05). The results show that Lactobacillus rhamnosus GYX-9 can significantly inhibit the fat accumulation of mice induced by a high-fat diet.

[0086] SOD in normal tissues is an important antioxidant metalloenzyme that removes ROS. A high-fat diet can induce an increase in the level of oxidative stress in the liver of mice and reduce the SOD content in the liver. Figure 8 As shown in A, the SOD activity in the liver of mice in the Moedel group was significantly lower than that in the Control group (P<0.05), which may be due to the rapid increase in oxidative stress in the liver, which led to the consumption of SOD enzymes in the liver that play an antioxidant role. After oral administration of Lactobacillus rhamnosus GYX-9, the SOD activity in the liver of mice increased, but there was no significant difference, and it still did not return to the level of the Control group.

[0087] Malondialdehyde (MDA) is a degradation product of polyunsaturated fatty acid peroxides in living organisms. The higher the MDA content in the body, the more severe the damage to the body. Figure 8 As shown in B, after 16 weeks of high-fat diet feeding, the MDA content in the liver of the Model group mice was significantly higher than that of the Control group (P<0.05), indicating that the body has produced obvious lipid peroxidation. After intervention with Lactobacillus rhamnosus GYX-9 and orlistat, although there was no significant difference in the MDA content in liver cells, it decreased, indicating that the level of intracellular lipid peroxidation decreased.

[0088] CAT is a marker enzyme of glutathione peroxisomes. CAT can work together with GPx to remove the toxic substance hydrogen peroxide produced by cell metabolism and reduce its damage to cells. Figure 8 As shown in C, compared with the Control group, the CAT activity in the liver of the Model group was significantly decreased, and compared with the Model group, the CAT activity in the liver of the SLT group was significantly increased (P<0.05).

[0089] The above results show that after 16 weeks of high-fat diet feeding, the SOD and CAT activities in the liver of mice were significantly lower than those in the Control group, while the MDA content was significantly higher than that in the Control group. The possible reason is that long-term feeding of a high-fat diet continuously accumulates in the body, and metabolism produces a large number of reactive oxygen free radicals, which leads to a large consumption of antioxidant enzymes such as SOD and CAT in the body, and the body's antioxidant defense capacity is reduced. In addition, free radicals react with unsaturated fatty acids on the cell membrane, accelerate the lipid peroxidation process, increase the MDA content, and cause serious damage to the cell membrane. Compared with the Model group, the activity of the antioxidant SOD enzyme in the liver of mice after intervention with Lactobacillus rhamnosus GYX-9 was enhanced, the CAT activity was significantly increased, and the MDA content was reduced, which enhanced the body's antioxidant capacity and reduced the damage of the high-fat diet to the liver of mice.

[0090] The level of free fatty acids (FFA) in the liver is an important indicator for evaluating the degree of lipid accumulation in mouse liver. FFA, as a product of IL-6 decomposition of fat, can induce inflammatory response in cells. Fig. 9 It can be seen that compared with the Control group, the FFA level in the Model group was significantly increased (P<0.05), and compared with the Model group, the FFA levels in the SLT group and the Orlistat group were significantly decreased (P<0.05). This shows that continuous high-fat diet feeding can significantly increase the level of FFA in the liver, and Lactobacillus rhamnosus GYX-9 has a significant inhibitory effect on the increase of FFA levels in mouse liver.

[0091] Example 4 Lactobacillus rhamnosus GYX-9 regulates colon immune disorders caused by high-fat diet

[0092] 1. Determination of inflammatory factor levels in mouse colon

[0093] After killing the mice, the colon tissue (from the cecum to the anus) was removed, washed with saline, and the length of the colon was measured and recorded. Then the colon contents were scraped with forceps and stored in a -80°C refrigerator. The proximal colon tissue was taken, weighed, and 19 times the volume of saline (weight-to-volume ratio, e.g., 10 mg tissue 190 μL saline) was added and homogenized in a tissue homogenizer for 40 seconds. Then, the supernatant was taken and used for the determination of inflammatory factors (IL-6, IL-1β, IL-10, TNF-α) after centrifugation at 4°C and 3500×g for 15 minutes.

[0094] The proximal colon of mice was homogenized and the supernatant was used for ELISA analysis. Fig.10, the Model group fed with a high-fat diet showed obvious inflammation of the colon tissue. Specifically, the levels of pro-inflammatory TNF-α, IL-1β, and IL-6 increased significantly, while the level of anti-inflammatory IL-10 decreased significantly (P<0.05). Feeding rhamnosus lactobacillus GYX-9 had a certain inhibitory effect on high-fat diet-induced colon inflammation. Compared with the Model group, the SLT group and the Orlistat group significantly reduced the level of TNF-α, and the SLT group and the Orlistat group significantly increased the content of IL-10 (P<0.05). The above results show that rhamnosus lactobacillus GYX-9 can alleviate the increase in the level of inflammatory factors, thereby alleviating inflammation in the body.

[0095] Example 5 Lactobacillus rhamnosus GYX-9 regulates intestinal flora imbalance caused by high-fat diet

[0096] The total bacterial DNA in the intestinal contents of mice was extracted according to the instructions of the QIAamp DNA Stool Mini Kit. The DNA was quantified using Nanodrop, and the quality of DNA extraction was tested by 1.2% agarose gel electrophoresis. The conserved regions of bacterial 16S rDNA were amplified using polymerase chain reaction (PCR), and the corresponding primers were designed, and sample-specific Barcode sequences were added.

[0097] PCR conditions were: 95°C, 3 min; 24 cycles: 95°C, 5 s, 57°C, 90 s, 72°C, 10 s; 72°C, 5 min. The 20 μL reaction system included: 2.5 μL TransStart buffer, 2 μL dNTPs, 1 μL forward primer, 1 μL reverse primer, 0.5 μL Pfu polymerase FastPfu Polymerase (derived from 2×TransStart FastPfu kit of Quanshijin, catalog number AS221-02) and 20 ng template DNA. The PCR amplification recovery product was quantified by fluorescence using Quant-iT PicoGreen dsDNAAssay Kit, and the quantitative instrument was Microplate reader (BioTek, FLx800). According to the fluorescence quantification results, the samples were mixed in the corresponding proportions according to the sequencing requirements of each sample.

[0098] Then, the sequencing library was prepared using the TruSeq Nano DNA LT Library Prep Kit (Illumina). The positive and negative reads obtained by double-end sequencing were first spliced ​​in pairs, and the sequences containing N in the splicing results were filtered, and the sequences with a length greater than 200 bp were retained. After quality filtering and purification of chimeric sequences, the obtained sequences were clustered into operational taxonomic units (OTUs) with 97% sequence homology using the QIIME2 dada2 analysis process. These OTUs were aligned with the Greengenes database using PyNAST software, and the classification information was annotated. QIIME2 software (2019.4) and R software (V3.4.1) were used to analyze α-diversity and β-diversity. The principal coordinates analysis (PCoA) of each group of samples was performed using the Jaccard method, and the distance box plot of the samples was drawn. Using the Galaxy online analysis platform, the LEfSe analysis method simultaneously performs differential analysis on all classification levels, draws a bar chart of the LDA value distribution of significantly different species and a species taxonomy branch diagram, and searches for robustly different species between groups.

[0099] 1. Mouse intestinal flora species OTU

[0100] Fig.11 The Venn diagram illustrates the effect of Lactobacillus rhamnosus GYX-9 on the OTUs contained in obese mice induced by a high-fat diet, indicating the similarity of the intestinal flora contained in different samples. OTU clustering was performed on non-repetitive sequences (excluding single sequences) at 97% similarity to obtain representative sequences of OTUs. The Venn diagram shows the diversity relationship of microbial composition between the Control group, Model group, Orlistat group, and SLT group. A total of 671 OTUs were obtained, of which 145 OTUs were in the Control group, of which 39 were unique OTUs; 7 were unique in the Model group; 7 were unique in the Orlistat group; 68 were unique in the SLT group; and 460 OTUs were shared by the four groups, accounting for about 68% of the total OTUs. The intervention of Lactobacillus rhamnosus GYX-9 affected the OUT composition of the intestinal flora of mice to a certain extent, among which the SLT group had the most unique OTUs.

[0101] 2. Alpha diversity analysis of mouse intestinal flora species

[0102] from Fig.12It can be seen that there is no significant difference in Chao index among the groups. Compared with the Control group, the Simpson index of the SLT group decreased, and the Shannon index increased significantly (P<0.05), indicating that the SLT group can improve the diversity of intestinal flora in high-fat mice. Compared with the Control and Model groups, the Orlistat group significantly reduced the Simpson index, and the Shannon index of the SLT and Orlistat groups increased significantly, with higher community diversity.

[0103] 3. Analysis of Beta Diversity of Mouse Intestinal Microbiota

[0104] In order to better reflect the diversity of microbial colony results between groups, PCoA analysis was used. Jaccard analysis method was used to analyze the Beta diversity of intestinal flora of mice in each group. Fig.13 The profiles of the intestinal flora of the four groups of samples were different. It can be considered that there were differences in the flora structure of the four groups. The sample composition between the Model group and the Control group was significantly different. The sample composition between the SLT group and the Orlistat group overlapped. The sample composition between the treatment groups and the Model group did not overlap, and the difference was obvious. The differences in the sample composition between all groups indicated that the high-fat diet intervention caused a significant change in the structure of the microbial community. The microbial community also changed after the intervention of Lactobacillus rhamnosus GYX-9. The microorganisms in the SLT group and the Orlistat group were more closely clustered.

[0105] 4. Composition analysis of mouse intestinal flora at the phylum level

[0106] Fig.14 ACompared the intestinal flora of mice in the Control group, Model group, SLT group, and Orlistat group at the phylum and genus classification levels to analyze the differences in flora. Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria were the dominant phyla in the cecal microbial community. The results of the analysis based on the phylum classification level are as follows Fig.14As shown in the figure, the intestinal flora of experimental mice was mainly Firmicutes, Bacteroidetes, Actionobacteria, Desulfobacterota, Verrucomicrobiota, Patescibacteria, etc. at the phylum level. Firmicutes and Bacteroidetes were the main intestinal flora of mice, accounting for more than 70%. Compared with the Control group, there was no significant difference in the relative abundance of Firmicutes and Bacteroidetes in the Model group, and the relative abundance of Actionobacteria was significantly reduced (P<0.05); compared with the Model group, the relative abundance of Firmicutes in the Orlistat group was significantly reduced, and there was no significant difference in the relative abundance of Bacteroidetes and Actionobacteria. The relative abundance of Patescibacteria in the SLT group was significantly upregulated.

[0107] Fig.14 B. When the ratio of Firmicutes / Bacteroidetes (F / B) in the intestine is imbalanced, the body will produce a large amount of glycoside hydrolases, which promote the fermentation and decomposition of food in the gastrointestinal tract, turning it into monosaccharides or short-chain fatty acids, causing the body to absorb excessive energy, thereby leading to weight gain. Compared with the Control group and the Model group, the F / B ratio in the SLT group and the ORT group was significantly reduced, indicating that Lactobacillus rhamnosus GYX-9 can prevent and improve obesity by reducing the ratio of Firmicutes to Bacteroidetes.

[0108] 5. Composition analysis of mouse intestinal flora at the genus level

[0109] The changes in intestinal flora were further analyzed at the genus level. Fig.15 It can be seen that the intestinal flora of the four groups of mice at the genus level mainly include beneficial bacteria such as norank_f_Muribaculaceae, Dubosiella, Lachnospiraceae_NK4A136 (Lachnospiraceae_NK4A136_group), Lactobacillus, Enterobacterium, and Bifidobacterium; and harmful bacteria such as Ileibacterium, Desulfovibrio, and norank_f__Oscillospiraceae.

[0110] Treatment with inactivated Bifidobacterium longum (IBL) in the genus Bifidobacterium resulted in a significant reduction in adipose tissue mass and blood glucose levels in obese diabetic model mice, as well as a significant reduction in blood glucose. IBL may have the potential to prevent obesity and diabetes. Although Colidextribacter may help regulate systemic inflammatory responses and maintain the integrity of the intestinal mucosa, the relative abundance of Colidextribacter increased significantly in some non-alcoholic liver injury models. The expansion of Candidatus_Saccharimonas is associated with inflammatory diseases. In this embodiment, the abundance of Candidatus_Saccharimonas was significantly upregulated, which may be due to its abundance being negatively correlated with the expression of TLR2, which plays an important role in regulating insulin sensitivity and glucose homeostasis. A high-fat diet can cause a significant increase in the relative abundance of Romboutsia, but there are also opposite results. For example, Romboutsia plays a vital role in the overall beneficial effects of certain polyphenols in alleviating obesity, hepatic steatosis and dyslipidemia. In some studies, the relative abundance of Erysipelatoclostridium was found to increase in the treatment group of ulcerative colitis, and its relative abundance was positively correlated with inflammatory factors in some non-alcoholic liver injury models. Fig.16 Compared with the Control group, the Model group significantly reduced the relative abundance of beneficial bacteria such as Bifidobacterium and Colidextribacter (P < 0.05). Compared with the Model group, the SLT group significantly increased the relative abundance of Bifidobacterium, Candidatus_Saccharimonas, Romboutsia, Erysipelatoclostridium, Colidextribacter, etc. (P < 0.05), and other relative abundances of bacteria at the level of body weight-related genera, thereby alleviating obesity in high-fat mice.

[0111] Studies have shown that in obesity and high-fat models, the abundance of Ileibacterium can increase 10 times. Ileibacterium is likely to be associated with metabolic diseases, and its specific role needs further study. In different disease models, norank_f_Ruminococcaceae has different roles. Its abundance in the DSS-induced colitis model is lower than that in the treatment group; in the diabetes model, g_Ruminococcaceae_UCG-014 is positively correlated with blood sugar; and it can also improve the intestinal flora disorder caused by a high-fat diet by increasing its relative abundance.

[0112] g__norank_f__norank_o__RF39 abundance was positively correlated with IL-6. Fig.17 , compared with the Model group,

[0113] The relative abundance of Ileibacterium, norank_f_Ruminococcaceae, norank_f__norank_o__RF39, and Gordonibacter in the SLT group was significantly decreased (P<0.05).

[0114] Therefore, a high-fat diet can cause intestinal flora disorders in mice to a certain extent. Lactobacillus rhamnosus GYX-9 intervention can regulate some intestinal flora, and there are certain differences in the regulated flora in different groups. Among them, Lactobacillus rhamnosus GYX-9 can increase the relative abundance of Bifidobacterium, Candidatus_Saccharimonas, Romboutsia, Colidextribacter, and Erysipelatoclostridium, and reduce the relative abundance of Ileibacterium, norank_f__norank_o__RF39, Gordonibacter, and norank_f__Ruminococcaceae, with significant regulatory effects.

[0115] 6. Analysis of species differences in the effects of Lactobacillus rhamnosus GYX-9 on high-fat diet mice

[0116] Fig.18 It can be seen that the species with significant differences between the groups were further characterized based on linear discriminant analysis (Linear discriminant analysisEffect Size, LEfSe), that is, LDA Effect Size analysis (LDA>2; P<0.05). The mice in the Control group were mainly enriched in Bifidobacterium, Staphylococcus, Lachnoclostridium, etc. The SLT group was mainly enriched in genera such as Colidextribacter and Parasutterella. In the Model group, the genera with higher LDA scores included norank_f__norank_o__Clostridia_vadinBB60_group and Faecalibaculum. The biomarkers of the Orlistat group were norank_f__Ruminococcaceae, Enterorhabdus, etc.

[0117] norank_f__norank_o__Clostridia_vadinBB60 is an unclassified Clostridia bacterial group. There may be a lack of detailed research data at present. Some may be related to the occurrence of diseases, such as some types of Clostridia may produce toxins or cause infection. In the Model group, except for the increase in the relative abundance of Faecalibaculum, the SLT group significantly reduced the main enriched genera in the Model group compared with the Model group, and the results of the enriched genera and genus level analysis were consistent.

[0118] 7. Correlation analysis between intestinal microorganisms and biochemical indicators

[0119] The correlation between intestinal microorganisms and biochemical indicators was analyzed by Spearman correlation coefficient, and the main enriched bacterial genera and intestinal microorganisms significantly regulated by the SLT group were combined, such as Fig.19 We found that the relative abundance of Ileibacterium was positively correlated with proinflammatory factors IL-1β, IL-6, AST, and leptin; the relative abundance of norank_f__norank_o__RF39 was positively correlated with IL-1β, showing a harmful effect. The relative abundance of Erysipelatoclostidium and Lachnoclostidium was negatively correlated with IL-6, TNF-α, LPS, TG, LDLC, etc., and positively correlated with HDLC, adiponectin, and CAT; the relative abundance of Romboutsia was negatively correlated with TNF-α and fasting blood glucose. In addition, the proinflammatory factors IL-1β, IL-6, and TNF-α were positively correlated with TC, LDLC, leptin, AST, ALT, and FFA; the anti-inflammatory factor IL-10 was negatively correlated with TC, TG, LDLC, and FFA, and positively correlated with antioxidant enzyme activity. Lactobacillus rhamnosus GYX-9 alleviates obesity and colon inflammation by downregulating Ileibacterium, norank_f__norank_o__RF39 and upregulating Erysipelatoclostidium, Lachnoclostidium, and Romboutsia.

[0120] Example 5 Encapsulation of Lactobacillus rhamnosus GYX-9

[0121] Freeze-drying method for embedding rhamnosus casei GYX-9: whey protein powder rich in milk fat globule membrane (M-WPI, Hilmar-7500 MFGM protein powder, purchased from Hilmar, USA) was selected as a freeze-drying protective agent, and the freeze-drying protective agent was dissolved in pure water to prepare a 14% freeze-drying protective agent solution (m / v, g / mL). 0.1 g of rhamnosus casei bacterial sludge (rhamnosus casei GYX-9 liquid culture to a bacterial concentration of 1×1011 cfu / mL, centrifuged at 6000 rpm for 10 min and discarded the supernatant) and reconstituted in 1 mL of lyophilization protectant solution. Pure water was used as a blank control group and vacuum freeze-dried for 20 h to obtain lyophilized bacterial powder.

[0122] Embedding rhamnosus casei GYX-9 by emulsification method: Take a certain amount of 10% (m / v, g / mL) M-WPI freeze-dried protective agent solution and mix it with rhamnosus casei bacterial mud (the liquid culture of rhamnosus casei GYX-9 reaches a bacterial concentration of 1×10 11 cfu / mL, centrifuge at 6000rpm for 10min, discard the supernatant, dissolve the bacterial sludge in distilled water (mass ratio of bacterial sludge to whey protein powder is 1:1), adjust the pH to 7.0, and emulsify for 60s using a high-speed shear dispersing emulsifier to obtain an emulsion. Weigh an appropriate amount of phospholipids and heat and dissolve them in soybean oil, add the above-prepared emulsion (water-oil ratio is 9:1), and emulsify for 60s. Then mix the two, and homogenize them using a high-pressure homogenizer at 30Mpa to obtain a M-WPI-phospholipid composite emulsion encapsulating lactic acid bacteria.

[0123] The MRS plate was used as the counting medium and the plate counting method was used to count the viable bacteria. The calculation method is as follows:

[0124]

[0125] In the formula: N1-the number of viable bacteria after treatment, lg (cfu / g); N0-the number of viable bacteria under normal conditions, lg (cfu / g).

[0126] Depend on Fig. 20 It can be seen that both methods can significantly improve the survival rate of Lactobacillus rhamnosus GYX-9. The survival rate before embedding was (66±0.02)%, and the survival rates after freeze-drying and emulsification embedding reached 92.63% and 96.4%, respectively, and there was no significant difference between the two embedding methods.

[0127] Example 6 Preparation of M-WPI pasteurized milk

[0128] 30 mL of raw milk was used as the raw material, 5% (m / v) M-WPI was added for sterilization, and then the above-mentioned freeze-dried Lactobacillus casei GYX-9 was inoculated, and commercial starter (including Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus, purchased from Suzhou Weikang Probiotics Co., Ltd., product standard code Q / WKSW) was used.

[0129] 0001S) was fermented at 42°C for 4h, wherein the addition ratio of Lactobacillus rhamnosus GYX-9 to commercial starter was 5:1 (m / m), and then the milk was pasteurized at 72°C for 30s to form M-WPI pasteurized milk.

[0130] Example 7 Preparation of M-WPI postbiotic yogurt + bacterial powder room temperature live bacteria fermented milk

[0131] Add 1% (m / v) freeze-dried Lactobacillus rhamnosus GYX-9 to pasteurized milk to produce a product with a viable cell count of more than 1×10 10 cfu / mL of M-WPI postbiotic yogurt + bacterial powder fermented milk with live bacteria at room temperature.

[0132] Example 8 Preparation of M-WPI postbiotic yogurt + emulsion room temperature live bacteria fermented milk

[0133] 10% (v / v) of Lactobacillus rhamnosus GYX-9 embedded by emulsification was added to M-WPI pasteurized milk to produce a culture with a viable cell count of more than 1×10 8 cfu / mL of M-WPI postbiotic yogurt + milk fermented with live bacteria at room temperature. At this time, the amount of milk added has no obvious effect on the appearance of yogurt.

[0134] Example 9 Determination of lipid-lowering properties of fermented milk prepared in Examples 6 to 8

[0135] Determination of the degradation ability of fermented milk cholesterol micelles: 2mmol / L cholesterol, 1mmol / L oleic acid, 0.5mmol / L monoacylglycerol, 6.6mmol / L sodium taurocholate, and 0.1mmol / L soybean lecithin were prepared and dissolved in a PBS solution, the pH was adjusted to 7.2, and stored at 37°C for use. 0.1mL of the three fermented milks prepared in Examples 6 to 8 were diluted 10 times and added to the cholesterol micelles respectively, and after oscillation and mixing, they were ultrasonically cleaned in an ultrasonic cleaner for 1h and placed in a 37°C incubator for 24h. The cholesterol content was detected using a total cholesterol determination kit.

[0136] Determination of the ability of fermented milk to bind taurocholate: Take 0.5 mL of fermented milk prepared in Examples 6 to 8 and 0.5 mL of simulated gastric juice in a test tube, vortex and mix, digest in a water bath at 37°C for 1 hour, adjust the pH value to 6.3, and then add 2 mL of simulated intestinal juice to continue digestion in a water bath at 37°C for 1 hour. Then add 2 mL of taurocholate solution to each test tube, react in a water bath at 37°C for 1 hour, centrifuge at 4000r / min for 10 minutes, take 1.25 mL of the supernatant, add 3 times the volume of 60% sulfuric acid solution, mix, and treat in a water bath at 70°C for 20 minutes. After the water bath ends, take out the ice bath for 5 minutes, measure its absorbance at a wavelength of 387nm, and calculate the taurocholate binding rate.

[0137] Determination of the inhibitory rate of pancreatic lipase activity in fermented milk: Use Tri-HCl buffer (0.01 mol / L, pH 8.0) to prepare a 0.5 mg / mL pancreatic lipase solution. Use acetonitrile to make up 10 μL of 2,4-dinitrophenol butyrate (PNPB) to 5 mL, centrifuge at 2000 r / min for 5 min, take 300 μL of the supernatant and mix with equal volumes of fermented milk prepared in Examples 6 to 8, and after 37°C water bath for 30 min, add 20 μL of PNPB solution and continue to water bath at 37°C for 10 min. After the reaction is completed, measure the absorbance at a wavelength of 405 nm.

[0138] Depend on Fig.21 It can be seen that the yogurt has relatively low binding ability with cholesterol micelles, taurocholate binding rate and pancreatic lipase activity inhibition rate after pasteurization, but still has a certain lipid-lowering ability, which indicates that the rhamnosus lactobacillus GYX-9 postbiotic also has lipid-lowering effect. Adding freeze-dried bacterial powder and emulsion to postbiotic yogurt can significantly improve the binding ability of postbiotic yogurt with cholesterol micelles, taurocholate binding rate and pancreatic lipase activity inhibition rate. Among them, the pancreatic lipase activity of M-WPI postbiotic yogurt + bacterial powder and M-WPI postbiotic yogurt + emulsion room temperature live bacteria fermented milk has significant differences, which are 67.06% and 57.60%, respectively. This may be because the live bacteria of rhamnosus lactobacillus GYX-9 have higher lipid-lowering activity.

Claims

1. A strain of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) GYX-9, characterized in that The Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) GYX-9 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 14, 2024, with the deposit number: CGMCC No. 30946.

2. A microbial preparation, food or medicine, characterized in that: It contains the Lactobacillus rhamnosus described in claim 1 ( Lacticaseibacillus rhamnosus ) GYX-9, its fermentation liquid or supernatant; preferably, the microbial preparation, food or medicine also includes a lyophilization protective agent, preferably, the lyophilization protective agent includes whey protein powder rich in milk fat globule membrane; preferably, the microbial preparation also includes a commercial starter, the microbial preparation or medicine includes a single or compound preparation, and the preparation type includes a liquid preparation or a solid preparation; preferably, the mass ratio of the whey protein powder rich in milk fat globule membrane to the rhamnosus casei bacteria mud GYX-9 is 1:1; preferably, the food is room temperature live bacteria fermented milk, and the mass ratio of the rhamnosus casei bacteria GYX-9 to the commercial starter is 5:1; preferably, the content of the rhamnosus casei bacteria GYX-9 is not less than 1×10 8 cfu / mL; preferably, the room temperature live bacteria fermented milk includes embedded Lactobacillus rhamnosus GYX-9 live bacteria and Lactobacillus rhamnosus GYX-9 postbiotics.

3. The Lactobacillus rhamnosus of claim 1 ( Lacticaseibacillus rhamnosus ) Use of GYX-9, the microbial preparation, food or medicine according to claim 2 in the preparation of medicines or fermented milk for improving blood lipid metabolism disorders, liver fat accumulation, improving intestinal flora imbalance and / or regulating colon immune disorders in obese animals caused by high-fat diet.

4. The Lactobacillus rhamnosus of claim 1 ( Lacticaseibacillus rhamnosus ) Use of GYX-9, the microbial preparation, food or medicine according to claim 2 in the preparation of a product for alleviating obesity or colitis caused by a high-fat diet.

5. A method for preparing room temperature fermented milk, characterized in that: The following steps are involved: 1) Encapsulating Lactobacillus rhamnosus GYX-9; 2) Using raw milk as raw material, inoculating Lactobacillus rhamnosus GYX-9 live bacteria for fermentation and then sterilizing to obtain sterilized milk; 3) adding the embedded Lactobacillus rhamnosus GYX-9 from step 1) to the sterilized milk from step 2); Preferably, the embedding method in step 1) includes freeze-drying or emulsification embedding; Preferably, step 2) further comprises inoculating a commercial starter culture.

6. Room temperature live bacteria fermented milk obtained by the preparation method according to claim 5.

7. The use according to claim 3 or 4, characterized in that: The application includes any one of the following (a) to (e): (a) preparing products for regulating indicators of blood lipid metabolism; (b) preparing a product for improving fat accumulation and oxidative stress in liver tissue caused by a high-fat diet; (c) preparing products that reduce pro-inflammatory factors and increase anti-inflammatory factors in the colon; (d) preparing products that increase the diversity of intestinal flora and modulate the abundance of intestinal microbiome communities; (e) Preparing products that lower cholesterol, increase taurocholate binding rate and / or pancreatic lipase activity inhibition rate.

8. The use according to claim 7, characterized in that: The indicators for regulating blood lipid metabolism include regulating TG, TC, HDL-C, LDL-C, leptin, adiponectin, ALT and / or AST; improving the oxidative stress of the liver tissue includes a significant increase in the activity of antioxidant enzymes; improving the fat accumulation of the liver tissue is manifested by a significant decrease in the free fatty acid FFA in the liver, and a significant decrease in the liver coefficient and the epididymal fat coefficient.

9. The use according to claim 7, characterized in that: The pro-inflammatory factors in the colon include TNF-α, IL-1β and / or IL-6, and the anti-inflammatory factors mainly include IL-10.

10. The use according to claim 9, characterized in that: The method of increasing the diversity of intestinal flora and regulating the abundance of intestinal microbial community includes increasing Candidatus_Saccharimonas, Romboutsia, Erysipelatoclostridium, Colidextribacte Relative abundance, decreased norank_f__norank_o__RF39, Gordonibacter, norank_f__Ruminococcaceae Relative abundance.

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