Yeast and probiotic compound for treating non-alcoholic fatty liver as well as preparation method and application of yeast and probiotic compound

Abstract

The invention discloses a yeast and probiotic compound for treating non-alcoholic fatty liver disease as well as a preparation method and application of the yeast and probiotic compound. The yeast and probiotic compound is prepared from probiotics such as Blauraceae and Blauraceae, Roseburia intestinae and Roseburia hominis and Saccharomyces cerevisiae Meyen ex E.C.Hansen, and the volume ratio of the probiotics such as the Blauraceae, the Roseburia intestinae and the Saccharomyces cerevisiae to the Saccharomyces cerevisiae is (0.1-10): (0.1-7): (0.2-5). According to the present invention, the key links of the non-alcoholic fatty liver disease are regulated, such that the occurrence and the development of the non-alcoholic fatty liver disease course can be effectively controlled, and the insulin resistance can be improved and the lipid metabolism abnormality can be adjusted; meanwhile, the vacancy that no marketed medicine for treating NAFLD exists is made up, and the medicine is convenient to use, safe and effective.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a yeast-probiotic complex for treating non-alcoholic fatty liver disease and an application thereof. Background Art

[0002] Nonalcoholic fatty liver disease (NAFLD) is a complex metabolic-related disease that affects approximately 1 / 4 of the world's population, posing a significant health and economic burden to society. NAFLD is closely associated with T2DM and abdominal obesity, and the global prevalence of NAFLD has increased dramatically with changes in lifestyle and dietary habits. NAFLD is common in all continents, with the highest incidence in South America (31%) and the Middle East (32%), followed by Asia (27%), the United States (24%) and Europe (23%), while the least prevalent in Africa (14%). In particular, its histological phenotype, NASH, may develop into advanced liver disease, cirrhosis and hepatocellular carcinoma (HCC). The prevalence of NAFLD continues to increase (15% in 2005 to 25% in 2010), and similarly, the incidence of NASH has almost doubled in the same time period (5.91% to 33%). NASH is now considered the second most common indication for liver transplantation in the United States after chronic viral hepatitis, and the number of cases continues to grow. NAFLD stands for Non-alcoholic Fatty Liver Disease.

[0003] Other studies have found that intestinal flora can affect the course of NAFLD. Intestinal microorganisms and bacterial metabolites may lead to the occurrence and development of NAFLD through multiple mechanisms such as increased lipid absorption, energy absorption, increased intestinal permeability, and induction of chronic inflammation. Feces from metabolic integrated model mice fed an HFD diet were transplanted into germ-free recipient mice, inducing de novo lipid synthesis in the liver of recipient mice, increasing triglyceride content in the liver of recipient mice by 3 times, and increasing the expression of lipogenic genes in the liver. In addition, intestinal flora microorganisms can regulate the progression of NAFLD-HCC. The intestinal microbial structure of NAFLD-HCC is more disordered than that of healthy people, and bacterial extracts from NAFLD-HCC microorganisms can induce T cell proliferation and a decrease in CD8+T cells.

[0004] Currently, there are no drugs on the market that can improve liver fibrosis, and new clinical bacterial therapy strategies, namely the use of specific prebiotics, probiotics, synbiotics and fecal transplants from healthy volunteers to restore the balance of intestinal microbial communities, thereby preventing and treating metabolic diseases such as NASH, are attracting attention. The research team of Academician Li Lanjuan of the Chinese Academy of Engineering wrote in the fifth issue of Engineering, the journal of the Chinese Academy of Engineering, in 2021 that the most popular probiotics in the treatment of non-alcoholic fatty liver disease mainly include traditional probiotics represented by Bifidobacterium and Lactobacillus, as well as the next generation of emerging probiotics, such as Akkermansia muciniphila and Faecalibacterium prausnitzii.

[0005] In a study on human intestinal bacteria, it was shown that fecal transplantation from healthy lean donors to obese patients could control the weight of obese patients, which may be related to a significant increase in the proportion of butyrate producer Roseburia intestinalis. In a weight loss study, it was found that the abundance of Blautia wexlerae (MGS0575) and Bacteroides dorei (MGS0187) bacteria was significantly positively correlated with the weight loss of patients, suggesting that these two bacteria are potential probiotics for weight loss. Summary of the invention

[0006] The present invention provides a yeast-probiotics complex for treating non-alcoholic fatty liver disease, and a preparation method and application thereof, in order to solve the above-mentioned problems in the prior art.

[0007] The scheme of the present invention is: A yeast-probiotic complex for treating non-alcoholic fatty liver disease, characterized in that it comprises Blautia ovata, probiotic Blautia ovata, Rosenblum, obligate Gram-positive anaerobic bacteria and Saccharomyces cerevisiae, wherein the volume ratio of Blautia ovata, probiotic Blautia ovata, Rosenblum, obligate Gram-positive anaerobic bacteria and Saccharomyces cerevisiae is 0.1-10:0.1-10:0.1-7:0.1-7:0.2-5, the Blautia ovata is preserved by the German Collection of Microorganisms and Cell Cultures with a preservation number of DSM 25238, the probiotic Blautia ovata is preserved by the German Collection of Microorganisms and Cell Cultures with a preservation number of DSM 19850, the Roseberys species is deposited by the German Collection of Microorganisms and Cell Cultures with the accession number DSM14610, the obligate Gram-positive anaerobic bacteria is deposited by the German Collection of Microorganisms and Cell Cultures with the accession number DSM16839, and the Saccharomyces cerevisiae is deposited by the German Collection of Microorganisms and Cell Cultures with the accession number DSM1333.

[0008] The volume ratio of Blautia ovale, probiotic Blautia, Roseburia, obligate Gram-positive anaerobic bacteria and Saccharomyces cerevisiae is 1:1:1:1:0.5.

[0009] The suspensions of Blautia ovata, probiotic Blautia ovata, Rosenblumeria, obligate Gram-positive anaerobic bacteria and Saccharomyces cerevisiae were prepared at a density of 1×10^8 cfu / ml respectively, and then mixed according to a volume ratio.

[0010] At the same time, the present invention also provides a method for preparing the yeast probiotics complex for treating non-alcoholic fatty liver disease, which is characterized by comprising the following steps: 1). Metagenomic testing was performed on the feces of patients with non-alcoholic steatohepatitis (NASH) and the control group (NO-NASH), and the probiotics significantly enriched in NO-NASH were screened, and significantly enriched Blautia ovata, probiotic Blautia, Rosenblumella, and obligate Gram-positive anaerobic bacteria were obtained; 2). The Blautia ovata was placed in a modified YCFA medium, the probiotic Blautia was placed in a modified YCFA medium, the Roseberry was placed in a modified PYG medium, the obligate Gram-positive anaerobic bacteria was placed in a modified PYG medium, and the Saccharomyces cerevisiae was placed in a YM medium, and then all of the above five were cultured in an anaerobic workstation, and the bacterial concentration was detected by the OD600 method during the logarithmic growth period, and diluted to a concentration of 1×10^8 cfu / ml; 3). Mix Blautia ovata, probiotic Blautia, Rosenblum, obligate Gram-positive anaerobic bacteria and Saccharomyces cerevisiae in a volume ratio of 0.1-10:0.1-10:0.1-7:0.1-7:0.2-5, and store the obtained suspension in a refrigerator at -80°C for later use.

[0011] The modified YCFA medium consists of 10.0 g of tryptic peptone, 10.0 g of beef extract powder, 3.0 g of yeast extract powder, 5.0 g of glucose, 5.0 g of sodium chloride, 1.0 g of soluble starch, 0.5 g of L-cysteine ​​hydrochloride, 3.0 g of sodium acetate, and 0.001 g of resazurin. The modified PYG medium consists of 5.0 g of tryptic peptone, 5.0 g of peptone, 10.0 g of alcohol mother extract powder, 5.0 g of beef extract powder, 5.0 g of glucose, 2.0 g of K2HPO4, 1.0 ml of Tween 80, and 1.0 mg of resazurin. The YM medium consists of 3.0 g of alcohol mother extract powder, 3.0 g of malt extract powder, 5.0 g of peptone, and 10.0 g of glucose.

[0012] In step 3), Blautia ovale, probiotic Blautia, Roseburia, obligate Gram-positive anaerobic bacteria and Saccharomyces cerevisiae are mixed in a volume ratio of 1:1:1:1:0.5.

[0013] At the same time, the present invention also provides an application of the yeast probiotic complex for treating non-alcoholic fatty liver disease, characterized in that the probiotic complex is used to treat non-alcoholic fatty liver disease, improve insulin resistance and adjust abnormal lipid metabolism.

[0014] The present invention performs metagenomic analysis on feces collected from NO-NASH and NASH patients, screens out probiotics that are significantly enriched in NO-NASH and combines them to provide a yeast-probiotic complex for treating non-alcoholic fatty liver disease. The present invention regulates key links of non-alcoholic fatty liver disease, which can not only effectively control the occurrence and development of the course of non-alcoholic fatty liver disease, but also improve insulin resistance and adjust abnormal lipid metabolism; at the same time, it also makes up for the lack of marketed drugs for the treatment of NAFLD, and is easy to use, safe and effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Comparison of mouse body weight, liver weight and fat weight in the experiment of the present invention; (A) mouse body weight (B) mouse liver weight (C) mouse fat weight; Ctrl is the normal diet group, HFD is the high-fat diet group, HFD+Probiotic is the high-fat diet + probiotic colonization group; N=6, * is P <0.05,** P <0.01; # is P <0.05,## is P <0.01.

[0016] Figure 2 Comparison of serum ALT, AST, TC and TG levels in the experiment of the present invention; (A) serum ALT content (B) serum AST content (C) serum TC content (D) serum TG content; Ctrl is the normal diet group, HFD is the high-fat diet group, HFD+Probiotic is the high-fat diet + probiotic colonization group; N=6, * is P <0.05,** P <0.01.

[0017] Figure 3 Figure 1 shows the experimental diagram of insulin tolerance and glucose tolerance of mice in the experiment of the present invention; (A) Insulin tolerance of mice (B) Glucose tolerance of mice, Ctrl is the normal diet group, HFD is the high-fat diet group, HFD+Probiotic is the high-fat diet + probiotic colonization group; N=6, * is P <0.05,**P <0.01.

[0018] Figure 4 Figure 2 shows the hematoxylin-eosin staining and oil red O staining of mouse liver in the experiment of the present invention; (A) hematoxylin-eosin staining of mouse liver tissue (B) oil red O staining of mouse liver, Ctrl is the normal diet group, HFD is the high-fat diet group, and HFD+Probiotic is the high-fat diet + probiotic colonization group; N=6, * is P <0.05,** P <0.01. DETAILED DESCRIPTION

[0019] In order to make up for the above deficiencies, the present invention provides a yeast-probiotic complex for treating non-alcoholic fatty liver disease and its application to solve the problems in the above background technology.

[0020] A yeast-probiotic complex for treating non-alcoholic fatty liver disease, characterized in that it includes Blautia obeum, probiotic Blautia wexlerae, Roseburia intestinalis, obligate Gram-positive anaerobic bacteria (Roseburia hominis) and Saccharomyces cerevisiae Meyen ex EC Hansen, wherein the volume ratio of Blautia obeum, probiotic Blautia wexlerae, Roseburia intestinalis, obligate Gram-positive anaerobic bacteria and Saccharomyces cerevisiae Meyen ex EC Hansen is 0.1-10:0.1-10:0.1-7:0.1-7:0.2-5, and the Blautia obeum is preserved by the German Collection of Microorganisms and Cell Cultures (dsmz) and the preservation number is DSM 25238, the probiotic bacterium Blautia is deposited by the German Collection of Microorganisms and Cell Cultures (dsmz) with the accession number DSM 19850, the Rosenblumeria is deposited by the German Collection of Microorganisms and Cell Cultures (dsmz) with the accession number DSM14610, the obligate Gram-positive anaerobic bacteria is deposited by the German Collection of Microorganisms and Cell Cultures (dsmz) with the accession number DSM16839, and the Saccharomyces cerevisiae is deposited by the German Collection of Microorganisms and Cell Cultures (dsmz) with the accession number DSM1333.

[0021] The volume ratio of Blautia ovale, probiotic Blautia, Roseburia, obligate Gram-positive anaerobic bacteria and Saccharomyces cerevisiae is 1:1:1:1:0.5.

[0022] The suspensions of Blautia ovata, probiotic Blautia ovata, Rosenblumeria, obligate Gram-positive anaerobic bacteria and Saccharomyces cerevisiae were prepared at a density of 1×10^8 cfu / ml respectively, and then mixed according to a volume ratio.

[0023] At the same time, the present invention also provides a method for preparing the yeast probiotics complex for treating non-alcoholic fatty liver disease, which is characterized by comprising the following steps: 1) Metagenomic testing was performed on the feces of patients with non-alcoholic steatohepatitis (NASH) and the control group (NO-NASH), and probiotics significantly enriched in NO-NASH were screened, obtaining significantly enriched Blautia ovata, probiotic Blautia, Roseberyces, and obligate Gram-positive anaerobic bacteria; 2) Placing the Blautia ovata in a modified YCFA medium, the probiotic Blautia ovata in a modified YCFA medium, the Roseberry in a modified PYG medium, the obligate Gram-positive anaerobic bacteria in a modified PYG medium, and the Saccharomyces cerevisiae in a YM medium, and then culturing all five in an anaerobic workstation, taking the logarithmic growth phase, detecting the bacterial concentration by the OD600 method, and diluting to a concentration of 1×10^8 cfu / ml; 3) Mix Blautia ovata, probiotic Blautia, Rosenblum, obligate Gram-positive anaerobic bacteria and Saccharomyces cerevisiae in a volume ratio of 0.1-10:0.1-10:0.1-7:0.1-7:0.2-5, and store the obtained suspension in a -80°C refrigerator for later use.

[0024] Modified YCFA medium: culture medium formula (per liter), Tryptose (trypticase peptone) 10.0g, Beef Extract (beef extract powder) 10.0g, Yeast Extract (yeast extract powder) 3.0g, Dextrose (glucose) 5.0g, NaCl (sodium chloride) 5.0g, Soluble Starch (soluble starch) 1.0g, L-Cysteine ​​HC1 (L-cysteine ​​hydrochloride) 0.5g, Sodium Acetate (sodium acetate) 3.0g, Resazurin (resazurin) 0.001g, pH 6.8±0.2 (25℃).

[0025] Instructions for use: Weigh 33.6 g of this product in 1% distilled water or deionized water, heat to boil (it is recommended to continue boiling for 3-5 minutes), cool under 002 environment, add 85605 (0.5 mg hemin), S5606 (0.1 mg vitamin K1) and S1505 (0.05 g cysteine ​​hydrochloride) 1 bottle each per 100 ml, divide into test tubes under N2 environment, sterilize at 121℃ for 15 minutes, and set aside.

[0026] YM medium; medium formula (per liter), Yeast Extract (alcohol extract powder) 3.0g, Malt Extract (malt extract powder) 3.0g, Peptone (peptone) 5.0g, Dextrose (glucose) 10.0g, pH 6.2±0.2 (25℃)).

[0027] Instructions for use: Weigh 21.0 g of this product into 1% distilled water or deionized water, heat and boil until completely dissolved, divide into portions, sterilize under high pressure at 121℃ for 15 minutes, and set aside.

[0028] In step 3), Blautia ovale, probiotic Blautia, Roseburia, obligate Gram-positive anaerobic bacteria and Saccharomyces cerevisiae are mixed in a volume ratio of 1:1:1:1:0.5.

[0029] The present invention also discloses an application of a yeast-probiotic complex for treating non-alcoholic fatty liver disease, and an application of the probiotic complex in treating non-alcoholic fatty liver disease, improving insulin resistance and regulating abnormal lipid metabolism.

[0030] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments.

[0031] Embodiment 1: 1) Perform metagenomic testing on the feces of NO-NASH and NASH patients, and screen for probiotics that are significantly enriched in NO-NASH; 2) The Blautia obeum, probiotic Blautia spp., Roseburia intestinalis, obligate Gram-positive anaerobic bacteria Roseburia hominis and Saccharomyces cerevisiae are cultured in an anaerobic workstation according to the official culture medium, and the logarithmic growth phase is taken, and the mixture is diluted to a density of 1×10^8 cfu / ml and then mixed; 3) Mix Blautia ovata, probiotic Blautia, Roseburia, obligate Gram-positive anaerobic bacteria and Saccharomyces cerevisiae in a volume ratio of 1:1:1:1:0.5, and store the resulting suspension in a -80°C refrigerator for future use.

[0032] Embodiment 2: 1) Perform metagenomic testing on the feces of NO-NASH and NASH patients, and screen for probiotics that are significantly enriched in NO-NASH; 2) Cultivate Blautia ovata, probiotic Blautia ovata, Rosenblum, obligate Gram-positive anaerobic bacteria and Saccharomyces cerevisiae in an anaerobic workstation according to the official culture medium, take the logarithmic growth phase, and mix at a density of 1×10^8 cfu / ml; 3) Blautia ovata, probiotic Blautia ovata, Roseburia rosea, obligate Gram-positive anaerobic bacteria and Saccharomyces cerevisiae were mixed at a volume ratio of 0.1:0.1:0.1:0.1:0.2, and the obtained suspension was stored in a -80°C refrigerator for later use.

[0033] Embodiment 3: 1) Perform metagenomic testing on the feces of NO-NASH and NASH patients, screen for probiotics that are significantly enriched in NO-NASH, and obtain significantly enriched probiotics Blautia and Roseberys; 2) Blautia ovata, probiotic Blautia ovata, Rosenblum, obligate Gram-positive anaerobic bacteria and Saccharomyces cerevisiae were cultured in an anaerobic chamber according to the official culture medium, and the logarithmic growth period was selected and mixed at a density of 1×10^8 cfu / ml; 3) Mix Blautia ovata, probiotic Blautia, Roseburia, obligate Gram-positive anaerobic bacteria and Saccharomyces cerevisiae in a volume ratio of 10:10:7:7:5, and store the resulting suspension in a -80°C refrigerator for later use.

[0034] Experiment: 1. Probiotics ratio: According to previous work, stool samples of NO-NASH and NASH patients were collected for metagenomic testing, and probiotics significantly enriched in NO-NASH were screened. The significantly enriched Blautia obeum (DSM 25238) and probiotics Blautia wexlerae (DSM 19850), Roseburia intestinalis (DSM14610) and obligate Gram-positive anaerobic bacteria (Roseburia hominis DSM16839) were compared with Saccharomyces cerevisiae Hansen DSM1333) was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ), and cultured in an anaerobic box according to the official culture medium, and a suspension was prepared at a density of 1*10^8cfu / ml. The mixture was combined according to the ratio in Example 1, and it was found that the five probiotics were mixed in a ratio of 1:1:1:1:0.5 to treat NAFLD mice with the most obvious effect. The five probiotics were inoculated in a liquid culture medium, and the logarithmic growth period was taken. The bacterial density was detected by the OD600 method, and the bacterial liquid was diluted according to a density of 1*10^8cfu / ml, and a probiotic complex suspension (Probiotics) was prepared in a ratio of 1:1:1:1:0.5 according to the volume, and stored in a refrigerator at -80°C for later use.

[0035] 2. Establishment of NAFLD mice: C57BL / 6J male mice, 6-8 weeks old, SPF grade, were purchased from Shanghai Slake Experimental Animal Center. After 1 week of adaptive feeding, they were randomly divided into a normal diet group (Ctrl, 10% calories from fat, 12450B, Research Diets) and a high-fat diet group (HFD, 60% calories from fat, D12492, Research Diets) according to a random number table, and the NAFLD model was established for 8 weeks.

[0036] 3. Establishment of Pseudo-germ-free Mice: According to the internationally recognized guidelines for the use of antibiotics (ABX Guidelines): the broad-spectrum antibiotic ampicillin (Am P Mice were treated with 1g / L icillin, 1g / L), 1g / L neomycin, 0.5g / L vancomycin (ABX) in drinking water for 2 weeks to establish a pseudo-germ-free mouse model, in which the intestinal flora was widely suppressed.

[0037] 4. Mouse grouping and drug administration: After 8 weeks of HFD induction, ABX was given to deplete intestinal bacteria for 2 weeks, and then probiotics were transplanted. The experiment was terminated after 6 weeks. The specific groups were as follows: 1) Normal diet group + saline (Ctrl); 2) high-fat diet group + ABX + saline (HFD); 3) high-fat diet group + ABX + probiotic colonization (HFD+Probiotics); The experiment was ended after 6 weeks, and the body weight, liver weight, glucose tolerance and insulin tolerance, serum biochemical indexes of mice were tested. The degree of fatty degeneration of mouse liver was observed by histopathology, and the effect of probiotics on NAFLD mouse model was observed.

[0038] The animal experiment process of the present invention is as follows: Through experiments, the efficacy of the probiotic complex on non-alcoholic fatty liver disease in C57 mice induced by a high-fat diet was preliminarily confirmed. The present invention has the following beneficial effects: the administration of the probiotic complex for colonization can significantly inhibit the growth of body weight, liver weight and epididymal fat weight of NAFLD mice induced by a high-fat diet. Serological indicators show that probiotic transplantation improves insulin tolerance and glucose tolerance in NAFLD mice induced by a high-fat diet, reduces serum liver enzyme activity, serum cholesterol and liver triglyceride content, and further liver hematoxylin-eosin and oil red O staining show that probiotics can inhibit liver fatty degeneration and lipid droplet deposition.

[0039] Probiotic supplementation can significantly improve the progression of NAFLD mice, which may be related to the restoration of intestinal flora disorders induced by high-fat diet in NAFLD mice through probiotic colonization, inhibition of lipid absorption in the intestine, correction of metabolic disorders of intestinal microorganisms, restoration of intestinal barrier damage, and inhibition of harmful substances from entering the liver through the intestinal barrier and portal vein, causing liver inflammatory response.

[0040] The results of mouse body weight showed that compared with the normal group, the weight of mice in the high-fat diet group was significantly increased ( P <0.01), and the body weight of mice in the probiotic group was significantly reduced compared with that in the high-fat diet group ( P <0.01) Figure 1 A in the figure); The results of mouse liver weight showed that the body weight of mice in the high-fat diet group was significantly increased compared with the normal group ( P <0.01), and the body weight of mice in the probiotic group was significantly reduced compared with that in the high-fat diet group ( P <0.05) ( Figure 1 B in the figure); The results of mouse fat weight showed that the body weight of mice in the high-fat diet group was significantly increased compared with the normal group ( P <0.01), and the body weight of mice in the probiotic group was significantly reduced compared with that in the high-fat diet group ( P<0.05) ( Figure 1 C in.

[0041] Table 1 Mouse body weight, liver weight and fat weight:

[0042] Note: The control group is a normal diet group, the model group is a high-fat diet group, and the model + probiotics group is a high-fat diet + probiotics colonization group; N=6, * is P <0.05,** P <0.01, compared with the model group.

[0043] The results of mouse biochemical indicators showed that compared with the normal group, the serum ALT of mice in the high-fat diet group was significantly increased ( P <0.01), compared with the high-fat diet group, the serum ALT of mice in the probiotic group was significantly reduced ( P <0.01) ( Figure 2 A in the figure); compared with the normal group, the serum AST of mice in the high-fat diet group was significantly increased ( P <0.01), compared with the high-fat diet group, the serum AST of mice in the probiotic group was significantly reduced ( P <0.05) ( Figure 2 B); compared with the normal group, the serum TC of mice in the high-fat diet group was significantly increased ( P <0.01), compared with the high-fat diet group, the serum TC of mice in the probiotic group was significantly reduced ( P <0.01) ( Figure 2 C in the figure); Compared with the normal group, the liver TG of mice in the high-fat diet group was significantly increased ( P <0.01), compared with the high-fat diet group, the liver TG of the mice in the probiotic group was significantly reduced ( P <0.05) ( Figure 2 D in.

[0044] Table 2 Serum ALT, AST, TC and TG levels:

[0045] Note: The control group is a normal diet group, the model group is a high-fat diet group, and the model + probiotics group is a high-fat diet + probiotics colonization group; N=6, * is P <0.05,** P <0.01, compared with the model group.

[0046] The results of mouse insulin tolerance and glucose tolerance showed that compared with the normal group, the area under the insulin tolerance curve of mice in the high-fat diet group was significantly increased ( P <0.01), and the area under the curve of mice in the probiotic group was significantly lower than that in the high-fat diet group (P <0.05), suggesting that probiotics improved the insulin tolerance impairment induced by high-fat diet ( Figure 3 A in the figure); compared with the normal group, the area under the glucose tolerance curve of mice in the high-fat diet group was significantly increased ( P <0.01), compared with the high-fat diet group, the area under the glucose tolerance curve of the probiotic group was significantly reduced ( P <0.05), suggesting that probiotics improved the impairment of glucose tolerance caused by a high-fat diet ( Figure 3 B in the figure).

[0047] Table 3 Insulin tolerance and glucose tolerance test in mice:

[0048] Note: The control group is a normal diet group, the model group is a high-fat diet group, and the model + probiotics group is a high-fat diet + probiotics colonization group; N=6, * is P <0.05,** P <0.01, compared with the model group.

[0049] In order to further observe the effect of probiotics on fatty degeneration of liver tissue in NAFLD mice induced by high-fat diet, hematoxylin-eosin staining was performed on the liver tissues of mice in each group. H&E staining (200 times) of paraffin sections of liver tissues in each group showed that: the liver tissue structure of mice in the normal group was compact, arranged normally, with red-stained cytoplasm, centrally centered nuclei, normal morphology of hepatocytes, no widening of hepatic cords, and no inflammatory cell infiltration in the central veins and portal areas; the liver tissue structure of mice in the high-fat diet group was loose, with obvious swelling of hepatocytes, light-stained cytoplasm, centrally centered or to one side of the nucleus, containing a large number of lipid droplets, diffuse fatty degeneration of hepatocytes, and scattered inflammatory cell infiltration in the central veins and portal areas; the liver cell structure of the probiotic group was compact, with red-stained cytoplasm, no obvious widening of the liver plate, and the hepatocytes were arranged radially around the liver plate, with occasional scattered lipid droplets ( Figure 4 A in the figure).

[0050] Oil red staining of mouse liver cells showed that only a small amount of oil droplets were evenly scattered in the cytoplasm in the normal group. Compared with the normal diet group, the liver cells of mice in the high-fat diet group had loose structure and obvious swelling, diffuse fatty degeneration of the liver parenchyma, and a large number of bright red lipid droplets were deposited in the liver cells. Compared with the high-fat diet group, the lipid droplets in the liver cells of the probiotic group were smaller and reduced in number, and the lipid droplets were scattered in the cytoplasm ( Figure 4 B in the figure).

[0051] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A yeast-probiotic complex for treating non-alcoholic fatty liver disease, characterized in that: The invention comprises Blautia ovata, probiotic Blautia ovata, Rosenblum, obligate Gram-positive anaerobic bacteria and Saccharomyces cerevisiae, wherein the volume ratio of Blautia ovata, probiotic Blautia ovata, Rosenblum, obligate Gram-positive anaerobic bacteria and Saccharomyces cerevisiae is 0.1-10:0.1-10:0.1-7:0.1-7:0.2-5, the Blautia ovata is preserved by the German Collection of Microorganisms and Cell Cultures and has a preservation number of DSM 25238, the probiotic Blautia ovata is preserved by the German Collection of Microorganisms and Cell Cultures and has a preservation number of DSM 19850, the Roseberys species is deposited by the German Collection of Microorganisms and Cell Cultures with the accession number DSM14610, the obligate Gram-positive anaerobic bacteria is deposited by the German Collection of Microorganisms and Cell Cultures with the accession number DSM16839, and the Saccharomyces cerevisiae is deposited by the German Collection of Microorganisms and Cell Cultures with the accession number DSM1333.

2. The yeast-probiotic complex for treating non-alcoholic fatty liver disease according to claim 1, characterized in that: The volume ratio of the Blautia ovata, the probiotic Blautia ovata, the Roseburia, the obligate Gram-positive anaerobic bacteria and the Saccharomyces cerevisiae is 1:1:1:1:0.

5.

3. The yeast-probiotic complex for treating non-alcoholic fatty liver disease according to claim 1 or 2, characterized in that: The suspensions of Blautia ovata, probiotic Blautia ovata, Rosenblumeria, obligate Gram-positive anaerobic bacteria and Saccharomyces cerevisiae were prepared at a density of 1×10^8 cfu / ml respectively, and then mixed according to a volume ratio.

4. A method for preparing the yeast-probiotic complex for treating non-alcoholic fatty liver disease as claimed in claim 1, characterized in that: The following steps are involved: Metagenomic testing was performed on the feces of patients with non-alcoholic steatohepatitis (NASH) and the control group (NO-NASH), and probiotics significantly enriched in NO-NASH were screened, obtaining significantly enriched Blautia ovata, probiotic Blautia, Rosenblumella, and obligate Gram-positive anaerobic bacteria; The Blautia ovata was placed in a modified YCFA medium, the probiotic Blautia was placed in a modified YCFA medium, the Roseberry was placed in a modified PYG medium, the obligate Gram-positive anaerobic bacteria was placed in a modified PYG medium, and the Saccharomyces cerevisiae was placed in a YM medium, and then all the five bacteria were cultured in an anaerobic workstation, and the bacterial concentration was detected by the OD600 method during the logarithmic growth period, and diluted to a concentration of 1×10^8 cfu / ml; Blautia ovata, probiotic Blautia ovata, Rosenblum, obligate Gram-positive anaerobic bacteria and Saccharomyces cerevisiae are mixed in a volume ratio of 0.1-10:0.1-10:0.1-7:0.1-7:0.2-5, and the obtained suspension is stored in a -80°C refrigerator for later use.

5. The method for preparing the yeast-probiotic complex for treating non-alcoholic fatty liver disease according to claim 4, characterized in that: The modified YCFA medium is 10.0 g of tryptic peptone, 10.0 g of beef extract powder, 3.0 g of yeast extract powder, 5.0 g of glucose, 5.0 g of sodium chloride, 1.0 g of soluble starch, 0.5 g of L-cysteine ​​hydrochloride, 3.0 g of sodium acetate, and 0.001 g of resazurin. The modified PYG medium is 5.0 g of tryptic peptone, 5.0 g of peptone, and 3.0 g of yeast extract powder. 10.0g, beef extract powder 5.0g, glucose 5.0g, K2HPO4 2.0g, Tween 80 1.0ml, resazurin 1.0mg, the YM medium is alcohol mother extract powder 3.0g, malt extract powder 3.0g, peptone 5.0g, glucose 10.0g.

6. The method for preparing the yeast-probiotic complex for treating non-alcoholic fatty liver disease according to claim 4, characterized in that: In the step 3), Blautia ovata, probiotic Blautia ovata, Roseburia rosea, obligate Gram-positive anaerobic bacteria and Saccharomyces cerevisiae are mixed in a volume ratio of 1:1:1:1:0.5.

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