Blauria winteriae bacterial agent and application thereof in preparation of products for preventing gall-stone

Abstract

The invention relates to the technical field of microorganisms, in particular to a Blauria winteriae bacterial agent and application of the Blauria winteriae bacterial agent in preparation of products for preventing gall-stone. The preparation method of the microbial agent comprises the following steps: (1) dissolving a PYG culture medium in a solvent, sterilizing to obtain a sterilized culture medium, adding a vitamin K1 solution and a hemin solution, and uniformly mixing to obtain an improved PYG liquid culture medium; (2) inoculating the improved PYG liquid culture medium with Blauria winteriae bacteria, and carrying out anaerobic culture, so as to obtain a culture solution; and (3) centrifuging the culture solution, adding normal saline, and uniformly mixing to obtain the microbial inoculum. According to the application disclosed by the invention, the formation of the gall-stone is prevented and the blood fat level is regulated by utilizing the Blauria winteriae (B.W) for intervention, and a new thought and a new method are provided for a gall-stone prevention strategy.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a Blautia wexlerae bacterial agent and application thereof in preparing a product for preventing gallstones. Background Art

[0002] As a digestive system disease, gallstones are divided into asymptomatic and symptomatic gallstone diseases according to whether they are accompanied by clinical symptoms such as nausea, vomiting, and biliary colic. Among them, if gallstones with clinical symptoms seriously affect the patient's quality of life, laparoscopic cholecystectomy should be considered for treatment, and surgical treatment often brings a large economic burden to patients and society. Although asymptomatic gallstone patients often use non-surgical conservative treatment, they cannot avoid frequent medication caused by the progression of the disease. If timely intervention can be carried out on people at high risk of gallstones to prevent the formation of gallstones in their bodies or delay the progression of the disease in patients who have already formed stones, the burden of gallstone-related diseases and the economic burden will be greatly reduced.

[0003] The clinical practice guidelines for gallstones point out that the main methods for preventing gallstones are to improve lifestyle (such as quitting smoking and limiting alcohol, appropriate physical activity, reducing the intake of high-calorie foods, and supplementing vitamin C and taking ursodeoxycholic acid. However, the use of ursodeoxycholic acid to prevent gallstone formation is only recommended in high-risk groups (such as people who lose weight rapidly), and the effect of ursodeoxycholic acid in preventing gallstones in the general population is still unclear. Previous studies have shown that intestinal flora participates in the metabolism of bile acids and may affect the occurrence and development of gallstones. Among these flora, there are probiotics that can confer health benefits when consumed in sufficient amounts. In recent years, these probiotics have been widely added to foods such as yogurt, cheese and ice cream to promote gastrointestinal health in the general population. However, the application of probiotics in the prevention or treatment of gallstones has not been deeply studied at present, and the development of strains that have therapeutic effects on gallstones has great application value. Summary of the invention

[0004] In order to solve the defects in the prior art, the present invention provides a Blautiawexlerae bacterial agent and its application in the preparation of a gallstone prevention product. Based on case-control studies and animal experiments, the present invention uses Blautiawexlerae bacteria (BW bacteria) to intervene to prevent the formation of gallstones and regulate blood lipid levels, providing new ideas and methods for gallstone prevention strategies.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a bacterial agent comprising Blautia wexlerae, wherein the preparation method of the bacterial agent comprises:

[0007] (1) dissolving a PYG medium in a solvent and sterilizing the medium to obtain a sterilized medium, and then adding a vitamin K1 solution and a hemin chloride solution to mix well to obtain a modified PYG liquid medium;

[0008] (2) inoculating Blautia wexlerae into a modified PYG liquid medium for anaerobic culture to obtain a culture solution;

[0009] (3) After centrifuging the culture solution, physiological saline is added and mixed to obtain a bacterial agent.

[0010] Preferably, the mass volume ratio of the vitamin K1 solution to the sterilized culture medium is 0.1 mg / 100 mL.

[0011] Preferably, the volume ratio of the hemin solution to the sterilized culture medium is 1:20.

[0012] Preferably, the anaerobic culture condition is constant temperature culture at 37° C. for 2 to 3 days.

[0013] Preferably, the concentration of Blautiawexlerae in the bacterial agent is 5×10 8 CFU.

[0014] In a second aspect, the present invention provides a use of Blautiawexlerae bacteria or the bacterial agent in the preparation of a product for preventing gallstones.

[0015] Preferably, the product for preventing gallstones is a product for improving dyslipidemia associated with gallstone disease.

[0016] Preferably, the product for preventing gallstones is a product for lowering triglyceride levels and total cholesterol levels.

[0017] Preferably, the product for preventing gallstones is a product that reduces IL-6 levels and TNF-α levels.

[0018] Preferably, the concentration of Blautiawexlerae in the product for preventing gallstones is 5×10 8 CFU.

[0019] The beneficial effects of the present invention are:

[0020] The present invention found that Blautiawexlerae bacteria can improve the formation of gallstones, improve dyslipidemia during gallstone formation, inhibit the increase of IL-6 and TNF-α levels caused by lithotripsy to a certain extent, and play a positive role in inhibiting gallstone formation. The present invention provides a new idea for the prevention and treatment of gallstones with microorganisms as the entry point, and provides a theoretical and experimental basis for the use of probiotics to improve dyslipidemia in patients with gallstones in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a diagram showing the gallstone formation in each group of mice tested in Example 1;

[0022] Figure 2 This is a graph showing the difference in gallbladder mass among the groups of mice tested in Example 1;

[0023] Figure 3 This is a graph showing the differences in blood lipid indexes of each group of mice tested in Example 1;

[0024] Figure 4 This is a graph showing the differences in inflammatory factor levels in each group of mice tested in Example 1. DETAILED DESCRIPTION

[0025] In order to enable those skilled in the art to better understand the technical solution of the invention, the present invention is further described in detail below in conjunction with specific implementation methods.

[0026] Example 1

[0027] This example intends to establish an animal model of gallstone disease based on a lithogenic diet, and to observe the effect of BW bacteria in preventing gallstone formation in mice fed a lithogenic diet through intervention with a BW bacterial suspension, thereby providing a new probiotic intervention program for the prevention of gallstones.

[0028] 1. Preparation of Improved PYG Liquid Medium

[0029] PYG medium: purchased from Ningbo Mingzhou Biotechnology Co., Ltd., product number KDM144, specific ingredients are shown in Table 1.

[0030] Preparation of improved PYG liquid culture medium: Weigh 34.09 g of culture medium powder and dissolve it in 990 ml of distilled water. Sterilize it with high-pressure steam at 121°C for 15 minutes. After cooling to 50°C, add 1 bottle of KDM167 vitamin K1 solution (0.1 mg / bottle) and 1 bottle of KDM152 hemin chloride solution (0.1 mg / ml, 5 ml / bottle) per 100 ml of culture medium, mix well, and place in a 4°C refrigerator for use.

[0031] Table 1. PYG medium composition

[0032]

[0033]

[0034] 2. Bacterial solution preparation

[0035] The Blautia wexlerae strain (Blautia wexlerae DSM 19850) was purchased from Ningbo Testo Biological Company (No. TS528109).

[0036] The original bacterial solution containing the Blautia wexlerae strain and the improved PYG liquid medium was shaken and 3 ml was inoculated into 100 ml of the improved PYG liquid medium. The culture was cultured in an anaerobic culture tank and placed at 37°C for 2-3 days. After that, the culture medium was taken out and shaken and about 30 ml of the culture medium was taken into a centrifuge tube under sterile conditions. The culture medium was centrifuged at 4°C and 5000 rpm for 10 min. After the centrifugation, the culture medium was discarded, and about 30 ml of sterile physiological saline was added and mixed by pipetting. The concentration of the bacterial solution was roughly measured using a wheat test turbidity tube, so that the final concentration was 5×10 8 CFU / 0.3ml of BW bacterial suspension.

[0037] 3. Experimental Procedure

[0038] Thirty healthy 8-week-old C57BL / 6J male mice were adaptively fed with ordinary feed for 2 weeks and then randomly divided into 3 groups (normal diet group, high-fat diet group, BW bacteria intervention group), with 10 mice in each group; then, the normal diet group was fed with ordinary feed, and the high-fat diet group and BW bacteria intervention group were fed with lithogenic feed (45% fat energy supply + 1.25% cholesterol and 0.5% bile acid). The feeding and intervention of the BW bacteria intervention group were carried out simultaneously. The intervention was administered by intragastric administration, once a day, with an intragastric dose of 0.3 ml and a concentration of 5×10 8 CFU / 0.3ml; at the end of 12 weeks, the mice were fasted for 8 hours and then killed.

[0039] 4. Experimental Results

[0040] (1) Stone-forming effect

[0041] The gallbladders of mice were removed by cholecystectomy and the gallstones in each group of mice were observed. Figure 1 As shown, compared with the normal diet group, the high-fat diet group and the BW bacteria intervention group had a significant effect on gallbladder stone formation in mice.

[0042] (2) Comparison of gallbladder quality

[0043] The gallbladder weights of the three groups of mice were tested by KW rank sum test, and the Wilcoxon rank sum test was used for pairwise comparison between the groups and corrected by the Bonferroni method. Figure 2 As shown, although it did not reach a significant level, BW bacteria intervention can improve the formation of gallstones in the gallbladder of mice induced by a high-fat diet to a certain extent.

[0044] (3) Comparison of blood lipid levels

[0045] Blood was collected from the eyeball vein of mice, and the blood samples were centrifuged to detect the levels of triglycerides, total cholesterol, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol in serum. The levels of blood lipid indicators between groups were tested using the Wilcoxon rank sum test and corrected using the Bonferroni method. The results are shown in the figure. Figure 3 As shown, it can be seen that BW bacteria intervention can significantly inhibit the increase in triglyceride levels and total cholesterol levels caused by a lithogenic diet, that is, BW bacteria can improve dyslipidemia associated with gallstone disease.

[0046] (4) Comparison of inflammatory factors

[0047] Whole blood samples were placed at room temperature for 2 hours or at 4°C overnight and then centrifuged at 2-8°C at 1000 g for 15 minutes. The supernatant was collected and IL-6 and TNF-α were detected using ELISA kits. The levels of inflammatory factors between groups were tested using the Wilcoxon rank sum test and corrected using the Bonferroni method. The results are shown in the figure. Figure 4 As shown in the figure, although it did not reach a significant level, BW bacteria intervention can inhibit the increase in IL-6 and TNF-α levels caused by the lithogenic diet to a certain extent.

[0048] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A bacterial agent comprising Blautiawexlerae bacteria, characterized in that The preparation method of the bacterial agent comprises: (1) dissolving a PYG medium in a solvent and sterilizing the medium to obtain a sterilized medium, and then adding a vitamin K1 solution and a hemin chloride solution to mix well to obtain a modified PYG liquid medium; (2) inoculating Blautia wexlerae into the modified PYG liquid culture medium for anaerobic culture to obtain a culture solution; (3) After centrifuging the culture solution, physiological saline is added and mixed to obtain a bacterial agent.

2. The bacterial agent according to claim 1, characterized in that The mass volume ratio of the vitamin K1 solution to the sterilized culture medium is 0.1 mg / 100 mL.

3. The bacterial agent according to claim 1 or 2, characterized in that The volume ratio of the hemin solution to the sterilized culture medium is 1:

20.

4. The bacterial agent according to claim 1, characterized in that The anaerobic culture condition is constant temperature culture at 37° C. for 2 to 3 days.

5. The bacterial agent according to claim 1, characterized in that The concentration of Blautia wexlerae in the bacterial agent is 5×10 8 CFU.

6. Use of Blautia wexlerae or the bacterial agent according to any one of claims 1 to 5 in the preparation of a product for preventing gallstones.

7. The use according to claim 6, characterized in that: The product for preventing gallstones is a product for improving dyslipidemia associated with gallstone disease.

8. The use according to claim 6 or 7, characterized in that: The product for preventing gallstones is a product for lowering triglyceride levels and total cholesterol levels.

9. The use according to claim 6, characterized in that: The product for preventing gallstones is a product for reducing IL-6 level and TNF-α level.

10. The use according to claim 6, characterized in that: The concentration of Blautiawexlerae in the product for preventing gallstones is 5×10 8 CFU.

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