Cristinia parvata microbial inoculum 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 Cristinia microphylla microbial agent and application thereof 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 the Cristinia parvata and carrying out anaerobic culture to obtain a culture solution; and (3) centrifuging the culture solution, adding normal saline, and uniformly mixing to obtain the microbial inoculum. On the basis of case contrast research and animal experiments, the formation of the gall-stone is prevented and the blood fat level is regulated by using the interference of the Cristinia microphylla, and a new thought and a new method are provided for a gall-stone prevention strategy.

Description

Technical Field

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

[0002] Gallstones are a common digestive system disease. The treatment methods are mainly divided into conservative treatment with symptomatic medication and laparoscopic cholecystectomy. The choice of treatment is often based on the severity of the patient's clinical symptoms, whether there are other serious diseases, and the patient's own economic level. However, drug treatment and surgical treatment are often accompanied by high treatment costs and discomfort and pain caused by postoperative syndrome. Preventing the formation of gallstones and reducing the incidence of the disease can fundamentally reduce the economic and disease burden.

[0003] 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. These probiotics have been widely added to foods such as yogurt, cheese, and ice cream in recent years 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, 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 Christensenella microphylla agent and its application in the preparation of a gallstone prevention product. Based on case-control studies and animal experiments, the present invention uses Christensenella microphylla intervention 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 Christensenella microphylla, and a 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 Christensenella microphylla 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, in step (1), the mass volume ratio of the vitamin K1 solution to the sterilized culture medium is 0.1 mg / 100 mL.

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

[0012] Preferably, in step (2), a hemin solution and a vitamin K1 solution are added to the culture medium after inoculation.

[0013] More preferably, the mass ratio of the active ingredients of the hemin solution and the vitamin K1 solution is 5:1.

[0014] Preferably, the anaerobic culture condition of step (2) is constant temperature culture at 37° C. for 2 to 3 days.

[0015] Preferably, the concentration of Christensen bacteria in the bacterial agent is 5×10 8 CFU.

[0016] In a second aspect, the present invention provides a use of Christensenella parvum or the bacterial agent in the preparation of a product for preventing gallstones.

[0017] Preferably, the product for preventing gallstones is a product that reduces the formation of gallstones in the gallbladder.

[0018] Preferably, the product for preventing gallstones is a product for improving dyslipidemia associated with gallstone disease.

[0019] Preferably, the product for preventing gallstones is a product that inhibits triglyceride levels and / or reduces high-density lipoprotein cholesterol levels.

[0020] Preferably, the product for preventing gallstones is a product that reduces IL-6 levels and / or TNF-α levels.

[0021] Preferably, the concentration of Christensenella parvum in the product for preventing gallstones is 5×10 8 CFU.

[0022] The beneficial effects of the present invention are:

[0023] The present invention explores the abundance changes of Christensenella in the intestines of patients with gallstones based on a case-control study, and further establishes an animal model of gallstone disease. Through animal intervention experiments, the effect of Christensenella in preventing gallstone formation is explored, providing a new probiotic intervention solution for the prevention of gallstones. The present invention is the first to discover that Christensenella can improve dyslipidemia in the process of gallstone formation and reduce inflammatory factors such as IL-6 and / or TNF-α in inhibiting gallstone formation, providing a new idea for the prevention and treatment of gallstones with microorganisms as the entry point. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the difference in relative abundance of Christensenella microti between the gallstone group and the healthy control group;

[0025] Figure 2 This is a diagram showing the gallstone formation in each group of mice tested in Example 1;

[0026] Figure 3 This is a graph showing the differences in gallbladder mass among the groups of mice tested in Example 1;

[0027] Figure 4 This is a graph showing the differences in blood lipid indexes of each group of mice tested in Example 1;

[0028] Figure 5 This is a graph showing the differences in inflammatory factor levels in each group of mice tested in Example 1. DETAILED DESCRIPTION

[0029] 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.

[0030] Example 1

[0031] This example intends to conduct a case-control study based on a population, using 16S rRNA sequencing to analyze the abundance difference of Christensenella minor in the feces of 34 research subjects in the gallstone case group and 80 research subjects in the healthy control group, and further establish an animal model of gallstone disease using a lithogenic diet, and observe the effect of Christensenella minor in preventing the formation of gallstones in mice fed a lithogenic diet through intervention with a suspension of Christensenella minor, so as to provide a new probiotic intervention solution for the prevention of gallstones. The technical route is as follows:

[0032] Phase 1: Comparison of relative abundance of Christensenella microti in fecal samples between case group and control group

[0033] At least 2 g of fresh tail stool was collected from 34 subjects in the gallstone group and 80 subjects in the healthy control group. The fecal samples were sequenced for intestinal flora 16S rRNA. The sequences were clustered into OTUs based on 97% similarity using UPARSE software. The OTU species taxonomy was annotated using RDP classifier compared with the Silva 16S rRNA gene database (v138) with a confidence threshold of 70%. The community composition of each sample was statistically analyzed at the species level.

[0034] Wilcoxon rank sum test was used to compare the relative abundance of Christensenella minor in the gallstone group and the healthy control group, with a two-sided test level of α = 0.05. Figure 1The results showed that P < 0.05, and the relative abundance of Christensenella between the two groups was significantly different.

[0035] Phase II: Establishment of mouse gallstone disease model and intervention with Christensenella microti suspension

[0036] 1. Preparation of Improved PYG Liquid Medium

[0037] PYG medium: purchased from Ningbo Mingzhou Biotechnology Co., Ltd., product number KDM144, specific ingredients are shown in Table 1.

[0038] 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.

[0039] Table 1. PYG medium composition

[0040] Element Content (g / L) Trypticase 5.0 Peptone 5.0 Yeast extract powder 10.0 Beef dip powder 5.0 glucose 5.0 Twain 80 1.0 Resazurin 0.001 Sodium chloride 0.08 Cysteine ​​hydrochloride 0.5 Calcium chloride 0.01 Magnesium sulfate 0.02 Dipotassium hydrogen phosphate 2.04 Potassium dihydrogen phosphate 0.04 Sodium bicarbonate 0.4

[0041] 2. Bacterial solution preparation

[0042] Christensenela minuta (JCM16072) was purchased from Ningbo Testo Biological Company (No. TS269500).

[0043] After the original bacterial solution containing the Christensen strain and the modified PYG liquid medium is shaken, 3 ml of the original solution is inoculated into 100 ml of the modified PYG liquid medium. If the Christensen strain grows slowly, more inoculation can be appropriately performed, and its culture medium needs to be added with 1 bottle of hemin chloride solution (0.1 mg / ml, 5 ml / bottle) and 1 bottle of vitamin K1 solution (0.1 mg / bottle). The culture is cultured in a round-bottom vertical anaerobic culture bag and placed at 37°C for 2 to 3 days. After that, the culture medium is taken out, shaken and taken into a centrifuge tube under sterile conditions. Centrifuge at 4°C and 5000 rpm for 10 minutes. After the centrifugation, the culture medium is discarded, about 30 ml of physiological saline is added and mixed with a pipette, and the concentration of the bacterial solution is roughly measured using a wheat test turbidity tube, so that the final concentration is 5×10 8 CFU of Christensenella microti bacterial suspension.

[0044] 3. Experimental Procedure

[0045] 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, and Christensenella minor 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 Christensenella minor intervention group were fed with lithogenic feed (45% fat energy supply + 1.25% cholesterol and 0.5% bile acid). Among them, the Christensenella minor intervention group was fed and intervened 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; at the end of 12 weeks, the mice were fasted for 8 hours and then killed.

[0046] 4. Experimental Results

[0047] (1) Stone-forming effect

[0048] The gallbladders of mice were removed by cholecystectomy and the gallstones in each group of mice were observed. Figure 2 As shown, compared with the normal diet group, the high-fat diet group and the Christensenella microphylla intervention group had a significant effect on gallbladder stone formation in mice.

[0049] (2) Comparison of gallbladder quality

[0050] 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 3 As shown, it can be seen that Christensenella parvum intervention significantly improved the formation of gallstones in the gallbladder of mice induced by high-fat diet (P<0.05).

[0051] (3) Comparison of blood lipid levels

[0052] 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 4 As shown, it can be seen that Christensenella miniata intervention can significantly inhibit the increase in triglyceride levels and the decrease in high-density lipoprotein cholesterol levels caused by a lithogenic diet, that is, Christensenella miniata can improve the dyslipidemia associated with gallstone disease.

[0053] (4) Comparison of inflammatory factors

[0054] 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 5 As shown, it can be seen that Christensenella microti intervention can significantly inhibit the increase in IL-6 levels caused by a lithogenic diet and control TNF-α levels to a certain extent.

[0055] 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 containing Christensenella microphylla, 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 Christensenella microphylla into a modified PYG liquid 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 in step (1) is 0.1 mg / 100 mL.

3. The bacterial agent according to claim 1 or 2, characterized in that In the step (1), 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 In the step (2), the hemin solution and the vitamin K1 solution are added to the culture medium after inoculation.

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

6. Use of Christensenella microti 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 suppressing triglyceride levels and / or lowering high-density lipoprotein 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 / or TNF-α level.

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

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