Cleristinia marcescens SJ-1 and application of Cleristinia marcescens SJ-1

Abstract

The invention belongs to the technical field of biological medicines, and provides a strain of Christsenella masliensis SJ-1, which is preserved in the China General Microbiological Culture Collection Center on January 29, 2024, the China General Microbiological Culture Collection Center is called as CGMCC for short, the preservation number is CGMCC No.29796, and the address of the preservation unit is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The strain disclosed by the invention has the effects of reducing blood sugar, reducing blood fat or losing weight and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine. Background Art

[0002] In recent years, with the changes in people's lifestyles and the popularity of high-fat and high-sugar Western diets, the prevalence of common metabolic chronic diseases such as obesity, hyperglycemia, hyperlipidemia, and non-alcoholic fatty liver disease has increased rapidly, threatening human health.

[0003] Numerous studies have shown that obesity, abnormal changes in blood sugar and blood lipids are often accompanied by intestinal flora imbalance, which in turn exacerbates the progression of the disease and even affects the therapeutic efficacy of hypoglycemic and lipid-lowering drugs and the patient's prognosis [1-5]. Therefore, some researchers believe that targeting the intestinal flora and its metabolites is a new trend in the treatment of metabolic diseases [6]. Studies have also shown that intervention with probiotics and prebiotics can effectively alleviate the symptoms of chronic metabolic diseases [7].

[0004] Currently, oral medications are the primary means of lowering blood sugar and lipids in clinical practice. Commonly used hypoglycemic drugs include sulfonylureas and biguanides, while statins are the primary lipid-lowering medications. However, long-term use of these medications can cause varying degrees of side effects. For example, biguanides can cause digestive tract abnormalities or lactic acidosis [8,9]; long-term use of statins can lead to hyperglycemia, cognitive impairment, and liver and muscle damage [10,11]. Clinically, there is a lack of effective medications for the treatment of obesity, and severe obesity is generally treated through interventional methods such as surgery. In contrast, interventions and regulation of chronic metabolic diseases using microecological therapies, such as probiotics or inactivated probiotic components, are safer. Therefore, developing new probiotics that are effective for weight loss and lowering blood sugar and lipids has practical application value. Summary of the Invention

[0005] In order to effectively treat chronic metabolic diseases such as obesity, hyperglycemia and hyperlipidemia, the first purpose of the present invention is to provide a strain of Christensenella massiliensis SJ-1, which was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms on January 29, 2024. The center is abbreviated as CGMCC, with the deposit number: CGMCC No. 29796, and the deposit unit address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0006] A second object of the present invention is to provide a microbial agent, the active ingredients of which include but are not limited to live bacteria of Christensenella massiliensis SJ-1.

[0007] The third objective of the present invention is the use of a live bacterial agent of Christensenella Marseilles SJ-1. Administration of the agent has the effect of lowering total triglycerides, total cholesterol, low-density lipoprotein, fasting blood glucose and free blood glucose in the host's blood, reducing glucose tolerance, and exerting the therapeutic effects of weight loss and lowering the host's blood glucose and blood lipids through the above-mentioned regulatory effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 .SJ-1 colony morphology.

[0009] Figure 2 .Design flow chart of the SJ-1 live bacteria gavage animal experiment. Specific implementation method:

[0010] Example

[0011] Modified GAM liquid medium (g / L): 10 g casein peptone, 3 g soy peptone, 15 g tryptone, 13.5 g digested serum, 5 g yeast extract, 2 g beef meal, 1.2 g bovine liver extract, 3 g glucose, 0.3 g soluble starch, 0.5 g L-cysteine ​​hydrochloride, 0.5 g L-arginine, 0.3 g L-tryptophan, 2 g sodium bicarbonate, 2.5 g potassium dihydrogen phosphate, 3 g NaCl, 0.15 g sodium thioglycolate, 2.46 g sodium acetate, 0.01 g hemin, 0.001 g resazurin, 10% (v / v) clarified rumen fluid, distilled water to 1 L, pH 7.2 ± 0.1, sterilized by autoclaving at 115°C for 25 min before use. For solid medium, add an additional 15 g agar.

[0012] 1. Acquisition, isolation and identification of strains

[0013] The strain was obtained by the patent inventor on August 5, 2022, at Shandong University from fresh feces provided by the feces donor after signing an informed consent form.

[0014] Colony morphology: In modified GAM broth medium (each liter of modified GAM broth medium contains: casein peptone 10 g, soy peptone 3 g, tryptone 15 g, digested serum 13.5 g, yeast extract 5 g, beef powder 2 g, bovine liver extract 1.2 g, glucose 3 g, soluble starch 0.3 g, L-cysteine ​​hydrochloride 0.5 g, L-arginine 0.5 g, L-tryptophan 0.3 g, sodium bicarbonate 2 g, potassium dihydrogen phosphate 2.5 g, NaCl 3 g, sodium thioglycolate 0.15 g, sodium acetate 2.46 g, hemin 0.01 g, resazurin 0.001 g, clarified rumen fluid 10% (v / v), distilled water to 1 L, pH 7.2±0.1, sterilized at 115℃ for 25 minutes before use. 15g agar should be added to the solid medium. Cultured under anaerobic conditions at 37℃ for 2-3 days, the colony diameter is 1-2mm, white, smooth, and without water-soluble pigments ( Figure 1 ).

[0015] Molecular identification methods: Full 16S rRNA sequence analysis led to the identification and classification of the strain as Christensenella massiliensis, corresponding to the Chinese name for Christensenella massiliensis. The strain was named SJ-1. This strain was deposited with the General Microbiology Center of the China Culture Collection Administration of Microorganisms (CGMCC) on January 29, 2024, with the deposit number CGMCC No. 29796. The depository address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The patented strain deposit number is CGMCC No. 29796. The strain shares 99% homology with the annotated 16S rRNA sequence of Christensenella massiliensis, as shown in SEQ ID NO. 1 in the sequence listing.

[0016] 2. Cultivation and preservation methods:

[0017] Christensenella massiliensis SJ-1 was activated once in a modified GAM broth medium supplemented with 10% clarified rumen fluid, then inoculated into a modified GAM liquid medium with the same composition at a 0.1% to 10% inoculum and cultured anaerobically at 37°C for 3 to 7 days.

[0018] Long-term storage method for bacteria: Centrifuge the bacterial solution at 4°C, 10,000 rpm for 2 minutes to collect the precipitated cells, wash with PBS buffer solution, resuspend in a solution containing 15%-25% skim milk powder at a ratio of 5:1, freeze-dry and seal for storage, or freeze in liquid nitrogen in a preservation solution containing 15% glycerol and 85% serum and store at -80°C.

[0019] 3. Uses and effects of strains:

[0020] 3.1 Animal Experiments on Lowering Blood Sugar and Blood Lipids with SJ-1 Live Bacteria Agent

[0021] 3.1.1 Materials and Methods

[0022] 1) Modified GAM medium (g / L):

[0023] Casein peptone 10 g, soy peptone 3 g, tryptone 15 g, digested serum 13.5 g, yeast extract 5 g, beef meal 2 g, bovine liver extract 1.2 g, glucose 3 g, soluble starch 0.3 g, L-cysteine ​​hydrochloride 0.5 g, L-arginine 0.5 g, L-tryptophan 0.3 g, sodium bicarbonate 2 g, potassium dihydrogen phosphate 2.5 g, NaCl 3 g, sodium thioglycolate 0.15 g, sodium acetate 2.46 g, hemin 0.01 g, resazurin 0.001 g, clarified rumen fluid 10% (v / v), distilled water to 1 L, pH 7.2 ± 0.1, sterilized at 115°C for 25 minutes by autoclaving before use. For solid media, add an additional 15 g of agar.

[0024] 2) SJ-1 live bacteria culture method:

[0025] SJ-1 was activated on modified GAM solid medium and incubated anaerobically at 37°C for 2-3 days. A single colony was then selected and inoculated into modified GAM liquid medium for seed culture at 37°C for 2-3 days. Subsequently, the seed culture was inoculated into modified GAM liquid medium with the same composition at a 1-5% inoculum level and incubated anaerobically at 37°C for 2-3 days.

[0026] 3) Animal Experiment Design:

[0027] The experiment used C57BL / 6J mice (DIO mice) induced by high-fat diet, male, 15 weeks old. The frequency of gavage was once a day. The treatment group (5 mice) was gavaged 10 9 The control group (5 animals) was given an equal dose of PBS buffer solution daily, and their body weight and food intake were monitored every three days. The total experimental period was 5 weeks. Figure 2 shown.

[0028] Endpoint sampling: A. Plasma sample (anticoagulated blood was centrifuged and the supernatant was collected); B. Liver and other solid organs were fixed; the remaining liver was accurately weighed to 100 mg and quickly frozen in liquid nitrogen in three tubes for other indicator testing; C. Intestine and contents.

[0029] 4) Tissue sample testing indicators:

[0030] a) Blood glucose related indicators: venous free blood glucose, fasting blood glucose, and oral glucose tolerance test (OGTT);

[0031] b) Blood lipid-related indicators: plasma levels of low-density lipoprotein, total cholesterol, total triglycerides, and total bile acid;

[0032] c) Liver function related: aspartate aminotransferase (AST) level in the blood.

[0033] d) Stomach and ileum related indicators: ghrelin content in the stomach and ileum.

[0034] The above-mentioned blood sugar-related indicators are detected by blood glucose meter. Other physiological and biochemical indicators except blood glucose are detected by crude protein extraction from blood or homogenized liver tissue using commercial ELISA kits.

[0035] 3.1.2 Experimental results:

[0036] 1) SJ-1 live bacteria agent reduces the body weight and food intake of model mice:

[0037] The results in Table 1 show that the SJ-1 live bacteria agent can significantly control the weight gain and food intake of HFD mice, and has a significant effect of reducing the weight gain and food intake of HFD mice.

[0038] Table 1. Net weight gain of mice (g)

[0039]

[0040] Table 2. Average daily food intake of mice (g)

[0041]

[0042]

[0043] 2) SJ-1 live bacteria agent reduces free blood glucose in model mice:

[0044] The data results in Table 2 show that SJ-1 live bacteria agent has a significant effect of lowering free blood sugar and alleviating hyperglycemia symptoms.

[0045] Table 2. Free blood glucose concentration (mmol / L) in venous blood of model mice after 5 weeks of intervention

[0046]

[0047] 3) SJ-1 live bacteria agent reduces fasting blood glucose in model mice:

[0048] The data results in Table 3 show that the SJ-1 bacterial agent has a significant effect in lowering the fasting blood sugar of model mice and alleviating the symptoms of hyperglycemia.

[0049] Table 3. Fasting blood glucose concentration (mmol / L) of model mice after 5 weeks of intervention

[0050]

[0051] 4) SJ-1 live bacteria agent reduces glucose tolerance in model mice:

[0052] The results in Table 4 show that the SJ-1 bacterial agent can significantly increase the glucose tolerance of model mice and alleviate impaired glucose tolerance.

[0053] Table 4. Area under the curve (AUC) of glucose tolerance in model mice after 7 weeks of intervention

[0054]

[0055] 5) SJ-1 live bacteria agent reduces the total triglyceride content in the blood of model mice:

[0056] The results in Table 5 show that the SJ-1 bacterial agent can significantly reduce the total triglyceride content in the blood of model mice and alleviate the symptoms of hyperlipidemia.

[0057] Table 5. Endpoint blood total triglyceride concentration (mmol / L) of model mice after 5 weeks of intervention

[0058]

[0059] 6) SJ-1 live bacteria agent reduces total cholesterol levels in the blood of model mice:

[0060] The results in Table 6 show that the SJ-1 bacterial agent can significantly reduce the total cholesterol content in the blood of model mice and alleviate the symptoms of high cholesterol.

[0061] Table 6. Endpoint blood total cholesterol concentration (mmol / L) of model mice after 5 weeks of intervention

[0062]

[0063] 7) SJ-1 live bacteria agent reduces the low-density lipoprotein content in the blood of model mice:

[0064] The results in Table 7 show that the SJ-1 bacterial agent can significantly reduce the content of low-density lipoprotein in model mice and alleviate the symptoms of hyperlipidemia.

[0065] Table 7. Endpoint blood low-density lipoprotein (LDL) content (mmol / L) of model mice after 5 weeks of intervention

[0066]

[0067] 8) SJ-1 live bacteria agent increases the total bile acid content in the blood of model mice:

[0068] The results in Table 8 show that the SJ-1 bacterial agent can significantly increase the content of total bile acid in model mice and regulate bile acid changes.

[0069] Table 8. Endpoint blood total bile acid (TBA) content (μmol / L) of model mice after 5 weeks of intervention

[0070]

[0071] 9) SJ-1 live bacteria agent can significantly reduce the level of aspartate aminotransferase in the blood:

[0072] Table 9 shows that SJ-1 live bacteria intervention can effectively alleviate the AST content in the blood of high-fat diet model mice and prevent the occurrence of liver damage.

[0073] Table 9. Endpoint blood aspartate aminotransferase (AST) activity (U / L) of model mice after 5 weeks of intervention

[0074]

[0075] 10) SJ-1 live bacteria can regulate the level of ghrelin in the stomach and ileum of model mice:

[0076] Tables 10-11 show that SJ-1 live bacteria intervention can effectively reduce the content of ghrelin (Ghl) in the stomach and ileum of model mice and significantly reduce appetite.

[0077] Table 10. Ghrelin (Ghl) content in the stomach of model mice after 5 weeks of intervention (ng / mL)

[0078]

[0079] Table 11. Ghrelin (Ghl) content in the ileum of model mice after 5 weeks of intervention (ng / mL)

[0080]

Claims

1. A strain of Christensenella massiliensis SJ-1, whose deposit number is CGMCC No. 29796.

2. A microbial agent, the active ingredient of which is the Christensenella massiliense SJ-1 described in claim 1.

3. A composition comprising the Christensenella massiliense SJ-1 according to claim 1.

4. Use of the Christensenella marseille SJ-1 according to claim 1 in the preparation of medicines or foods for lowering blood sugar, lowering blood lipids or losing weight.

5. Use of the Christensenella marseille SJ-1 according to claim 1 in the preparation of medicines or foods for preventing liver damage.

6. Use of the Christensenella marseille SJ-1 according to claim 1 in the preparation of a medicine or food for increasing the total bile acid content.

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