Weizmannia coagulans with fat-reducing and weight-reducing functions

Abstract

The application discloses a Weizmannia coagulans with fat-reducing and weight-reducing functions, and belongs to the technical field of microorganisms. The Weizmannia coagulans screened by the application can inhibit the content and activity of fat synthesis enzyme on the basis of reducing blood fat and weight, can relieve liver damage caused by high-fat diet, has excellent acid tolerance, bile salt tolerance and high fat hydrolysis capacity, and is a multifunctional Weizmannia coagulans, and has a wide application prospect in the preparation of fat-reducing, weight-reducing and weight-losing products.

Description

TECHNICAL FIELD

[0001] The present application relates to a strain of Weizmannia congelens with fat-reducing and weight-reducing functions, belonging to the technical field of microorganisms. BACKGROUND

[0002] With the change of modern lifestyle, high-fat diet and lack of exercise have become the main reason for the rise of obesity worldwide. Obesity is not only a problem of overweight, but also closely related to a variety of metabolic diseases, such as type 2 diabetes, cardiovascular disease, non-alcoholic fatty liver disease, etc. Fat metabolism disorder caused by high-fat diet is an important factor leading to obesity, and excessive fat accumulation in the body, especially the increase of visceral fat, further increases the risk of these metabolic diseases.

[0003] In recent years, studies have shown that gut microbiota plays a key role in regulating fat metabolism and weight management. Certain beneficial strains, such as Weizmannia congelens ( Weizmannia coagulans ), Lactobacillus plantarum ( Lactiplantibacillus plantarum ), etc. probiotics, are found to have the potential to regulate intestinal flora and improve hyperlipidemia.

[0004] Weizmannia congelens is a gram-positive, acid-tolerant and heat-tolerant probiotic that can survive and colonize in the intestine, produce lactic acid and other beneficial metabolites, thereby inhibiting the growth of pathogenic bacteria and improving the intestinal environment. Patent CN116083326A discloses the use of Weizmannia congelens and its postbiotic preparation, which has the effect of reducing weight and lowering lipid; patent CN111278302A discloses the use of inactivated Weizmannia congelens to enhance physical fitness. The above-mentioned technologies are limited in the functional research of Weizmannia congelens, ignoring the functions of the strain itself.

[0005] Therefore, applying Weizmannia congelens as a functional probiotic for anti-obesity and lipid metabolism regulation has broad application prospects. Research in this field not only provides a new approach for obesity treatment, but also opens up a new way for the development of new probiotic products. SUMMARY

[0006] The present application solves the problem of single function and less research of existing technology Weizmannia congelens in weight loss and lipid reduction. The screened Weizmannia congelens can inhibit the content and activity of fat synthesis enzyme, has excellent acid and bile salt tolerance, and has high fat hydrolysis capacity, in addition to reducing blood lipid and weight loss. It is a multifunctional Weizmannia congelens, which has a wide application prospect in the preparation of lipid-lowering, weight-reducing and weight-loss products.

[0007] The Weizmannia congelens S511 of the present application has the following functions:

[0008] (1) Acid and bile salt tolerance;

[0009] (2) Inhibit the activity of fat synthase;

[0010] (3) Highly efficient fat hydrolysis;

[0011] (4) To lower blood lipids, take Weizmannella coagulans S511 10 7 CFU / day can significantly reduce the serum TG content in the high-fat diet, compared with the model group, the reduction reached 73.46%;

[0012] (5) Taking Weizmannella coagulans S511 10 7 CFU / day can significantly reduce serum ALT and AST indicators under high-fat diet, compared with the model group, by 70% and 57.8%, respectively;

[0013] (6) Reduce weight and take Weizmannella coagulans S511 10 7 CFU / day can significantly reduce body weight under high-fat diet, which is 29.0% lower than that of the model group;

[0014] (7) To improve glucose tolerance, take Weizmannella coagulans S511 10 7 CFU / day can significantly reduce body weight under high-fat diet, reducing it by 28% compared with the model group.

[0015] The first object of the present invention is to provide a strain of Weizmannella coagulans ( Weizmannia coagulans ), Weizmannella coagulans S511 was deposited in the China Center for Type Culture Collection on September 24, 2024, with the deposit number CCTCCNO: M 20242055.

[0016] The second object of the present invention is to provide a microbial agent containing the above-mentioned Weizmannella coagulans.

[0017] In one embodiment, the number of spores of Weizmannella coagulans in the bacterial agent is not less than 1×10 7 CFU / g.

[0018] In one embodiment, chitosan oligosaccharide, whey protein powder, sucrose or skim milk powder is used as a protective agent in the bacterial agent.

[0019] In one embodiment, the microbial agent is a liquid agent, a solid agent, or an immobilized agent.

[0020] The third object of the present invention is to provide the use of the above-mentioned Weizmannella coagulans in the preparation of medicines for reducing body weight, lowering blood lipids or inhibiting lipase activity.

[0021] In one embodiment, the product contains one or more of the following substances: Alisma orientalis, rhubarb, honeysuckle, wolfberry, cassia seed, lotus leaf, kudzu root, poria, hawthorn, white hyacinth bean, tsaoko, chrysanthemum, atractylodes macrocephala, tangerine peel, curcumin, hovenia dulcis fruit, emblica fruit, vitamin B1, vitamin B6, vitamin C, chitosan oligosaccharide, orlistat.

[0022] In one embodiment, the dosage form of the drug is powder, granules, capsules, tablets, pills or oral liquid.

[0023] In one embodiment, the product is characterized in that it has at least one of the following functions:

[0024] (a) Lowering blood lipids;

[0025] (b) weight loss;

[0026] (c) inhibiting the activity of lipase;

[0027] (d) Improved glucose tolerance.

[0028] Beneficial effects of the present invention

[0029] The Weizmannella coagulans screened out by the present invention can inhibit the content and activity of lipase on the basis of lowering blood lipids and reducing weight, and also has excellent acid resistance, bile salt resistance and efficient fat hydrolysis ability, and is a multifunctional Weizmannella coagulans.

[0030] Specifically:

[0031] (1) Acid and bile salt resistance; the strain can survive after 12 hours of culture under 1.5% bile salt conditions, and the survival rate of the strain reaches 74% and OD600 reaches above 0.84 after 24 hours of culture under pH 2.0 conditions;

[0032] (2) To lower blood lipids, take Weizmannella coagulans S511 10 7 CFU / day can significantly reduce the serum TG content in the high-fat diet, compared with the model group, the reduction reached 73.46%;

[0033] (3) Taking Weizmannella coagulans S511 10 7 CFU / day can significantly reduce serum ALT and AST indicators under high-fat diet, compared with the model group, by 70% and 57.8%, respectively;

[0034] (4) Reduce weight and take Weizmannella coagulans S511 10 7 CFU / day can significantly reduce body weight under high-fat diet, which is 29.0% lower than that of the model group;

[0035] (5) To improve glucose tolerance, take Weizmannella coagulans S511 10 7 CFU / day can significantly reduce body weight under high-fat diet, which is 28% lower than that of the model group;

[0036] (6) Inhibit the activity of fatty acid synthase. The metabolites of Weizmannella S511 can effectively inhibit the activity of fatty acid synthase, with an inhibition rate of 76.9%;

[0037] (7) It can efficiently hydrolyze fat and coagulate Weizmannella S511 on olive oil-MRS solid medium, with the clear zone of a single strain reaching 1.5 cm.

[0038] The Weizmannella coagulans S511 of the present invention is a multifunctional strain and has great application prospects in the preparation of lipid-lowering and weight-reducing products.

[0039] Biomaterial Deposit

[0040] Weizmannella coagulans S511, taxonomically designated Weizmannia coagulans S511 was deposited in the China Center for Type Culture Collection on September 24, 2024, with the deposit number CCTCC NO: M 20242055.

[0041] Weizmannella coagulans VC77 and Weizmannella coagulans BC77 were disclosed in patent CN118165890A on April 28, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0043] Figure 1 This is the streak pattern of the Weizmannia coagulans S511 plate; DETAILED DESCRIPTION

[0044] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0045] Example 1: Strain screening

[0046] (1) Isolation and purification of strains: First, add 15 mL of sterile water to the soil sample, shake vigorously for 20 min, and then incubate in an 80°C water bath for 20 min; then use the concentration gradient dilution method to spread 100 μL of the dilution solution on an agar plate (the culture medium formula is: yeast extract 10.0 g / L; beef extract 10.0 g / L; peptone 10.0 g / L; sodium bicarbonate 1.5 g / L; potassium dihydrogen phosphate 1.5 g / L; magnesium sulfate heptahydrate 1.0 g / L; manganese sulfate 0.1 g / L; calcium chloride 1.0 g / L; deionized water 1 L), culture at 37°C for 48 h, select colonies, further isolate and purify them by streaking, and store them in glycerol tubes.

[0047] (2) Strain screening: single colony strains were selected and inoculated into MRS medium for activation for 24 hours to obtain seed solution. The strains in the seed solution were inoculated at a 2% v / v inoculation amount into MRS medium containing different concentrations (g / 100mL) of pig bile salts (0.6%, 0.8%, 1%, 1.5%, 2%) and cultured at 37°C for 12 hours. 0.1mL was then spread on MRS solid medium and cultured at 37°C for 24 hours to test the growth ability of bile salts.

[0048] A bile salt-tolerant strain was screened and was able to survive after being cultured in MRS medium containing 1.5% porcine bile salts for 12 hours. The strain was named S511 and used in subsequent experiments.

[0049] Example 2: Identification of strains and preparation of bacterial agents

[0050] (1) Strain identification

[0051] The strain obtained in Example 1 was streaked onto MRS agar medium and cultured at 37°C for 48 hours. Figure 1 As shown, the colony morphology is flat, protruding, opaque white dots with a smooth surface.

[0052] PCR amplification of the 16S rRNA gene was performed using universal primers 27F and 1525R. For 16S rRNA PCR amplification and sequencing analysis, the PCR reaction system consisted of 12.5 μL of PCR Master Mix, 1 μL of each primer, 1 μL of DNA template, and the remainder made up with sterile water. PCR reaction conditions were: 95°C for 3 minutes, followed by 30 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 90 seconds, and 72°C for 10 minutes. The PCR product was verified by agarose gel electrophoresis and sent to GeneWeizhi for sequencing. Sequencing results were compared with the NCBI nucleic acid database, confirming that the strain was Weizmannella coagulans S511.

[0053] The Weizmannia coagulans S511 was deposited in the China Center for Type Culture Collection on September 24, 2024, with the deposit number CCTCC NO: M 20242055.

[0054] (2) Preparation of microbial agents

[0055] Chitosan oligosaccharide, whey protein powder or skim milk powder, and sucrose were mixed in a ratio of 1:2:2 in distilled water at 60°C, stirred evenly, and homogenized (160-180 Bar). After sterilization at 95°C for 300s, the mixture was cooled to 37±1°C and inoculated with 1×10 Weizmannella S511. 6 CFU / mL, ferment for 24 hours, cool and freeze-dry to obtain the coagulant Weizmannella S511 inoculum, and store at room temperature (20-25°C) for later use.

[0056] After testing, the number of live bacteria and spores of Weizmannella coagulans S511 in the inoculum reached 10 9 CFU / g.

[0057] Example 3: Performance testing of strain S511

[0058] (1) Acid resistance

[0059] The activated Weizmannella coagulans S511 seed solution was inoculated into MRS medium with pH 2.0, 3.0, 4.0, 9.0, and 10.0 at a 2% v / v inoculum. The MRS medium without pH adjustment was used as the control group. The culture was carried out at 37°C and 220 rpm for 24 h. The OD 600 Detect strain concentration and compare with the control group OD 600 The ratio is the survival rate.

[0060] The results showed that Weizmannella coagulans BC-Y09 had a good survival rate under the conditions of pH 2.0-9.0. After culturing for 24 h, OD 600 The survival rates of the strains reached 74% when the pH was 2.0, and 91% when the pH was 9.0.

[0061] (2) Fat hydrolysis ability test

[0062] Take 3 mL of olive oil and 6 mL of 4% polyvinyl alcohol, mix them evenly, stir and emulsify, add 10 mL of sterilized melted solid MRS medium, mix well, and pour into a plate to obtain olive oil-MRS solid medium.

[0063] Take 100 μL of seed solution of Weizmannella coagulans S511, Weizmannella coagulans VC77 (as control strain), and Weizmannella coagulans BC77 (as control strain) and dilute to 10 -6, take 200 μL and spread it on olive oil-MRS solid culture medium, culture it at 37℃ for 24h, and detect the diameter of the transparent zone. The larger the transparent zone, the stronger the fat hydrolysis ability.

[0064] The results showed that the transparent zones of Weizmannella coagulans VC77 and Weizmannella coagulans BC77 were very small, with diameters of 0.2 cm and 0.3 cm respectively; the transparent zone of Weizmannella coagulans S511 was the largest, with a diameter of 1.5 cm, and had excellent fat hydrolysis ability.

[0065] (3) Metabolite fatty acid synthase inhibitory activity detection

[0066] Seed liquids of Weizmannella coagulans VC77, Weizmannella coagulans BC77, and Weizmannella coagulans S511 were inoculated into MRS medium, cultured at 37°C for 12 h, centrifuged, and filtered through a 0.22 μm filter to prepare VC77 metabolites, BC77 metabolites, and S511 metabolites (pH 4.1 after testing), respectively.

[0067] The inhibitory activity of metabolites (50 μL) against fatty acid synthase was determined using a fatty acid synthase (FAS) kit (purchased from Nanjing Jiancheng, product number H231-1-2). An equal amount of MRS medium was used as a blank control, and MRS medium adjusted to pH 4.1 was used as a negative control. Inhibition rate (%) = enzyme activity in the test sample group / enzyme activity in the blank control group. The results are shown in Table 1.

[0068] Table 1 Fatty acid synthase inhibition rate

[0069]

[0070] The results showed that the metabolites of Weizmannella coagulans S511 have the function of efficiently inhibiting fatty acid synthase, and this effect is significantly better than that of other Weizmannella coagulans.

[0071] Example 4: Detection of lipid-lowering effect of strains

[0072] (1) Animal experiment groups

[0073] Forty healthy SPF male C57BL / 6J mice were fed an adaptive diet for one week, with free access to food and water. They were then randomly divided into four groups of 10 mice each and treated as follows:

[0074] Blank group: The rats were given basal feed and free access to food and water from the 2nd to the 8th week. From the 9th to the 16th week, normal saline (0.2 mL / rat) was gavaged once a day and the body weight was measured every week.

[0075] Model group: The rats were fed a high-fat diet and 5% fructose in drinking water ad libitum from the 2nd to the 16th week. From the 9th to the 16th week, normal saline (0.2 mL / rat) was gavaged once a day. The body weight was measured every week.

[0076] The treatment group was given a high-fat diet and 5% fructose in drinking water from the 2nd to the 16th week, and was gavaged once a day with Weizmannella coagulans S511 solution (0.2 mL / animal, viable count 10 7 CFU / animal), and the body weight was measured weekly.

[0077] Positive control group: The mice were fed a high-fat diet and given 5% fructose in drinking water ad libitum from week 2 to week 16. Orlistat (90 mg / kg) was administered orally once a day from week 9 to week 16, and body weight was measured weekly.

[0078] On the day after the experiment, an oral glucose tolerance test was performed, and then the experimental animals were sacrificed and serum, liver tissue, and epididymal adipose tissue were collected.

[0079] Oral glucose tolerance test method: After the last oral gavage, the mice were fasted but not watered for 12 hours, and then gavaged with glucose solution (dose of 2 g / kg per mouse). The blood glucose levels of the mice were measured at five time points: 0 min, 30 min, 60 min, 90 min, and 120 min, and the area under the blood glucose curve (AUC) was calculated according to the following formula.

[0080] (2) Weight changes

[0081] The average initial weight of the mice was 14.6g, with no significant differences between groups. After modeling, the average weight of the blank group was 21.1g, the average weight of the model group was 32.7g, the average weight of the treatment group was 23.2g, and the average weight of the positive control group was 27.5g. The results showed that administration of Weizmannella coagulans S511 bacterial solution effectively alleviated abnormal weight gain in mice, demonstrating a weight loss effect, with an average weight reduction of 29.0% compared to the model group.

[0082] Table 2 Mouse body weight

[0083]

[0084] (3) Oral glucose tolerance test

[0085] The results of oral glucose tolerance test are shown in Table 3. The results showed that the AUC of the model group was significantly higher than that of the normal group, while the AUC of the drug-treated group was significantly lower than that of the model group, and the average AUC was reduced by 28% compared with that of the model group.

[0086] Table 3 AUC averages

[0087]

[0088] (4) Mouse serum indicators

[0089] The TG content in the serum of mice, the fat content in the epididymal adipose tissue, and the fatty acid synthase (FAS) content in the liver tissue were detected. The results are shown in Table 4.

[0090] The results showed that the serum TG, ALT, AST, fat content in epididymal fat and FAS content in the liver of the drug-treated group were significantly lower than those in the model group, that is, oral administration of coagulant Weizmannella has the effect of lowering blood lipids, reducing fat and inhibiting lipase activity.

[0091] Table 4 Average serum indexes

[0092]

[0093] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Weizmannella coagulans ( Weizmannia coagulans ) S511, characterized in that, The Weizmannella coagulans S511 was deposited in the China Center for Type Culture Collection on September 24, 2024, with the deposit number CCTCC NO: M20242055.

2. A microbial agent, characterized in that: Contains the Weizmannia coagulans according to claim 1.

3. The microbial agent according to claim 2, characterized in that The number of spores of Weizmannella coagulans in the bacterial agent is not less than 1×10 7 CFU / g.

4. The microbial agent according to claim 2, characterized in that In the bacterial agent, chitosan oligosaccharide, whey protein powder, sucrose or skimmed milk powder is used as a protective agent.

5. The microbial agent according to claim 2, characterized in that The microbial agent is a liquid agent, a solid agent or an immobilized agent.

6. Use of the Weizmannella coagulans according to claim 1 in the preparation of medicines for reducing body weight and lowering blood lipids.

7. The application according to claim 6, characterized in that The product contains one or more of the following substances: Alisma orientale, rhubarb, honeysuckle, wolfberry, cassia seed, lotus leaf, kudzu root, poria, hawthorn, white hyacinth bean, tsaoko, chrysanthemum, atractylodes macrocephala, tangerine peel, curcumin, hovenia dulcis fruit, emblica fruit, vitamin B1, vitamin B6, vitamin C, chitosan oligosaccharide, and orlistat.

8. The use according to claim 6, characterized in that The dosage form of the medicine is powder, granule, capsule, tablet, pill or oral solution.

9. The use according to any one of claims 6 to 8, characterized in that: The product has at least one of the following functions: (a) Lowering blood lipids; (b) weight loss; (c) inhibiting the activity of lipase; (d) Improved glucose tolerance.

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