Strain of Witzia ciliaris with fat-reducing and weight-reducing functions

Abstract

The invention discloses a strain of Witzia ciliaris with fat-reducing and weight-reducing functions, and belongs to the technical field of microorganisms. On the basis of reducing blood fat and weight, the screened Witzia ciliaris can inhibit the content and activity of fat synthase and relieve liver injury caused by high-fat diet, also has excellent acid resistance, bile salt resistance and efficient fat hydrolysis ability, is a strain of multifunctional Witzia ciliaris, and has good application prospects. The method has a wide application prospect in preparation of lipid-lowering, weight-losing and weight-losing products.

Description

Technical Field

[0001] The invention relates to a strain of Weizmannella coagulans with fat-reducing and weight-reducing functions, belonging to the technical field of microorganisms. Background Art

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

[0003] In recent years, studies have shown that intestinal microorganisms play a key role in regulating fat metabolism and weight management. Certain beneficial strains, such as probiotics such as Weizmannia coagulans and Lactiplantibacillus plantarum, have been found to have the potential to regulate intestinal flora and improve hyperlipidemia.

[0004] Weizmannella coagulans is a Gram-positive, acid-resistant, and heat-resistant probiotic that can survive and colonize in the intestines, producing lactic acid and other beneficial metabolites, thereby inhibiting the growth of pathogenic bacteria and improving the intestinal environment. Patent CN116083326A discloses a method of using Weizmannella coagulans and its postbiotic preparations, which have the effect of weight loss and lipid reduction; Patent CN111278302A discloses the use of inactivated Weizmannella coagulans to enhance physical fitness. The above-mentioned technology is relatively limited to the functional research of Weizmannella coagulans, ignoring the function of the strain itself.

[0005] Therefore, the application of Weizmannella coagulans as a functional probiotic in anti-obesity and lipid metabolism regulation has broad application prospects. Research in this field not only provides new ideas for obesity treatment, but also opens up new ways for the development of new probiotic products. Summary of the invention

[0006] The prior art has the problem that the Weizmannella coagulans has a single function in weight loss and lipid reduction and has been less studied. The Weizmannella coagulans screened out by the present invention can inhibit the content and activity of lipase and alleviate liver damage caused by a high-fat diet on the basis of lowering blood lipids and reducing weight. It also has excellent acid and bile salt resistance and efficient fat hydrolysis ability. It is a multifunctional Weizmannella coagulans and has broad application prospects in the preparation of lipid-lowering, weight-reducing and slimming products.

[0007] The coagulant Weizmannella S511 of the present invention has the following functions:

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

[0009] (2) Inhibit the activity of lipase;

[0010] (3) Highly efficient fat hydrolysis;

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

[0012] (5) To relieve liver damage, take Weizmannella coagulans S511 10 7 CFU / day can significantly reduce the ALT and AST indexes in serum under high-fat diet, compared with the model group, the reductions were 70% and 57.8%, respectively;

[0013] (6) Reduce weight by taking 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, compared with the model group, by 28%.

[0015] The first object of the present invention is to provide a strain of Weizmannia coagulans. Weizmannia coagulans S511 has been deposited in the China Center for Type Culture Collection on September 24, 2024, with a deposit number of 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, in the bacterial agent, chitosan oligosaccharide, whey protein powder, sucrose or skimmed milk powder is used as a protective 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 products for reducing body weight, lowering blood lipids, protecting the liver or inhibiting fat synthesis.

[0021] In one embodiment, the product is a food, a health product or a medicine.

[0022] In one embodiment, the product contains one or more of the following substances: oriental rhizome, rhubarb, honeysuckle, wolfberry, cassia seed, lotus leaf, kudzu root, poria, hawthorn, white hyacinth bean, amomum villosum, chrysanthemum, atractylodes macrocephala, tangerine peel, curcumin, hovenia dulcis, emblica fruit, vitamin B1, vitamin B6, vitamin C, chitosan oligosaccharide, and orlistat.

[0023] In one embodiment, the dosage form of the drug is powder, granules, capsules, tablets, pills or oral liquid.

[0024] In one embodiment, the product has at least one of the following functions:

[0025] (a) Lowering blood lipids;

[0026] (b) Alleviate liver damage;

[0027] (c) reduce body weight;

[0028] (d) inhibiting the activity of lipase;

[0029] (e) Improve glucose tolerance.

[0030] Beneficial effects of the present invention

[0031] The Weizmannella coagulans screened out by the present invention can inhibit the content and activity of lipase and alleviate liver damage caused by a high-fat diet on the basis of lowering blood lipids and reducing weight. It also has excellent acid and bile salt resistance and efficient fat hydrolysis ability, and is a multifunctional Weizmannella coagulans.

[0032] Specific:

[0033] (1) Acid and bile salt resistance; the strain can survive when cultured under 1.5% bile salt for 12 hours, and the survival rate of the strain reaches 74% and the OD600 reaches above 0.84 when cultured under pH 2.0 for 24 hours;

[0034] (2) To lower blood lipids, take Weizmannella coagulans S511 10 7 CFU / day can significantly reduce the TG content in serum under high-fat diet, and the reduction reaches 73.46% compared with the model group;

[0035] (3) To relieve liver damage, take Weizmannella coagulans S511 10 7 CFU / day can significantly reduce the ALT and AST indexes in serum under high-fat diet, compared with the model group, the reductions were 70% and 57.8%, respectively;

[0036] (4) Reduce weight, 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;

[0037] (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;

[0038] (6) Inhibition of fatty acid synthase activity: The metabolites of Weizmannella S511 can effectively inhibit fatty acid synthase activity, with an inhibition rate of 76.9%;

[0039] (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.

[0040] 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.

[0041] Biomaterial Deposit

[0042] Weizmannia coagulans S511, taxonomically named 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.

[0043] Weizmannella coagulans VC77 and Weizmannella coagulans BC77 were disclosed in patent CN118165890A on April 28, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] 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.

[0045] Figure 1 This is the streak pattern of the coagulated Weizmannella S511 plate; DETAILED DESCRIPTION

[0046] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0047] Example 1: Strain screening

[0048] (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 uL of the dilution 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 by streaking, and store in glycerol tubes.

[0049] (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 / 100 mL) of porcine bile salts (0.6%, 0.8%, 1%, 1.5%, 2%) and cultured at 37°C for 12 hours. 0.1 mL was then spread on MRS solid medium and cultured at 37°C for 24 hours to monitor the growth capacity of the bile salts.

[0050] A bile salt-tolerant strain was screened and was able to survive in MRS medium containing 1.5% porcine bile salts for 12 hours. The strain was named S511 and used in subsequent experiments.

[0051] Example 2: Identification of strains and preparation of bacterial agents

[0052] (1) Strain identification

[0053] 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.

[0054] PCR amplification was performed using universal primers 27F and 1525R of the bacterial 16S rRNA gene; PCR amplification and sequencing analysis of 16S rRNA: PCR reaction system was 25mL: PCR Master Mix 12.5μL, 1μL of upper and lower primers, 1μL of DNA template, and the rest was filled with sterile water. PCR reaction conditions: 95℃3min; 95℃30s, 55℃30s, 72℃90s, 30 cycles; 72℃10min. The PCR product was sent to GENEWISE for sequencing after agarose gel electrophoresis. The sequencing results were compared and analyzed in the nucleic acid database on the NCBI website, confirming that the strain was Weizmannella coagulans, namely Weizmannella coagulans S511.

[0055] The coagulant Weizmannella S511 was deposited in the China Center for Type Culture Collection on September 24, 2024, with the deposit number CCTCC NO: M 20242055.

[0056] (2) Preparation of bacterial agents

[0057] 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), sterilized at 95°C for 300s, and then cooled to 37±1°C. Weizmannella S511 was coagulated and inoculated with 1×10 6 CFU / mL, ferment for 24 hours, cool and freeze-dry to obtain the coagulant Weizmannella S511 inoculum, and place it at room temperature (20-25°C) for later use.

[0058] After testing, the number of live bacteria and spores of Weizmannella S511 in the bacterial agent reached 10 9 CFU / g.

[0059] Example 3: Performance testing of strain S511

[0060] (1) Acid resistance

[0061] 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 inoculation rate. 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.

[0062] The results showed that Weizmannella coagulans BC-Y09 had a good survival rate under the condition of pH 2.0-9.0. After culturing for 24 hours, OD 600 The survival rates of the strains reached 74% when the pH was 2.0 and 91% when the pH was 9.0.

[0063] (2) Fat hydrolysis ability test

[0064] Take 3 mL of olive oil and 6 mL of 4% polyvinyl alcohol and mix them evenly, stir and emulsify them, add 10 mL of sterilized melted solid MRS culture medium, mix evenly, and pour them into plates to obtain olive oil-MRS solid culture medium.

[0065] Take 100 μL of the seed solution of Weizmannella coagulans S511, Weizmannella coagulans VC77 (as a control strain), and Weizmannella coagulans BC77 (as a 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 24 hours, and detect the diameter of the transparent circle. The larger the transparent circle, the stronger the fat hydrolysis ability.

[0066] 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.

[0067] (3) Metabolite fatty acid synthase inhibitory activity detection

[0068] The 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 the supernatant was filtered through a 0.22 μm filter membrane to prepare VC77 metabolites, BC77 metabolites and S511 metabolites (pH was 4.1 after testing), respectively.

[0069] The inhibitory activity of metabolites (50 μL) on fatty acid synthase was detected using a fatty acid synthase kit (purchased from Nanjing Jiancheng, product number H231-1-2), with an equal amount of MRS medium as a blank control and MRS medium adjusted to pH 4.1 as a negative control; inhibition rate (%) = enzyme activity of the sample group to be tested / enzyme activity of the blank control group. The results are shown in Table 1.

[0070] Table 1 Fatty acid synthase inhibition rate

[0071] VC77 metabolites BC77 metabolites S511 metabolites Blank control Negative control Inhibition rate (%) 8.4％ 13.1％ 76.9％ 5.2％ 6.7％

[0072] The results showed that the metabolites of Weizmannella coagulans S511 had the function of efficiently inhibiting fatty acid synthase, and this effect was significantly better than that of other Weizmannella coagulans.

[0073] Example 4: Detection of the lipid-lowering effect of strains

[0074] (1) Animal experiment groups

[0075] 40 SPF healthy male C57BL / 6J mice were fed with adaptive feeding for one week, and the mice were allowed to eat and drink freely. Then they were randomly divided into 4 groups, with 10 mice in each group, and the treatments were as follows:

[0076] Blank group: The mice were given basic feed from the 2nd to the 8th week and were allowed to eat and drink freely. From the 9th to the 16th week, the mice were gavaged with normal saline (0.2 mL / mouse) once a day and their body weight was measured every week.

[0077] Model group: The mice were given high-fat diet and 5% fructose in drinking water freely from the 2nd to the 16th week, and normal saline (0.2 mL / mouse) was gavaged once a day from the 9th to the 16th week. The body weight was measured every week.

[0078] The treatment group was given a high-fat diet and 5% fructose in drinking water from the 2nd to the 16th week, and a coagulant Weizmannella S511 solution (0.2 mL / animal, viable count 10 7 CFU / animal), and the body weight was measured weekly.

[0079] Positive control group: The mice were given high-fat diet and 5% fructose in drinking water freely from the 2nd to the 16th week, and orlistat (90 mg / kg) was gavaged once a day from the 9th to the 16th week. The body weight was measured every week.

[0080] An oral glucose tolerance test was performed on the day the experiment ended, and then the experimental animals were killed and serum, liver tissue, and epididymal adipose tissue were collected.

[0081] Experimental method for oral glucose tolerance test: fasting but not water deprivation for 12h after the last oral gavage, and oral gavage of glucose solution (dose of 2g / kg per mouse), and blood glucose levels of mice were measured at five time points: 0min, 30min, 60min, 90min, and 120min, respectively. The area under the blood glucose curve (AUC) was calculated according to the following formula.

[0082] (2) Weight changes

[0083] The initial average weight of mice was 14.6g, and there was no significant difference between each group of mice; after the modeling, the average weight of mice in the blank group was 21.1g, the average weight of mice in the model group was 32.7g, the weight of mice in the drug group was 23.2g, and the weight of mice in the positive control group was 27.5g. The results showed that the administration of Weizmannella coagulans S511 bacterial liquid can effectively alleviate the abnormal weight gain of mice and has a weight loss effect, which is 29.0% lower than the average weight of mice in the model group.

[0084] Table 2 Mouse body weight

[0085] Blank group Model Group Drug group Positive control group Average weight (g) 21.1 32.7 23.2 27.5

[0086] (3) Oral glucose tolerance test

[0087] The results of the oral glucose tolerance test are shown in Table 3. The results show 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 28% lower than that of the model group.

[0088] Table 3 AUC average

[0089] Blank group Model Group Drug group Positive control group AUC (min·mmol / L) 1097 1671 1203 1384

[0090] (4) Mouse serum indicators

[0091] The TG in serum, the fat content in epididymal adipose tissue, and the fatty acid synthase (FAS) content in liver tissue of mice were detected. The results are shown in Table 4.

[0092] 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 of the model group, that is, oral administration of Weizmannella coagulans has the effects of lowering blood lipids, reducing fat, alleviating liver damage and reducing fat synthesis.

[0093] Table 4 Average serum indexes

[0094]

[0095] Although the present invention has been disclosed as above in the form of a preferred embodiment, 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 Weizmannia coagulans S511, characterized in that: The coagulant Weizmannella 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 Weizmannella coagulans according to claim 1.

3. The microbial agent according to claim 2, characterized in that: In the bacterial agent, the number of spores of Weizmannella coagulans 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 described in claim 1 in preparing products for reducing body weight, lowering blood lipids, protecting the liver or inhibiting fat synthesis.

7. The use according to claim 6, characterized in that: The product is a food, a health product or a medicine.

8. The use according to claim 6, characterized in that: 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, amomum villosum, chrysanthemum, atractylodes macrocephala, tangerine peel, curcumin, hovenia dulcis, emblica fruit, vitamin B1, vitamin B6, vitamin C, chitosan oligosaccharide, and orlistat.

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

10. The use according to any one of claims 6 to 9, characterized in that: The product has at least one of the following functions: (a) Lowering blood lipids; (b) Alleviate liver damage; (c) reduce body weight; (d) inhibiting the activity of lipase; (e) Improve glucose tolerance.

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