A strain of Clostridium butyricum for alleviating obesity and its application

Abstract

The present invention discloses a strain of Clostridium butyricum for alleviating obesity and its application, belonging to the fields of microbial technology and medical technology. The Clostridium butyricum CCFM1299 disclosed in the present invention has been deposited in the Guangdong Provincial Microbial Culture Collection Center on January 8, 2023, with a deposit number of GDMCC No: 63125. After the Clostridium butyricum CCFM1299 provided by the present invention is used to act on obese mice, the obesity of mice can be significantly alleviated, which is specifically reflected in the slowing down of weight gain in obese mice, the inhibition of the accumulation of abdominal fat and epididymal fat in obese mice, the reduction of monocyte recruitment in epididymal fat of obese mice, the significant reduction of serum levels of LDL-C and TC, and the alleviation of liver fat accumulation and inflammation. Therefore, Clostridium butyricum CCFM1299 has great application prospects in the preparation of products for the prevention and / or treatment of obesity.

Description

Technical Field

[0001] The invention discloses a Clostridium butyricum strain for alleviating obesity and application thereof, and belongs to the technical field of microorganisms and medicine. Background Art

[0002] Obesity, as a non-communicable chronic disease, is associated with an increased risk of 21 diseases, including cardiovascular, gastrointestinal, respiratory and nervous system diseases, and seriously threatens people's health. According to the definition of the World Health Organization, a body mass index (BMI) greater than 25 is considered overweight, and a value of 30 or above is considered obese. With the improvement of quality of life and changes in people's lifestyles, the number of obese people is increasing day by day. From 1975 to the present, the prevalence of obesity has increased at least 7 times, from 0.7% to 5.6% for women, and from 0.9% to 7.8% for men.

[0003] Genetic and environmental factors are the main factors that lead to obesity. Among them, environmental factors mainly include bad eating habits and lifestyle habits. For example, long-term intake of high-calorie foods and decreasing exercise will promote fat accumulation and lead to obesity. However, genetic differences can also cause differences in individual susceptibility to obesity. The absence of leptin and melanocortin 4 receptor (MC4R) genes is associated with increased susceptibility to obesity. In addition to environmental and genetic factors, sleep quality, stress and intestinal flora have also been found to be related to the onset of obesity.

[0004] Hundreds of millions of microorganisms live in the human intestine, forming an organic micro-ecosystem. The number of intestinal microorganisms in an adult is about 10 14 The number of intestinal microorganisms in the intestine is about 10 times that of the human body, and the total weight accounts for 1-3% of the human body weight. Intestinal microorganisms not only affect the intestine itself, but also play a vital role in many physiological activities of the human body, such as metabolism, nutrition and immunity. In 2006, Gordon et al. found that the occurrence of obesity was related to changes in the relative abundance of Bacteroidetes and Firmicutes. The intestinal flora of obese individuals has not only changed in species diversity and composition, but also differed from the flora of normal-weight people in terms of function. The ability of the intestinal flora of obese individuals to obtain energy from food is greatly enhanced. In addition to affecting energy consumption, the microbiome also changes the composition of substances in the intestine, thereby affecting the occurrence and development of obesity. Among them, microbial metabolites such as short-chain fatty acids, indole derivatives and secondary bile acids have been shown to have anti-obesity benefits. Therefore, the importance of the flora in the occurrence and development of obesity cannot be ignored.

[0005] In recent years, the relationship between intestinal flora and obesity has been continuously revealed, and intestinal flora has become one of the intervention targets for obesity treatment. At present, the main methods for treating / preventing obesity include surgical intervention, dietary intervention, drug therapy, and prebiotic / probiotic intervention, all of which will have a certain impact on intestinal flora to a certain extent. Surgery and dietary intervention have certain adverse effects on intestinal flora. Both methods may reduce the abundance of flora, especially butyrate-producing bacteria, which may have adverse effects on the colon. As for drug intervention, most drugs will have certain side effects, such as Semaglutide, a GLP-1R agonist, which can cause symptoms such as nausea, vomiting, and diarrhea. Compared with the above interventions, probiotic therapy has fewer side effects and can positively regulate intestinal flora. From a mechanistic point of view, probiotic intervention can resist obesity by: (a) changing the microbiota to reduce energy absorption and intestinal permeability, (b) regulating satiety to reduce energy intake, and (c) inhibiting inflammation and promoting thermogenesis.

[0006] Therefore, developing probiotics that can fight obesity may be a safe and harmless way to improve obesity-related symptoms. Summary of the invention

[0007] The present invention provides a Clostridium butyricum CCFM1299, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on January 8, 2023, with a deposit number of GDMCC No: 63125, and a deposit address of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] The Clostridium butyricum CCFM1299 was isolated from human feces. The strain was sequenced and analyzed, and its 16S rDNA sequence was shown in SEQ ID NO.1. The sequence obtained by sequencing was compared with the nucleic acid sequence of Clostridium butyricum in Genbank, and the result showed that the similarity with the nucleic acid sequence of Clostridium butyricum was as high as 99.59%. The strain was named Clostridium butyricum CCFM1299.

[0009] The Clostridium butyricum CCFM1299 was inoculated into RCM solid culture medium, inverted and cultured at 37°C for 24 hours, and then its colonies were observed and its bacterial bodies were observed under a microscope. It was found that the bacteria were milky white, irregular, round-protruding, and smooth. The bacterial body shape was slightly irregular, with rounded ends, and was usually present alone, in pairs, and in small clusters.

[0010] The present invention also provides a microbial preparation, which contains the Clostridium butyricum CCFM1299 or its fermentation liquid, or contains the lysate of the Clostridium butyricum CCFM1299.

[0011] In one embodiment of the present invention, the number of Clostridium butyricum CCFM1299 in the microbial preparation is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0012] The present invention also provides a bacterial powder, wherein the bacterial powder contains the Clostridium butyricum CCFM1299 or its fermentation liquid.

[0013] The present invention also provides a product, wherein the product contains the above-mentioned Clostridium butyricum CCFM1299 or the above-mentioned microbial preparation.

[0014] In one embodiment of the present invention, the product comprises a food, a feed additive or a medicine.

[0015] In one embodiment of the present invention, the products include but are not limited to common foods, special foods, feed additives and medicines.

[0016] In one embodiment of the present invention, the special food includes food for specific (special) groups of people, such as infants, young children, diabetics, and malnourished people.

[0017] In one embodiment of the present invention, the drug can be used to prevent and alleviate obesity. In the product, the number of Clostridium butyricum CCFM1299 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g

[0018] In one embodiment of the present invention, the drug has at least one of the effects (a) to (c):

[0019] (a) reduce body weight and fat mass;

[0020] (b) Improve dyslipidemia and reduce serum TC and LDL-C levels;

[0021] (c) Improve fat accumulation in the liver.

[0022] In one embodiment of the present invention, the food is a dairy product, a bean product or a fruit and vegetable product produced using Clostridium butyricum CCFM1299 or a starter containing Clostridium butyricum CCFM1299.

[0023] In one embodiment of the present invention, the food is a solid beverage containing Clostridium butyricum CCFM1299.

[0024] In one embodiment of the present invention, the medicine contains Clostridium butyricum CCFM1299 and pharmaceutical excipients.

[0025] In one embodiment of the present invention, the pharmaceutical excipient comprises an excipient and an additive.

[0026] In one embodiment of the present invention, the pharmaceutical excipients include anti-adhesive agents, penetration enhancers, buffers, plasticizers, surfactants, defoamers, thickeners, inclusion agents, absorbents, humectants, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, pH regulators, adhesives, disintegrants, fillers, lubricants, wetting agents, integrators, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, foaming agents, suspending agents, coating materials, fragrances, diluents, flocculants and deflocculating agents, filter aids and release retardants.

[0027] In one embodiment of the present invention, the additive comprises microcrystalline cellulose, hydroxypropyl methylcellulose and refined lecithin.

[0028] In one embodiment of the present invention, the dosage form of the drug comprises granules, capsules, tablets, pills or oral liquids.

[0029] The present invention also provides the use of the above-mentioned Clostridium butyricum CCFM1299 or the above-mentioned microbial preparation in the preparation of products for alleviating obesity.

[0030] In one embodiment of the present invention, the product comprises a feed additive or a drug, wherein the number of Clostridium butyricum CCFM1299 in the product is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g

[0031] In one embodiment of the present invention, the drug can be used to prevent and alleviate obesity.

[0032] In one embodiment of the present invention, the drug has at least one of the effects (a) to (c):

[0033] (d) reduce body weight and fat mass;

[0034] (e) Improve dyslipidemia and reduce serum TC and LDL-C levels;

[0035] (f) Improve fat accumulation in the liver.

[0036] In one embodiment of the present invention, the medicine contains Clostridium butyricum CCFM1299 and pharmaceutical excipients.

[0037] In one embodiment of the present invention, the pharmaceutical excipient comprises an excipient and an additive.

[0038] In one embodiment of the present invention, the pharmaceutical excipients include anti-adhesive agents, penetration enhancers, buffers, plasticizers, surfactants, defoamers, thickeners, inclusion agents, absorbents, humectants, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, pH regulators, adhesives, disintegrants, fillers, lubricants, wetting agents, integrators, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, foaming agents, suspending agents, coating materials, fragrances, diluents, flocculants and deflocculating agents, filter aids and release retardants.

[0039] In one embodiment of the present invention, the additive comprises microcrystalline cellulose, hydroxypropyl methylcellulose and refined lecithin.

[0040] In one embodiment of the present invention, the dosage form of the drug comprises granules, capsules, tablets, pills or oral liquids.

[0041] The present invention also provides the use of the above-mentioned Clostridium butyricum CCFM1299 or the above-mentioned microbial preparation in the production of fermented food and feed additives.

[0042] Beneficial Effects

[0043] The present invention provides a Clostridium butyricum CCFM1299 that can alleviate obesity and related metabolic disorders. The Clostridium butyricum CCFM1299 provided by the present invention can significantly inhibit the occurrence and development of obesity in mice under a high-fat diet, as shown in the following:

[0044] (1) The body weight of obese mice decreased from 43.58±2.086g to 34.66±2.61g, and there was no significant difference in body weight compared with the blank group mice;

[0045] (2) The amount of epididymal fat in obese mice decreased from 2.61±0.37g to 1.49±0.30g;

[0046] (3) The amount of abdominal fat in obese mice decreased from 1.68±0.34g to 0.67±0.14g;

[0047] (4) The serum LDL-C of obese mice decreased from 0.38±0.06mmol / L to 0.20±0.08mmol / L, and

[0048] There was no significant difference in body weight between the mice in the blank group and those in the control group.

[0049] (5) TC in the serum of obese mice decreased from 1.94±0.10mmol / L to 1.54±0.45mmol / L;

[0050] (6) The ratio of LDL-C to HDL-C in the serum of obese mice decreased from 0.28 to 0.16, and there was no significant difference in the body weight of mice in the blank group;

[0051] (7) The level of adipose tissue inflammation in obese mice was significantly reduced, as evidenced by a decrease in the staining intensity of the monocyte surface molecule F4 / 80 from 148.3±30.33 to 32.24±17.01, and there was no significant difference in body weight compared with the blank group mice;

[0052] (8) Fat accumulation in the liver of obese mice was significantly reduced, and the score of non-alcoholic fatty liver disease was significantly decreased;

[0053] (9) The levels of TNF-α and IL-1β in the liver of obese mice decreased significantly, and there was no significant difference between them and the blank control group mice;

[0054] Therefore, Clostridium butyricum CCFM1299 has great application prospects in the preparation of products for preventing and / or treating obesity and related metabolic diseases.

[0055] Biomaterial Deposit

[0056] A strain of Clostridium butyricum CCFM1299, taxonomically named Clostridiumbutyricum, was deposited in the Guangdong Provincial Microbiological Culture Collection on January 8, 2023, with the collection number GDMCCNo: 63125. The collection address is the 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 : Flowchart of animal experiments.

[0058] Figure 2 : Body weight of experimental mice in different groups.

[0059] Figure 3 : Abdominal fat mass of experimental mice in different groups.

[0060] Figure 4 :The amount of epididymal fat in different groups of experimental mice.

[0061] Figure 5 :The results of F4 / 80 immunohistochemistry in epididymal adipose tissue of different groups of experimental mice.

[0062] Figure 6 :Quantitative results of F4 / 80 immunohistochemistry in epididymal adipose tissue of different groups of experimental mice.

[0063] Figure 7 : LDL-C levels in serum of different groups of experimental mice.

[0064] Figure 8 : LDL-C / HDL-C ratio in serum of different groups of experimental mice.

[0065] Fig. 9 : TC levels in serum of different groups of experimental mice.

[0066] Fig.10 : HE staining results of livers of mice in different groups.

[0067] Fig.11 : Disease scores of nonalcoholic fatty liver disease in mice of different groups.

[0068] Fig.12 : TNF-α levels in liver of mice in different groups.

[0069] Fig.13 :IL-1β levels in mice of different groups.

[0070] In the above pictures: *, **, ***, **** respectively indicate that the p values ​​are less than 0.05, 0.01, 0.001, 0.0001 compared with the model group; #, ##, ###, #### respectively indicate that the p values ​​are less than 0.05, 0.01, 0.001, 0.0001 compared with the blank group; ns indicates that there is no significant difference compared with the model group; NS indicates that there is no significant difference compared with the blank group. DETAILED DESCRIPTION

[0071] The C57BL / 6 male mice involved in the following examples were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and the 60% high-fat diet TP23300 and low-fat control diet TP23301 involved were purchased from Nantong Trophy Co., Ltd. TC, HDL-C and LDL-C kits were purchased from Beckman Coulter, USA. TNF-α and IL-1β Elisa detection kits were purchased from RD.

[0072] The culture medium involved in the following examples is as follows:

[0073] RCM liquid culture medium (L): peptone 10 g, beef extract 10 g, yeast powder 3 g, anhydrous glucose 5 g, soluble starch 1 g, sodium chloride 5 g, anhydrous sodium acetate 3 g, L-cysteine ​​hydrochloride 0.5 g, resazurin 4 mL (0.025%), pH 6.8±0.1.

[0074] RCM solid medium (L): peptone 10g, beef extract 10g, yeast powder 3g, anhydrous glucose 5g, soluble starch 1g, sodium chloride 5g, anhydrous sodium acetate 3g, L-cysteine ​​hydrochloride 0.5g, resazurin 4mL (0.025%), agar 15g, pH value is 6.8±0.1.

[0075] Example 1: Screening and identification of Clostridium butyricum CCFM1299

[0076] (1) Clostridium butyricum CCFM1299 screening:

[0077] A spoonful of feces from healthy people in Wuxi, Jiangsu Province was taken as a sample. It was added to 5 mL of PBS (with 0.05% cysteine) and mixed for gradient dilution. -5 ~10 -7The gradient dilutions were spread on the above RCM solid medium and cultured at 37°C for 48. Typical colonies were picked and placed in RCM solid medium for streak purification, placed in a 37°C constant temperature incubator and inverted for 48h, a single colony was picked and inoculated into 5mL liquid RCM medium, placed in a 37°C constant temperature incubator and cultured for 16-18h, 1.5mL of bacterial solution was centrifuged at 6000r / min for 3min, the supernatant was removed, and 1mL of 30% sterile glycerol was added for storage, and 1.5mL of bacterial body was centrifuged, the supernatant was removed, and resuspended in sterile water for strain identification.

[0078] The strain was sequenced and analyzed, and its 16S rDNA sequence was shown in SEQ ID NO.1. The sequence obtained was compared with the nucleic acid sequence of Clostridium butyricum in Genbank, and the result showed that the similarity with the nucleic acid sequence of Clostridium butyricum was as high as 99.59%. The strain was named Clostridium butyricum CCFM1299.

[0079] (2) Screening of Clostridium butyricum NXYCHL3M3:

[0080] A spoonful of feces from healthy people in Wuxi, Jiangsu Province was taken as a sample. It was added to 5 mL of PBS (with 0.05% cysteine) and mixed for gradient dilution. -5 ~10 -7 The gradient dilutions were spread on the above RCM solid medium and cultured at 37°C for 48h. Typical colonies were picked and placed in RCM solid medium for streaking purification, placed in a 37°C constant temperature incubator and inverted for 48h, single colonies were picked and inoculated into 5mL liquid RCM medium, placed in a 37°C constant temperature incubator and cultured for 16-18h, 1.5mL of bacterial solution was centrifuged at 6000r / min for 3min to remove the supernatant, and 1mL of 30% sterile glycerol was added for preservation. At the same time, a portion of 1.5mL of bacteria was centrifuged, the supernatant was removed, and then resuspended in sterile water for strain identification. The sequence obtained by sequencing was compared with the nucleic acid sequence in Genbank, and the results showed that the nucleic acid sequence similarity with Clostridium butyricum was as high as 99.42%, and it was named Clostridium butyricum NXYCHL3M3.

[0081] Example 2: Cultivation of Clostridium butyricum CCFM1299

[0082] The specific steps are as follows:

[0083] Clostridium butyricum CCFM1299 and Clostridium butyricum NXYCHL3M3 were respectively inoculated into RCM liquid culture medium and cultured at 37°C for 16 hours. The bacterial liquid was then transferred into fresh RCM liquid culture medium at an inoculum volume of 4% (v / v). After culturing under the same conditions for 12 hours, the bacteria were centrifuged at 8000 r / min for 15 minutes, washed with 0.9% saline (containing cysteine ​​hydrochloride), and centrifuged again at 8000 r / min for 10 minutes. The bacteria were collected, resuspended with 30% glycerol solution, and resuspensions were prepared respectively, which were frozen at -80°C for use.

[0084] Preparation of bacterial suspension for intragastric administration:

[0085] The prepared resuspension was taken out from -80°C and centrifuged at 4°C, 8000 r / min for 10 min. The supernatant was discarded and resuspended with 0.9% saline (containing cysteine ​​hydrochloride) to obtain a bacterial suspension for gavage. The strain concentration in the bacterial suspension was 5×10 8 CFU / mL.

[0086] Example 3: Effect of Clostridium butyricum CCFM1299 on the body weight of obese mice

[0087] The specific steps are as follows:

[0088] Four-week-old SPF C57BL / 6J male mice were divided into four groups, namely, a blank group, a model group, and an experimental group, wherein the experimental group included the CCFM1299 group gavaged with Clostridium butyricum CCFM1299 and the NXYCHL3M3 group gavaged with Clostridium butyricum NXYCHL3M3. Eight mice in each group were kept in the Experimental Animal Center of Jiangnan University, with a constant temperature of 21-26°C, humidity of 40-70%, noise less than or equal to 60dB, and animal illumination of 15-20LX (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

[0089] The experimental period is 13 weeks in total:

[0090] The first week was the adaptation period, during which mice in each cage had free access to water and were given normal growth and reproduction feed.

[0091] After the adaptation period, from the 2nd to the 13th week:

[0092] Model group: 60% high-fat diet + 0.2 mL normal saline was given by intragastric administration every day;

[0093] CCFM1299 group: 60% high-fat diet and intragastric administration of 0.2 mL of bacterial concentration of 5×10 8 CFU / mL of CCFM1299 bacterial suspension;

[0094] NXYCHL3M3 group: 60% high-fat diet and intragastric administration of 0.2 mL of bacterial concentration of 5×10 8 CFU / mL of NXYCHL3M3 bacterial suspension;

[0095] Blank group: The mice were given low-fat control feed and 0.2 mL of normal saline by intragastric administration every day.

[0096] The specific procedures of animal experiments are shown in Figure 1 shown.

[0097] The weight of mice was tested every week during the experiment, and the final weight changes were as follows: Figure 2 shown.

[0098] like Figure 2 As shown, the body weight of the model group reached 43.58±2.086g under a high-fat diet for 12 weeks, while the body weight of the blank group was only 30.10±1.466g. The CCFM1299 group, which also took a high-fat diet, weighed 34.66±2.61g, a 20% decrease compared to the model group, while after the intervention of Clostridium butyricum NXYCHL3M3, the body weight was 39.78±6.23g, only 8.7% lower than the model group. In addition, there was no significant difference in the body weight of mice after CCFM1299 intervention and the blank group (p=0.07). Therefore, Clostridium butyricum CCFM1299 has the effect of inhibiting weight gain under a high-fat diet.

[0099] Example 4: Effect of Clostridium butyricum CCFM1299 on abdominal fat mass in obese mice The specific steps are as follows:

[0100] Four-week-old SPF C57BL / 6J male mice were divided into four groups, namely, a blank group, a model group, and an experimental group, wherein the experimental group included the CCFM1299 group gavaged with Clostridium butyricum CCFM1299 and the NXYCHL3M3 group gavaged with Clostridium butyricum NXYCHL3M3. Eight mice in each group were kept in the Experimental Animal Center of Jiangnan University, with a constant temperature of 21-26°C, humidity of 40-70%, noise less than or equal to 60dB, and animal illumination of 15-20LX (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

[0101] The experimental period is 13 weeks in total:

[0102] The first week was the adaptation period, during which mice in each cage had free access to water and were given normal growth and reproduction feed.

[0103] After the adaptation period, from the 2nd to the 13th week:

[0104] Model group: 60% high-fat diet + 0.2 mL normal saline was given by intragastric administration every day;

[0105] CCFM1299 group: 60% high-fat diet and intragastric administration of 0.2 mL of bacterial concentration of 5×10 8 CFU / mL of CCFM1299 bacterial suspension;

[0106] NXYCHL3M3 group: 60% high-fat diet and intragastric administration of 0.2 mL of bacterial concentration of 5×10 8 CFU / mL of NXYCHL3M3 bacterial suspension;

[0107] Blank group: The mice were given low-fat control feed and 0.2 mL of normal saline by intragastric administration every day.

[0108] The specific procedures of animal experiments are shown in Figure 1 shown.

[0109] After the experiment, the mice were dissected in accordance with ethical requirements, and the abdominal fat was taken and weighed.

[0110] Abdominal fat weight Figure 3 As shown in the figure, compared with the blank group's abdominal fat weight of 0.25±0.08g, the abdominal fat weight of the model group increased significantly to 1.68±0.34g. Under CCFM1299 intervention, the amount of abdominal fat was significantly reduced to 0.67±0.14g; under NXYCHL3M3 intervention, it was reduced to 1.12±0.45g. Therefore, CCFM1299 can more effectively inhibit the accumulation of abdominal fat than NXYCHL3M3.

[0111] Example 5: Effect of Clostridium butyricum CCFM1299 on epididymal fat mass in obese mice The specific steps are as follows:

[0112] Four-week-old SPF C57BL / 6J male mice were divided into four groups, namely, a blank group, a model group, and an experimental group, wherein the experimental group included the CCFM1299 group gavaged with Clostridium butyricum CCFM1299 and the NXYCHL3M3 group gavaged with Clostridium butyricum NXYCHL3M3. Eight mice in each group were kept in the Experimental Animal Center of Jiangnan University, with a constant temperature of 21-26°C, humidity of 40-70%, noise less than or equal to 60dB, and animal illumination of 15-20LX (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

[0113] The experimental period is 13 weeks in total:

[0114] The first week was the adaptation period, during which mice in each cage had free access to water and were given normal growth and reproduction feed.

[0115] After the adaptation period, from the 2nd to the 13th week:

[0116] Model group: 60% high-fat diet + 0.2 mL normal saline was given by intragastric administration every day;

[0117] CCFM1299 group: 60% high-fat diet and intragastric administration of 0.2 mL of bacterial concentration of 5×10 8 CFU / mL of CCFM1299 bacterial suspension;

[0118] NXYCHL3M3 group: 60% high-fat diet and intragastric administration of 0.2 mL of bacterial concentration of 5×10 8 CFU / mL of NXYCHL3M3 bacterial suspension;

[0119] Blank group: The mice were given low-fat control feed and 0.2 mL of normal saline by intragastric administration every day.

[0120] The specific procedures of animal experiments are shown in Figure 1 shown.

[0121] After the experiment, the mice were dissected according to ethical requirements, and the epididymal fat was taken and weighed.

[0122] Epididymal fat weight Figure 4 As shown in the figure, compared with the blank group abdominal fat weight of 0.43±0.11g, the abdominal fat weight of the model group increased significantly to 2.61±0.37g. Under CCFM1299 intervention, the amount of epididymal fat was significantly reduced to 1.49±0.3g; under NXYCHL3M3 intervention, it was reduced to 2.15±0.76g. Therefore, CCFM1299 can more effectively inhibit the accumulation of epididymal fat than NXYCHL3M3.

[0123] Example 6: Effect of Clostridium butyricum CCFM1299 on the alleviation of epididymal fat inflammation in obese mice

[0124] The specific steps are as follows:

[0125] Four-week-old SPF C57BL / 6J male mice were divided into four groups, namely, a blank group, a model group, and an experimental group, wherein the experimental group included the CCFM1299 group gavaged with Clostridium butyricum CCFM1299 and the NXYCHL3M3 group gavaged with Clostridium butyricum NXYCHL3M3. Eight mice in each group were kept in the Experimental Animal Center of Jiangnan University, with a constant temperature of 21-26°C, humidity of 40-70%, noise less than or equal to 60dB, and animal illumination of 15-20LX (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

[0126] The experimental period is 13 weeks in total:

[0127] The first week was the adaptation period, during which mice in each cage had free access to water and were given normal growth and reproduction feed.

[0128] After the adaptation period, from the 2nd to the 13th week:

[0129] Model group: 60% high-fat diet + 0.2 mL normal saline was given by intragastric administration every day;

[0130] CCFM1299 group: 60% high-fat diet and intragastric administration of 0.2 mL of bacterial concentration of 5×10 8 CFU / mL of CCFM1299 bacterial suspension;

[0131] NXYCHL3M3 group: 60% high-fat diet and intragastric administration of 0.2 mL of bacterial concentration of 5×10 8 CFU / mL of NXYCHL3M3 bacterial suspension;

[0132] Blank group: The mice were given low-fat control feed and 0.2 mL of normal saline by intragastric administration every day.

[0133] The specific procedures of animal experiments are shown in Figure 1 shown.

[0134] After the experiment, the mice were dissected according to ethical requirements, and the epididymal fat was removed and fixed in 4% paraformaldehyde solution for 36 h. After dehydration, embedding, sectioning, dewaxing, antigen retrieval, serum blocking, incubation with primary and secondary antibodies, color development, counterstaining, dehydration, and sealing, the degree of inflammation of the tissue was evaluated. The results of immunohistochemistry are shown in Figure 5 ,Depend on Figure 5 It can be seen that there are obviously a large number of F4 / 80 positive staining areas around the epididymal adipocytes in the model group.

[0135] Quantification of positive staining area is shown in Figure 6 .Depend on Figure 6 The quantitative results showed that the F4 / 80 positive staining intensity of the blank group was 25.02, while that of the model group was 148.30. The F4 / 80 positive staining intensity was significantly reduced to 32.34 under CCFM1299 intervention; the positive staining intensity was reduced to 86.62 under NXYCHL3M3 intervention. The F4 / 80 staining intensity of epididymal fat under CCFM1299 intervention was significantly decreased compared with the model group, and there was no significant difference compared with the blank group. Therefore, CCFM1299 can more advantageously inhibit the recruitment of macrophages in epididymal fat and thereby relieve tissue inflammation.

[0136] Example 7: Effect of Clostridium butyricum CCFM1299 on serum low-density lipoprotein cholesterol (LDL-C) levels in obese mice

[0137] The specific steps are as follows:

[0138] Four-week-old SPF C57BL / 6J male mice were divided into four groups, namely, a blank group, a model group, and an experimental group, wherein the experimental group included the CCFM1299 group gavaged with Clostridium butyricum CCFM1299 and the NXYCHL3M3 group gavaged with Clostridium butyricum NXYCHL3M3. Eight mice in each group were kept in the Experimental Animal Center of Jiangnan University, with a constant temperature of 21-26°C, humidity of 40-70%, noise less than or equal to 60dB, and animal illumination of 15-20LX (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

[0139] The experimental period is 13 weeks in total:

[0140] The first week was the adaptation period, during which mice in each cage had free access to water and were given normal growth and reproduction feed.

[0141] After the adaptation period, from the 2nd to the 13th week:

[0142] Model group: 60% high-fat diet + 0.2 mL normal saline was given by intragastric administration every day;

[0143] CCFM1299 group: 60% high-fat diet and intragastric administration of 0.2 mL of bacterial concentration of 5×10 8 CFU / mL of CCFM1299 bacterial suspension;

[0144] NXYCHL3M3 group: 60% high-fat diet and intragastric administration of 0.2 mL of bacterial concentration of 5×10 8 CFU / mL of NXYCHL3M3 bacterial suspension;

[0145] Blank group: The mice were given low-fat control feed and 0.2 mL of normal saline by intragastric administration every day.

[0146] The specific procedures of animal experiments are shown in Figure 1 shown.

[0147] After the experiment, blood was collected from the mice's eyeballs, and the blood was left to stand at room temperature for more than an hour before centrifugation to separate the serum. The content of low-density lipoprotein cholesterol (LDL-C) in the serum was measured by a biochemical analyzer. The results are shown in Figure 7 .

[0148] Depend on Figure 7It can be seen that the LDL-C content in the serum of the blank group was 0.11±0.03mmol / L, while the high-fat diet caused the LDL-C content in the serum of the model group to increase to 0.38±0.06mmol / L, which was significantly higher than the blank group. After mice were gavaged with Clostridium butyricum CCFM1299, the LDL-C content in the serum (the content was 0.20±0.08mmol / L) was significantly lower than that in the model group, and there was no significant difference with the blank group. However, after mice were gavaged with Clostridium butyricum NXYCHL3M3, the LDL-C content in the serum also decreased (the content was 0.26±0.08mmol / L), but there was no significant difference with the model group. The above experimental results show that Clostridium butyricum CCFM1299 can better reduce the LDL-C level in the serum of obese mice.

[0149] Example 8: Effect of Clostridium butyricum CCFM1299 on the ratio of serum low-density lipoprotein cholesterol (LDL-C) to high-density lipoprotein cholesterol (HDL-C) in obese mice

[0150] The specific steps are as follows:

[0151] Four-week-old SPF C57BL / 6J male mice were divided into four groups, namely, a blank group, a model group, and an experimental group, wherein the experimental group included the CCFM1299 group gavaged with Clostridium butyricum CCFM1299 and the NXYCHL3M3 group gavaged with Clostridium butyricum NXYCHL3M3. Eight mice in each group were kept in the Experimental Animal Center of Jiangnan University, with a constant temperature of 21-26°C, humidity of 40-70%, noise less than or equal to 60dB, and animal illumination of 15-20LX (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

[0152] The experimental period is 13 weeks in total:

[0153] The first week was the adaptation period, during which mice in each cage had free access to water and were given normal growth and reproduction feed.

[0154] After the adaptation period, from the 2nd to the 13th week:

[0155] Model group: 60% high-fat diet + 0.2 mL normal saline was given by intragastric administration every day;

[0156] CCFM1299 group: 60% high-fat diet and intragastric administration of 0.2 mL of bacterial concentration of 5×10 8 CFU / mL of CCFM1299 bacterial suspension;

[0157] NXYCHL3M3 group: 60% high-fat diet and intragastric administration of 0.2 mL of bacterial concentration of 5×10 8 CFU / mL of NXYCHL3M3 bacterial suspension;

[0158] Blank group: The mice were given low-fat control feed and 0.2 mL of normal saline by intragastric administration every day.

[0159] The specific procedures of animal experiments are shown in Figure 1 shown.

[0160] After the experiment, blood was collected from the mice's eyes, and the blood was left to stand at room temperature for more than an hour before centrifugation to separate the serum. The levels of low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) in the serum were measured using a biochemical analyzer, and the ratio of LDL-C / HDL-C was calculated. This ratio reflects the body's fat transport status, and an increase in this ratio often indicates the occurrence of obesity and metabolic syndrome. The results are shown in Figure 8 .

[0161] Depend on Figure 8 It can be seen that the ratio of LDL-C / HDL-C in the serum of the blank group was 0.15, while the high-fat diet caused the ratio of LDL-C / HDL-C in the serum of the model group to increase to 0.28, which was significantly higher than that of the blank group. After mice were gavaged with Clostridium butyricum CCFM1299, the ratio of LDL-C / HDL-C in serum (the ratio was 0.16) was significantly lower than that of the model group, and there was no significant difference with the blank group. However, after mice were gavaged with Clostridium butyricum NXYCHL3M3, the LDL-C content in the serum also decreased (the ratio was 0.22), but there was no significant difference with the model group. The above experimental results show that Clostridium butyricum

[0162] CCFM1299 more balanced the levels of LDL-C and HDL-C in the serum of obese mice.

[0163] Example 9: Effect of Clostridium butyricum CCFM1299 on serum total cholesterol (TC) levels in obese mice

[0164] The specific steps are as follows:

[0165] Four-week-old SPF C57BL / 6J male mice were divided into four groups, namely, a blank group, a model group, and an experimental group, wherein the experimental group included the CCFM1299 group gavaged with Clostridium butyricum CCFM1299 and the NXYCHL3M3 group gavaged with Clostridium butyricum NXYCHL3M3. Eight mice in each group were kept in the Experimental Animal Center of Jiangnan University, with a constant temperature of 21-26°C, humidity of 40-70%, noise less than or equal to 60dB, and animal illumination of 15-20LX (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

[0166] The experimental period is 13 weeks in total:

[0167] The first week was the adaptation period, during which mice in each cage had free access to water and were given normal growth and reproduction feed.

[0168] After the adaptation period, from the 2nd to the 13th week:

[0169] Model group: 60% high-fat diet + 0.2 mL normal saline was given by intragastric administration every day;

[0170] CCFM1299 group: 60% high-fat diet and intragastric administration of 0.2 mL of bacterial concentration of 5×10 8 CFU / mL of CCFM1299 bacterial suspension;

[0171] NXYCHL3M3 group: 60% high-fat diet and intragastric administration of 0.2 mL of bacterial concentration of 5×10 8 CFU / mL of NXYCHL3M3 bacterial suspension;

[0172] Blank group: The mice were given low-fat control feed and 0.2 mL of normal saline by intragastric administration every day.

[0173] The specific procedures of animal experiments are shown in Figure 1 shown.

[0174] After the experiment, blood was collected from the eyeballs of the mice, and the blood was allowed to stand at room temperature for more than one hour before centrifugation to separate the serum. The content of cholesterol TC in the serum was measured by a biochemical analyzer. The results are shown in Fig. 9 .

[0175] Depend on Fig. 9It can be seen that the TC content in the blank group serum was 0.96±0.12mmol / L, while the high-fat diet caused the TC content in the model group serum to increase to 1.94±0.10mmol / L, which was significantly higher than the blank group. After mice were gavaged with Clostridium butyricum CCFM1299, the TC content in the serum (1.54±0.45mmol / L) was significantly lower than that in the model group, and after mice were gavaged with Clostridium butyricum NXYCHL3M3, the TC content in the serum was also reduced (1.66±0.18mmol / L), but there was no significant difference between it and the model group. The above experimental results show that Clostridium butyricum CCFM1299 can better reduce the TC level in the serum of obese mice.

[0176] Example 10: Effects of Clostridium butyricum CCFM1299 on liver lipid accumulation and non-alcoholic fatty liver disease scores in obese mice

[0177] The specific steps are as follows:

[0178] Four-week-old SPF C57BL / 6J male mice were divided into four groups, namely, a blank group, a model group, and an experimental group, wherein the experimental group included the CCFM1299 group gavaged with Clostridium butyricum CCFM1299 and the NXYCHL3M3 group gavaged with Clostridium butyricum NXYCHL3M3. Eight mice in each group were kept in the Experimental Animal Center of Jiangnan University, with a constant temperature of 21-26°C, humidity of 40-70%, noise less than or equal to 60dB, and animal illumination of 15-20LX (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

[0179] The experimental period is 13 weeks in total:

[0180] The first week was the adaptation period, during which mice in each cage had free access to water and were given normal growth and reproduction feed.

[0181] After the adaptation period, from the 2nd to the 13th week:

[0182] Model group: 60% high-fat diet + 0.2 mL normal saline was given by intragastric administration every day;

[0183] CCFM1299 group: 60% high-fat diet and intragastric administration of 0.2 mL of bacterial concentration of 5×10 8 CFU / mL of CCFM1299 bacterial suspension;

[0184] NXYCHL3M3 group: 60% high-fat diet and intragastric administration of 0.2 mL of bacterial concentration of 5×10 8 CFU / mL of NXYCHL3M3 bacterial suspension;

[0185] Blank group: The mice were given low-fat control feed and 0.2 mL of normal saline by intragastric administration every day.

[0186] The specific procedures of animal experiments are shown in Figure 1 shown.

[0187] After the experiment, blood was collected and the mice were killed. The chest cavity was opened, the liver was removed and fixed in 4% paraformaldehyde solution for 36 hours, and then dehydrated with ethanol gradient, transparentized with xylene, embedded in paraffin, and stained with HE on sections. The pathological degree of the tissue was evaluated from three levels: fatty degeneration (fatty degeneration <5%, 0 points; 5%-33%, 1 point; 34%-66%, 2 points; >66%, 3 points), hepatic lobular inflammation (the number of inflammation-related lesions counted at 20× was 0, 0 points; <2, 1 point; 2-4, 2 points; >4, 3 points) and hepatocyte ballooning (no ballooning, 0 points; a few ballooning, 1 point; most ballooning, 2 points). The results of liver staining are shown in Figure 2. Fig.10 As shown. Fig.10 It can be seen that the high-fat diet caused the destruction of the liver lobule structure of mice, hepatocytes were enlarged and contained large fat droplets, some hepatocytes showed fatty degeneration mainly in the form of macrovesicles, and inflammatory cells aggregated. After the intervention of Clostridium butyricum CCFM1299, there were no macrovesicular fat droplets in the liver, the fat droplets were small and evenly distributed, and the pathological characteristics were almost similar to those of the blank group. After the intervention of NXYCHL3M3, although the number and area of ​​vacuoles in the liver of mice were significantly reduced, a small number of macrovesicular fat droplets still existed.

[0188] The pathological scores of each group were Fig.11 As shown by Fig.11 It can be seen that the high-fat diet caused the non-alcoholic fatty liver disease score of mice to increase from 0.33±0.5 in the blank group to 5.89±0.93. Both the intervention of Clostridium butyricum CCFM1299 and NXYCHL3M3 promoted the decrease of the score, which was 1.11±0.60 (CCFM1299) and 2.67±1.3 (NXYCHL3M3), respectively. Among them, there was no significant difference between the disease score under the intervention of Clostridium butyricum CCFM1299 and the blank group. Therefore, Clostridium butyricum CCFM1299 can also inhibit the onset of obesity-related non-alcoholic fatty liver.

[0189] Example 11: Effects of Clostridium butyricum CCFM1299 on the levels of TNF-α and IL-1β inflammatory factors in the liver of obese mice

[0190] The specific steps are as follows:

[0191] Four-week-old SPF C57BL / 6J male mice were divided into four groups, namely, a blank group, a model group, and an experimental group, wherein the experimental group included the CCFM1299 group gavaged with Clostridium butyricum CCFM1299 and the NXYCHL3M3 group gavaged with Clostridium butyricum NXYCHL3M3. Eight mice in each group were kept in the Experimental Animal Center of Jiangnan University, with a constant temperature of 21-26°C, humidity of 40-70%, noise less than or equal to 60dB, and animal illumination of 15-20LX (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

[0192] The experimental period is 13 weeks in total:

[0193] The first week was the adaptation period, during which mice in each cage had free access to water and were given normal growth and reproduction feed.

[0194] After the adaptation period, from the 2nd to the 13th week:

[0195] Model group: 60% high-fat diet + 0.2 mL normal saline was given by intragastric administration every day;

[0196] CCFM1299 group: 60% high-fat diet and intragastric administration of 0.2 mL of bacterial concentration of 5×10 8 CFU / mL of CCFM1299 bacterial suspension;

[0197] NXYCHL3M3 group: 60% high-fat diet and intragastric administration of 0.2 mL of bacterial concentration of 5×10 8 CFU / mL of NXYCHL3M3 bacterial suspension;

[0198] Blank group: The mice were given low-fat control feed and 0.2 mL of normal saline by intragastric administration every day.

[0199] The specific procedures of animal experiments are shown in Figure 1 shown.

[0200] After the experiment, blood was collected and the mice were killed. The chest cavity was opened, the liver was removed, and about 50 mg of liver lobule tissue was cut for total protein extraction. Subsequently, the two inflammatory factors TNF-α and IL-1β in the liver were detected using Elisa kits. The results are as follows: Figures 12-13 shown.

[0201] Depend on Fig.12 , Fig.13 It can be seen that the high-fat diet significantly led to the increase of two inflammatory factors in the liver of mice, among which TNF-α increased from 2255±214.5pg / mL in the blank group to 3417±189.4pg / mL, and IL-1β increased from 2114±146.4pg / mL to 3044±425.7pg / mL.

[0202] Although intervention with Clostridium butyricum NXYCHL3M3 reduced the levels of TNF-α (3238±135.4pg / mL) and IL-1β (2820±264.1pg / mL) in the liver, there was no significant difference compared with the model group.

[0203] In contrast, Clostridium butyricum CCFM1299 not only significantly reduced the levels of TNF-α (2514±381.3pg / mL) and IL-1β (2033±232.1pg / mL) in the liver compared with the model group, but also had no significant difference in the levels of the two inflammatory factors compared with the blank group. Therefore, Clostridium butyricum has the potential to inhibit liver inflammatory response under high-fat diet.

[0204] Example 12: Preparation of bacterial powder containing Lactobacillus plantarum CCFM1189

[0205] The specific steps are as follows:

[0206] (1) Preparation of Clostridium butyricum CCFM1299 seed solution

[0207] The Clostridium butyricum CCFM1299 was inoculated into RCM liquid culture medium and cultured at 37°C for 16 hours to prepare Clostridium butyricum CCFM1299;

[0208] (2) inoculating the prepared seed solution of Clostridium butyricum CCFM1299 into RCM medium at an inoculum amount of 3% of the total mass of the medium, and culturing at 37° C. for 30 h to obtain a culture solution;

[0209] The culture solution was centrifuged to collect the bacterial cells; the bacterial cells were washed three times with a phosphate buffer solution having a pH of 7.2, and then resuspended with a trehalose freeze-drying protective agent having a concentration of 100 g / L, and the mass ratio of the freeze-drying protective agent to the bacterial cells was controlled to be 2:1 to obtain a resuspension;

[0210] The resuspension was precooled at -80°C for 1.5 h and then immediately transferred to a freeze dryer for drying for 24 h to obtain Clostridium butyricum CCFM1299 bacterial powder.

[0211] Example 13: Preparation of yogurt containing Clostridium butyricum CCFM1299

[0212] The specific steps are as follows:

[0213] (1) Milk powder, inulin, stevioside and water were mixed in a weight ratio of 20:5:5:75, homogenized and prepared into fermentation raw materials; sterilized by ultra-high temperature sterilization at 121°C for 300s, then cooled to 42°C, inoculated with mixed bacterial powder of Lactobacillus bulgaricus and Streptococcus thermophilus, and fermented at 42°C for 12h until the bacterial concentration of Lactobacillus bulgaricus and Streptococcus thermophilus was controlled to 10 5 CFU / g and 10 7 CFU / g, and then blended; the fermentation product was cooled to 37°C;

[0214] (2) Adding the freeze-dried bacterial powder of Clostridium butyricum CCFM1299 prepared according to the method of Example 11 to the fermentation product after cooling in step (1), the feed amount of freeze-dried bacterial powder of Clostridium butyricum CCFM1299 is 10 9 The yogurt was stirred, canned, and stored at 4°C for 2 days to naturally complete post-ripening to make a probiotic yogurt.

[0215] 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 Clostridium butyricum CCFM1299 was deposited in Guangdong Provincial Microbiological Culture Collection Center on January 8, 2023, with the deposit number GDMCC No: 63125, and the deposit address is Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. A microbial preparation, characterized in that: The microbial preparation contains the Clostridium butyricum CCFM1299 or its fermentation broth according to claim 1.

3. The microbial preparation according to claim 2, characterized in that In the microbial preparation, the number of Clostridium butyricum CCFM1299 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

4. A product, characterized in that The product contains the Clostridium butyricum CCFM1299 described in claim 1 or the microbial preparation described in claim 2 or 3.

5. The product according to claim 4, characterized in that The products include but are not limited to ordinary food, special food, medicine, and feed additives.

6. The product according to claim 4 or 5, characterized in that In the product, the viable count of Clostridium butyricum CCFM1299 is at least: 1×10 6 CFU / mL or 1×10 6 CFU / g.

7. Use of the Clostridium butyricum CCFM1299 according to claim 1, or the microbial preparation according to claim 2 or 3, in the preparation of medicines or feed additives for alleviating obesity.

8. The use according to claim 7, characterized in that: In the medicine or feed additive, the viable count of Clostridium butyricum CCFM1299 is at least: 1×10 6 CFU / mL or 1×10 6 CFU / g.

9. Use of the Clostridium butyricum according to claim 1, or the microbial preparation according to claim 2 or 3 in the production of fermented food or feed additives.

Images