CN116004472A - Clostridium butyricum for relieving obesity and application thereof - Google Patents
Clostridium butyricum for relieving obesity and application thereof Download PDFInfo
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Abstract
The invention discloses clostridium butyricum for relieving obesity and application thereof, and belongs to the technical field of microorganisms and the technical field of medicines. Clostridium butyricum CCFM1299 disclosed herein was deposited at the microorganism strain collection in guangdong province at 2023, 01, and 08, under the accession number GDMCC No:63125. after the clostridium butyricum CCFM1299 provided by the invention acts on an obese mouse, the obesity of the mouse can be obviously relieved, and the method is particularly characterized in that the weight gain of the obese mouse is slowed down, the accumulation of abdominal fat and epididymal fat of the obese mouse is inhibited, the epididymal fat mononuclear cell recruitment of the obese mouse is reduced, the serum level of LDL-C and TC is obviously reduced, and the liver fat is accumulated and inflammation is relieved. Therefore, clostridium butyricum CCFM1299 has great application prospect in preparing products for preventing and/or treating obesity.
Description
Technical Field
The invention discloses clostridium butyricum for relieving obesity and application thereof, and belongs to the technical field of microorganisms and the technical field of medicines.
Background
Obesity is a non-infectious chronic disease, and is associated with an increased risk of 21 diseases such as cardiovascular, gastrointestinal, respiratory and nervous system diseases, which seriously threatens the physical health of people. According to the definition of the world health organization, a Body Mass Index (BMI) greater than 25 is overweight, and a value above 30 is considered obese. With the improvement of life quality and the change of life style of people, the number of obese people is increasing. From 1975 to now, obesity prevalence increased at least 7-fold, women increased from 0.7% to 5.6%, and men increased from 0.9% to 7.8%.
Genetic and environmental factors are the main factors responsible for the occurrence of obesity. Among them, environmental factors mainly include bad eating habits and living habits, such as long-term intake of high calorie foods and increasingly reduced exercise amount, promote fat accumulation, leading to obesity. However, differences in the genetic genes also lead to individuals with a different susceptibility to obesity, and the deletion of leptin and the melanocortin 4 receptor (MC 4R) 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 associated with the onset of obesity.
Hundreds of millions of microorganisms inhabit in the human intestinal tract, and an organic micro-ecological system is formed. The number of intestinal microorganisms in adult individuals is about 10 14 The number of the billions is about 10 times of the number of the cells of the human body, and the total weight of the billions accounts for 1-3% of the weight of the human body. The intestinal microorganisms not only affect the intestinal tract, but also play a vital role in a plurality of physiological activities such as metabolism, nutrition, immunity and the like of the human body. In 2006 Gordon et al found that the occurrence of obesity was associated with changes in the relative abundance of Bacteroides and Thick-walled bacteria. The intestinal flora of obese individuals is not only altered in species diversity and composition, but also functionally different from that of the normal population. The ability of the intestinal flora of obese individuals to extract energy from food is greatly enhanced. In addition to affecting energy expenditure, microbiota also alters the composition of substances in the gut and thus affects the development of obesity. Wherein 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 development of obesity is not neglected.
In recent years, the link between intestinal flora and obesity has been revealed, and intestinal flora has become one of the intervention targets for obesity treatment. Current approaches to the treatment/prevention of obesity mainly include surgical intervention, dietary intervention, medication and prebiotic/probiotic intervention, which have a more or less effect on the intestinal flora. Surgical and dietary intervention have a certain adverse effect on the intestinal flora, both of which may reduce the abundance of the flora, especially butyrate-producing bacteria, which may have an adverse effect on the colon. As for the intervention of medicines, most medicines have certain side effects, such as cable Ma Lutai (Semaglutide), a GLP-1R agonist, and the taking of the medicines can cause symptoms of nausea, vomiting, diarrhea and the like. Probiotic therapy has fewer side effects than the above-described interventions and can positively regulate the intestinal flora. From a mechanistic standpoint, probiotic intervention may counteract the occurrence of obesity by (a) altering microbiota to reduce energy absorption and intestinal permeability, (b) modulating satiety to reduce energy intake, and (c) inhibiting inflammation and promoting thermogenesis.
Thus, the symptoms associated with obesity can be safely and harmlessly ameliorated by developing probiotics or permissions that are resistant to obesity.
Disclosure of Invention
The invention provides clostridium butyricum (Clostridium butyricum) CCFM1299 which is deposited in the microorganism strain collection of Guangdong province at the year 08 of 2023, wherein the deposited number is GDMCC No:63125, the preservation address is Guangzhou Mr. first 100 college No. 59 building.
The clostridium butyricum (Clostridium butyricum) CCFM1299 is separated from human excrement, the 16S rDNA sequence of the strain is shown as SEQ ID NO.1 through sequencing analysis, the sequence obtained through sequencing is subjected to nucleic acid sequence comparison in Genbank, the result shows that the similarity with the nucleic acid sequence of clostridium butyricum is as high as 99.59%, and the strain is named clostridium butyricum (Clostridium butyricum) CCFM1299.
The clostridium butyricum CCFM1299 is inoculated into an RCM solid culture medium, and after the clostridium butyricum CCFM1299 is reversely cultured for 24 hours at 37 ℃, bacterial colonies are observed and bacterial bodies are observed under a microscope, and the bacterial bodies are found to be milky white, irregular, round convex and smooth, and the bacterial bodies are slightly irregular in shape and form campylobacter with round tail ends, and are usually in single, paired and small clusters.
The invention also provides a microbial preparation, which contains clostridium butyricum (Clostridium butyricum) CCFM1299 or a fermentation broth thereof or a lysate containing clostridium butyricum (Clostridium butyricum) CCFM1299.
In one embodiment of the present invention, the amount of clostridium butyricum (Clostridium butyricum) CCFM1299 in the microbial preparation is not less than 1X 10 6 CFU/mL or 1X 10 6 CFU/g。
The invention also provides a bacterial powder, which contains the clostridium butyricum (Clostridium butyricum) CCFM1299 or fermentation liquor thereof.
The invention also provides a product which contains the clostridium butyricum (Clostridium butyricum) CCFM1299 or the microbial preparation.
In one embodiment of the invention, the product comprises a food, feed additive or medicament.
In one embodiment of the invention, the products include, but are not limited to, general foods, special foods, feed additives, and pharmaceuticals.
In one embodiment of the invention, the special food product comprises a food product intended for a specific (special) group of people, such as infants, young children, diabetics, malnourished people.
In one embodiment of the invention, the medicament may be used to prevent, alleviate obesity. The clostridium butyricum (Clostridium butyricum) CCFM1299 is present in an amount of not less than 1X 10 in the product 6 CFU/mL or 1X 10 6 CFU/g
In one embodiment of the present invention, the medicament has at least one of the effects of (a) to (c):
(a) Weight and fat loss;
(b) Improving blood lipid disorder, and reducing TC and LDL-C levels in serum;
(c) Improving fat accumulation of liver.
In one embodiment of the invention, the food product is a dairy product, a soy product or a fruit and vegetable product produced using clostridium butyricum (Clostridium butyricum) CCFM1299 or a starter comprising clostridium butyricum CCFM1299.
In one embodiment of the invention, the food product is a solid beverage containing clostridium butyricum (Clostridium butyricum) CCFM1299.
In one embodiment of the present invention, the pharmaceutical product contains clostridium butyricum (Clostridium butyricum) CCFM1299, and a pharmaceutical excipient.
In one embodiment of the invention, the pharmaceutical excipients comprise excipients and additives.
In one embodiment of the invention, the pharmaceutical excipients comprise anti-adhesive, permeation enhancers, buffers, plasticizers, surfactants, defoamers, thickeners, inclusion agents, absorbents, humectants, solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, pH modifiers, adhesives, disintegrants, fillers, lubricants, wetting agents, integration agents, tonicity modifiers, stabilizers, glidants, flavoring agents, preservatives, foaming agents, suspending agents, coating materials, fragrances, diluents, flocculants and deflocculants, filter aids, and release retarders.
In one embodiment of the invention, the additive comprises microcrystalline cellulose, hydroxypropyl methylcellulose, and refined lecithin.
In one embodiment of the invention, the dosage form of the pharmaceutical product comprises granules, capsules, tablets, pills or oral liquids.
The invention also provides application of the clostridium butyricum (Clostridium butyricum) CCFM1299 or the microbial preparation in preparing a product for relieving obesity.
In one embodiment of the present invention, the product comprises a feed additive or a medicament, and the amount of clostridium butyricum (Clostridium butyricum) CCFM1299 in the product is not less than 1X 10 6 CFU/mL or 1X 10 6 CFU/g
In one embodiment of the invention, the medicament may be used to prevent, alleviate obesity.
In one embodiment of the present invention, the medicament has at least one of the effects of (a) to (c):
(d) Weight and fat loss;
(e) Improving blood lipid disorder, and reducing TC and LDL-C levels in serum;
(f) Improving fat accumulation of liver.
In one embodiment of the present invention, the pharmaceutical product contains clostridium butyricum (Clostridium butyricum) CCFM1299, and a pharmaceutical excipient.
In one embodiment of the invention, the pharmaceutical excipients comprise excipients and additives.
In one embodiment of the invention, the pharmaceutical excipients comprise anti-adhesive, permeation enhancers, buffers, plasticizers, surfactants, defoamers, thickeners, inclusion agents, absorbents, humectants, solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, pH modifiers, adhesives, disintegrants, fillers, lubricants, wetting agents, integration agents, tonicity modifiers, stabilizers, glidants, flavoring agents, preservatives, foaming agents, suspending agents, coating materials, fragrances, diluents, flocculants and deflocculants, filter aids, and release retarders.
In one embodiment of the invention, the additive comprises microcrystalline cellulose, hydroxypropyl methylcellulose, and refined lecithin.
In one embodiment of the invention, the dosage form of the pharmaceutical product comprises granules, capsules, tablets, pills or oral liquids.
The invention also provides application of the clostridium butyricum (Clostridium butyricum) CCFM1299 or the microbial preparation in the production of fermented food and feed additives.
Advantageous effects
The clostridium butyricum (Clostridium butyricum) CCFM1299 capable of relieving obesity and related metabolic disorders provided by the invention can obviously inhibit the occurrence and development of mouse obesity under high-fat diet by adopting the clostridium butyricum (Clostridium butyricum) CCFM1299, and is specifically characterized in that compared with a model group and a blank group:
(1) The weight of the obese mice is reduced from 43.58 +/-2.086 g to 34.66 +/-2.61 g, and the obese mice have no significant difference with the weight of the mice in the blank group;
(2) The epididymal fat amount of obese mice was reduced from 2.61.+ -. 0.37g to 1.49.+ -. 0.30g;
(3) The abdominal fat mass of the obese mice is reduced from 1.68+/-0.34 g to 0.67+/-0.14 g;
(4) The serum LDL-C of obese mice is reduced from 0.38+/-0.06 mmol/L to 0.20+/-0.08 mmol/L, and
no significant difference from the body weight of the mice in the blank group;
(5) TC in serum of obese mice is reduced from 1.94+/-0.10 mmol/L to 1.54+/-0.45 mmol/L;
(6) The ratio of LDL-C to HDL-C in the serum of obese mice was reduced from 0.28 to 0.16 without significant differences in the weight of the mice in the blank group;
(7) The fat tissue inflammation level of the obese mice is obviously reduced, the monocyte surface molecule F4/80 staining intensity is reduced from 148.3+/-30.33 to 32.24+/-17.01, and the fat tissue inflammation level is not obviously different from the body weight of the mice in the blank group;
(8) The fat hoarding in the liver of the obese mice is obviously reduced, and the non-alcoholic fatty liver disease score is obviously reduced;
(9) TNF-alpha and IL-1 beta levels were significantly reduced in the liver of obese mice, all without significant differences from the mice in the blank group;
therefore, clostridium butyricum (Clostridium butyricum) CCFM1299 has great application prospect in preparing products for preventing and/or treating obesity and related metabolic diseases thereof.
Preservation of biological materials
Clostridium butyricum (Clostridium butyricum) CCFM1299, taxonomic designation Clostridium butyricum, was deposited at the cantonese province microorganism strain collection at 2023, month 08, under accession number GDMCC No:63125 the preservation address is building 5 of Guangzhou Miao 100 # college of first-vogue 59, and the university of Guangdong province institute of microorganisms.
Drawings
Fig. 1: animal experiment flow chart.
Fig. 2: mice were tested for body weight status in different groups.
Fig. 3: the mice were tested for abdominal fat mass in different groups.
Fig. 4: the epididymal fat mass of mice was tested in different groups.
Fig. 5: results of F4/80 immunohistochemistry in epididymal adipose tissue of mice were tested in different groups.
Fig. 6: f4/80 immunohistochemical quantitative results in epididymal adipose tissues of mice in different groups.
Fig. 7: LDL-C levels in serum from mice were tested in different groups.
Fig. 8: LDL-C/HDL-C ratios in serum of mice were tested in different groups.
Fig. 9: the levels of TC in serum of mice were tested in different groups.
Fig. 10: liver HE staining results for mice of different groups.
Fig. 11: non-alcoholic fatty liver disease scores for different groups of mice.
Fig. 12: liver TNF- α levels in mice of different groups.
Fig. 13: IL-1β levels in mice of different groups.
In the above pictures: * The terms "p" and "p" refer to values less than 0.05, 0.01, 0.001, and 0.0001, respectively, compared to the model group; tables 7777777 of #, # and # # respectively show p values less than 0.05, 0.01, 0.001, 0.0001 compared to the blank group; ns indicates no significant difference from the model group; NS indicates no significant difference compared to the blank.
Detailed Description
The C57BL/6 male mice referred to in the examples below were purchased from Experimental animal technologies Inc. of Lewa, beijing, and the 60% high fat diet TP23300 and the low fat control diet TP23301 were purchased from Nantong Talaofe. TC, HDL-C and LDL-C kits were purchased from Beckman coulter, inc. of America. TNF-alpha and IL-1β Elisa detection kit were purchased from RD.
The following examples relate to the following media:
RCM liquid Medium (L): 10g of peptone, 10g of beef extract, 3g of yeast powder, 5g of anhydrous dextrose, 1g of soluble starch, 5g of sodium chloride, 3g of anhydrous sodium acetate, 0.5g of L-cysteine hydrochloride, 4mL (0.025%) of resazurin and the pH value of 6.8+/-0.1.
RCM solid medium (L): 10g of peptone, 10g of beef extract, 3g of yeast powder, 5g of anhydrous dextrose, 1g of soluble starch, 5g of sodium chloride, 3g of anhydrous sodium acetate, 0.5g of L-cysteine hydrochloride, 4mL (0.025%) of resazurin, 15g of agar and the pH value of 6.8+/-0.1.
Example 1: screening and identification of clostridium butyricum (Clostridium butyricum) CCFM1299
(1) Clostridium butyricum (Clostridium butyricum) CCFM1299 screening:
taking healthy human body feces from Jiangsu tin-free region as sample, taking one spoon of feces sample, adding into 5mL PBS (0.05% cysteine), mixing, gradient diluting, selecting 10 -5 ～10 -7 Is spread on the RCM solid medium and cultured at 37℃for 48. And (3) picking a typical colony into an RCM solid culture medium for streaking purification, placing the culture medium in a 37 ℃ constant temperature incubator for inverted culture for 48 hours, picking a single colony, inoculating the single colony into 5mL of liquid RCM culture medium, placing the culture medium in the 37 ℃ constant temperature incubator for culture for 16-18 hours, taking 1.5mL of bacterial liquid 6000r/min, centrifuging for 3 minutes to remove the supernatant, adding 1mL of 30% sterile glycerol for preservation, simultaneously leaving a 1.5mL of bacterial body for centrifugation, removing the supernatant, and re-suspending the bacterial body with sterile water for strain identification.
The sequence of 16S rDNA of the strain is shown as SEQ ID NO.1, the sequence obtained by sequencing is subjected to nucleic acid sequence comparison in Genbank, the result shows that the similarity with the nucleic acid sequence of clostridium butyricum is up to 99.59%, and the clostridium butyricum is named as clostridium butyricum (Clostridium butyricum) CCFM1299.
(2) Clostridium butyricum NXYCHL3M3 screening:
taking healthy human body feces from Jiangsu tin-free region as sample, taking one spoon of feces sample, adding into 5mL PBS (0.05% cysteine), mixing, gradient diluting, selecting 10 -5 ～10 -7 Is spread on the RCM solid medium and cultured at 37℃for 48 hours. And (3) picking a typical colony into an RCM solid culture medium for streaking purification, placing the culture medium in a 37 ℃ constant temperature incubator for inverted culture for 48 hours, picking a single colony, inoculating the single colony into 5mL of liquid RCM culture medium, placing the culture medium in the 37 ℃ constant temperature incubator for culture for 16-18 hours, taking 1.5mL of bacterial liquid 6000r/min, centrifuging for 3 minutes to remove the supernatant, adding 1mL of 30% sterile glycerol for preservation, simultaneously leaving a 1.5mL of bacterial body for centrifugation, removing the supernatant, and re-suspending the bacterial body with sterile water for strain identification. The sequence obtained by sequencing is subjected to nucleic acid sequence comparison in Genbank, and the result shows that the similarity with the nucleic acid sequence of clostridium butyricum is up to 99.42%, and the sequence is named clostridium butyricum NXYCHL3M3.
Example 2: culture of Clostridium butyricum (Clostridium butyricum) CCFM1299
The method comprises the following specific steps:
clostridium butyricum CCFM1299 and Clostridium butyricum NXYCHL3M3 are respectively inoculated into RCM liquid culture medium and cultured for 16 hours at 37 ℃, then bacterial liquid is absorbed and transferred into fresh RCM liquid culture medium according to the inoculation amount of 4% (v/v), after the same condition is cultured for 12 hours, bacterial cells are centrifuged for 15 minutes at 8000r/min, and after the bacterial cells are washed by 0.9% physiological saline (containing cysteine hydrochloride) and centrifuged for 10 minutes again at 8000r/min, bacterial cells are collected and resuspended by 30% glycerol solution, and resuspension is respectively prepared and stored at-80 ℃ for standby.
Preparation of bacterial suspension for gastric lavage:
taking out the obtained heavy suspension from-80deg.C, centrifuging at 4deg.C and 8000r/min for 10min, discarding supernatant, and re-suspending with 0.9% physiological saline (containing cysteine hydrochloride) to obtain bacterial suspension for gastric administration, wherein the concentration of strain in the bacterial suspension is 5×10 8 CFU/mL。
Example 3: effects of clostridium butyricum (Clostridium butyricum) CCFM1299 on weight of obese mice
The method comprises the following specific steps:
SPF-class C57BL/6J male mice of 4 weeks of age were divided into 4 groups, which were a blank group, a model group and an experimental group, respectively, wherein the experimental group included a CCFM1299 group of Clostridium gastrinicum CCFM1299 and a NXYCHL3M3 group of Clostridium gastrinicum NXYCHL3M3. 8 animals are fed in each group at the center of experimental animals of university of Jiangnan, the constant temperature is 21-26 ℃, the humidity is 40-70%, the noise is less than or equal to 60dB, and the animal illuminance is 15-20LX (all animal experimental procedures are examined and approved by the animal welfare and ethics management committee of university of Jiangnan).
The experimental period totaled 13 weeks:
and the 1 st week is the adaptation period, and each cage of mice is free to drink water in the adaptation period, and common growth and propagation feed is given.
After the adaptation period is finished, the following weeks 2 to 13:
model group: 60% high-fat feed and 0.2mL physiological saline for gastric lavage are administered daily;
CCFM1299 group: the bacterial concentration of 0.2mL of 60% high-fat feed and gastric lavage is 5×10 8 CFU/mL of CCFM1299 bacterial suspension;
NXYCHL3M3 group: the bacterial concentration of 0.2mL of 60% high-fat feed and gastric lavage is 5×10 8 CFU/mL of NXYCHL3M3 bacterial suspension;
blank group: low fat control feed plus lavage 0.2mL physiological saline was administered daily.
The specific flow of the animal experiment is shown in figure 1.
The body weight of the mice was examined weekly during the course of the experiment and the final body weight changes are shown in figure 2.
As shown in fig. 2, the model group reached 43.58 ±2.086g under a 12-week high fat diet, while the blank group had a weight of only 30.10±1.466g. The same was true for the CCFM1299 group on a high-fat diet, which had a weight of 34.66.+ -. 2.61g, which was reduced by 20% compared to the model group, whereas the Clostridium butyricum NXYCHL3M3, which had a dry prognosis, had a weight of 39.78.+ -. 6.23g, which was reduced by only 8.7% compared to the model group. In addition, there was no significant difference in body weight of mice with the CCFM1299 dry prognosis from the blank group (p=0.07). Thus, clostridium butyricum CCFM1299 has an effect of inhibiting weight gain on high fat diets.
Example 4: the effect of clostridium butyricum (Clostridium butyricum) CCFM1299 on fat mass in the abdominal cavity of obese mice was specifically as follows:
SPF-class C57BL/6J male mice of 4 weeks of age were divided into 4 groups, which were a blank group, a model group and an experimental group, respectively, wherein the experimental group included a CCFM1299 group of Clostridium gastrinicum CCFM1299 and a NXYCHL3M3 group of Clostridium gastrinicum NXYCHL3M3. 8 animals are fed in each group at the center of experimental animals of university of Jiangnan, the constant temperature is 21-26 ℃, the humidity is 40-70%, the noise is less than or equal to 60dB, and the animal illuminance is 15-20LX (all animal experimental procedures are examined and approved by the animal welfare and ethics management committee of university of Jiangnan).
The experimental period totaled 13 weeks:
and the 1 st week is the adaptation period, and each cage of mice is free to drink water in the adaptation period, and common growth and propagation feed is given.
After the adaptation period is finished, the following weeks 2 to 13:
model group: 60% high-fat feed and 0.2mL physiological saline for gastric lavage are administered daily;
CCFM1299 group:the bacterial concentration of 0.2mL of 60% high-fat feed and gastric lavage is 5×10 8 CFU/mL of CCFM1299 bacterial suspension;
NXYCHL3M3 group: the bacterial concentration of 0.2mL of 60% high-fat feed and gastric lavage is 5×10 8 CFU/mL of NXYCHL3M3 bacterial suspension;
blank group: low fat control feed plus lavage 0.2mL physiological saline was administered daily.
The specific flow of the animal experiment is shown in figure 1.
After the experiment is finished, the mice are dissected according to ethical requirements, and abdominal fat is taken for weighing.
The abdominal fat weight of the model group was significantly increased to 1.68.+ -. 0.34g as compared to the weight of the blank group of 0.25.+ -. 0.08g as shown in FIG. 3. The abdominal fat mass is significantly reduced to 0.67 + -0.14 g under CCFM1299 intervention; NXYCHL3M3 was reduced to 1.12.+ -. 0.45g under intervention. Thus, CCFM1299 may be more effective in inhibiting accumulation of abdominal fat than NXYCHL3M3.
Example 5: the effect of clostridium butyricum (Clostridium butyricum) CCFM1299 on epididymal fat mass of obese mice was specifically as follows:
SPF-class C57BL/6J male mice of 4 weeks of age were divided into 4 groups, which were a blank group, a model group and an experimental group, respectively, wherein the experimental group included a CCFM1299 group of Clostridium gastrinicum CCFM1299 and a NXYCHL3M3 group of Clostridium gastrinicum NXYCHL3M3. 8 animals are fed in each group at the center of experimental animals of university of Jiangnan, the constant temperature is 21-26 ℃, the humidity is 40-70%, the noise is less than or equal to 60dB, and the animal illuminance is 15-20LX (all animal experimental procedures are examined and approved by the animal welfare and ethics management committee of university of Jiangnan).
The experimental period totaled 13 weeks:
and the 1 st week is the adaptation period, and each cage of mice is free to drink water in the adaptation period, and common growth and propagation feed is given.
After the adaptation period is finished, the following weeks 2 to 13:
model group: 60% high-fat feed and 0.2mL physiological saline for gastric lavage are administered daily;
CCFM1299 group: daily administration of 60% high-fat feed and 0.2mL of fungus concentrate for gastric lavageIs 5 multiplied by 10 8 CFU/mL of CCFM1299 bacterial suspension;
NXYCHL3M3 group: the bacterial concentration of 0.2mL of 60% high-fat feed and gastric lavage is 5×10 8 CFU/mL of NXYCHL3M3 bacterial suspension;
blank group: low fat control feed plus lavage 0.2mL physiological saline was administered daily.
The specific flow of the animal experiment is shown in figure 1.
After the experiment is finished, the mice are dissected according to ethical requirements, and epididymal fat is taken and weighed.
As shown in figure 4, the weight of epididymal fat is remarkably increased to 2.61+ -0.37 g compared with the weight of the epididymal fat of the blank group which is 0.43+ -0.11 g. The epididymal fat mass is obviously reduced to 1.49+/-0.3 g under the intervention of CCFM1299; NXYCHL3M3 was reduced to 2.15.+ -. 0.76g under intervention. Therefore, CCFM1299 can more effectively inhibit epididymal fat accumulation than NXYCHL3M3.
Example 6: fusobacterium butyrate (Clostridium butyricum) CCFM1299 effect on relieving epididymal fat inflammation in obese mice
The method comprises the following specific steps:
SPF-class C57BL/6J male mice of 4 weeks of age were divided into 4 groups, which were a blank group, a model group and an experimental group, respectively, wherein the experimental group included a CCFM1299 group of Clostridium gastrinicum CCFM1299 and a NXYCHL3M3 group of Clostridium gastrinicum NXYCHL3M3. 8 animals are fed in each group at the center of experimental animals of university of Jiangnan, the constant temperature is 21-26 ℃, the humidity is 40-70%, the noise is less than or equal to 60dB, and the animal illuminance is 15-20LX (all animal experimental procedures are examined and approved by the animal welfare and ethics management committee of university of Jiangnan).
The experimental period totaled 13 weeks:
and the 1 st week is the adaptation period, and each cage of mice is free to drink water in the adaptation period, and common growth and propagation feed is given.
After the adaptation period is finished, the following weeks 2 to 13:
model group: 60% high-fat feed and 0.2mL physiological saline for gastric lavage are administered daily;
CCFM1299 group: the daily administration of 60% high-fat feed and 0.2mL of gastric lavage is that5×10 8 CFU/mL of CCFM1299 bacterial suspension;
NXYCHL3M3 group: the bacterial concentration of 0.2mL of 60% high-fat feed and gastric lavage is 5×10 8 CFU/mL of NXYCHL3M3 bacterial suspension;
blank group: low fat control feed plus lavage 0.2mL physiological saline was administered daily.
The specific flow of the animal experiment is shown in figure 1.
After the experiment is finished, the mice are dissected according to ethical requirements, epididymal fat is taken down and placed in 4% paraformaldehyde solution for fixing for 36 hours, and after dehydration, embedding, slicing, dewaxing, antigen retrieval, serum sealing, primary antibody incubation, color development, counterstaining, dehydration and sealing, the tissues are subjected to inflammation degree assessment. The results of immunohistochemistry are shown in FIG. 5, and as can be seen from FIG. 5, a large number of F4/80 positive staining areas are obviously arranged around epididymal adipocytes of the model group.
The quantification of the positive staining areas is shown in FIG. 6. As can be seen from the quantitative results in FIG. 6, the F4/80 positive staining intensity of the blank group was 25.02, while the positive staining intensity of the model group was 148.30. The F4/80 positive staining intensity is obviously reduced to 32.34 under the intervention of CCFM1299; the positive staining intensity was reduced to 86.62 with NXYCHL3M3 intervention. Epididymal fat F4/80 staining intensity was significantly reduced compared to the model group with CCFM1299 intervention, and there was no significant difference compared to the blank group. Therefore, CCFM1299 can more advantageously inhibit macrophage recruitment in epididymal fat and thereby alleviate tissue inflammation.
Example 7: effects of clostridium butyricum (Clostridium butyricum) CCFM1299 on Low Density lipoprotein cholesterol (LDL-C) levels in serum of obese mice
The method comprises the following specific steps:
SPF-class C57BL/6J male mice of 4 weeks of age were divided into 4 groups, which were a blank group, a model group and an experimental group, respectively, wherein the experimental group included a CCFM1299 group of Clostridium gastrinicum CCFM1299 and a NXYCHL3M3 group of Clostridium gastrinicum NXYCHL3M3. 8 animals are fed in each group at the center of experimental animals of university of Jiangnan, the constant temperature is 21-26 ℃, the humidity is 40-70%, the noise is less than or equal to 60dB, and the animal illuminance is 15-20LX (all animal experimental procedures are examined and approved by the animal welfare and ethics management committee of university of Jiangnan).
The experimental period totaled 13 weeks:
and the 1 st week is the adaptation period, and each cage of mice is free to drink water in the adaptation period, and common growth and propagation feed is given.
After the adaptation period is finished, the following weeks 2 to 13:
model group: 60% high-fat feed and 0.2mL physiological saline for gastric lavage are administered daily;
CCFM1299 group: the bacterial concentration of 0.2mL of 60% high-fat feed and gastric lavage is 5×10 8 CFU/mL of CCFM1299 bacterial suspension;
NXYCHL3M3 group: the bacterial concentration of 0.2mL of 60% high-fat feed and gastric lavage is 5×10 8 CFU/mL of NXYCHL3M3 bacterial suspension;
blank group: low fat control feed plus lavage 0.2mL physiological saline was administered daily.
The specific flow of the animal experiment is shown in figure 1.
After the completion of the experiment, the mice were subjected to eyeball blood collection, the blood was allowed to stand at room temperature for more than one hour, and serum was separated by centrifugation. The serum was assayed for low density lipoprotein cholesterol (LDL-C) by a biochemical analyzer, and the results are shown in fig. 7.
As can be seen from FIG. 7, the serum LDL-C content of the blank was 0.11.+ -. 0.03mmol/L, whereas the high-fat diet resulted in an increase in the serum LDL-C content of the model to 0.38.+ -. 0.06mmol/L, which is significantly higher than that of the blank. After mice were perfused with clostridium butyricum CCFM1299, the serum LDL-C content (0.20+ -0.08 mmol/L) was significantly reduced compared to the model group, and there was no significant difference in the blank group. However, after mice were perfused with clostridium butyricum NXYCHL3M3, the LDL-C content in serum was also reduced (content 0.26±0.08 mmol/L), but there was no significant difference from the model group. The above experimental results indicate that clostridium butyricum (Clostridium butyricum) CCFM1299 is more capable of reducing LDL-C levels in serum of obese mice.
Example 8: effects of Clostridium butyricum (Clostridium butyricum) CCFM1299 on the serum Low Density lipoprotein cholesterol (LDL-C)/high Density lipoprotein cholesterol (HDL-C) ratio of obese mice
The method comprises the following specific steps:
SPF-class C57BL/6J male mice of 4 weeks of age were divided into 4 groups, which were a blank group, a model group and an experimental group, respectively, wherein the experimental group included a CCFM1299 group of Clostridium gastrinicum CCFM1299 and a NXYCHL3M3 group of Clostridium gastrinicum NXYCHL3M3. 8 animals are fed in each group at the center of experimental animals of university of Jiangnan, the constant temperature is 21-26 ℃, the humidity is 40-70%, the noise is less than or equal to 60dB, and the animal illuminance is 15-20LX (all animal experimental procedures are examined and approved by the animal welfare and ethics management committee of university of Jiangnan).
The experimental period totaled 13 weeks:
and the 1 st week is the adaptation period, and each cage of mice is free to drink water in the adaptation period, and common growth and propagation feed is given.
After the adaptation period is finished, the following weeks 2 to 13:
model group: 60% high-fat feed and 0.2mL physiological saline for gastric lavage are administered daily;
CCFM1299 group: the bacterial concentration of 0.2mL of 60% high-fat feed and gastric lavage is 5×10 8 CFU/mL of CCFM1299 bacterial suspension;
NXYCHL3M3 group: the bacterial concentration of 0.2mL of 60% high-fat feed and gastric lavage is 5×10 8 CFU/mL of NXYCHL3M3 bacterial suspension;
blank group: low fat control feed plus lavage 0.2mL physiological saline was administered daily.
The specific flow of the animal experiment is shown in figure 1.
After the completion of the experiment, the mice were subjected to eyeball blood collection, the blood was allowed to stand at room temperature for more than one hour, and serum was separated by centrifugation. The contents of low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) in serum were measured by a biochemical analyzer, and the ratio of LDL-C/HDL-C was calculated. This ratio reflects the body fat transport and an increase in this ratio is often indicative of the occurrence of obesity and metabolic syndrome, as shown in figure 8.
As can be seen from FIG. 8, the ratio of LDL-C/HDL-C in the serum of the blank was 0.15, whereas the high fat diet resulted in an increase in the ratio of LDL-C/HDL-C in the serum of the model to 0.28, significantly higher than that of the blank. After mice were perfused with clostridium butyricum CCFM1299, the ratio of LDL-C/HDL-C in serum (ratio 0.16) was significantly reduced compared to the model group, and there was no significant difference in the blank group. However, after mice were perfused with clostridium butyricum NXYCHL3M3, the LDL-C content in serum was also reduced (ratio 0.22), but there was no significant difference from the model group. The experimental results show that clostridium butyricum (Clostridium butyricum)
CCFM1299 more balances LDL-C and HDL-C levels in serum of obese mice.
Example 9: effects of clostridium butyricum (Clostridium butyricum) CCFM1299 on Total Cholesterol (TC) levels in obese mouse serum
The method comprises the following specific steps:
SPF-class C57BL/6J male mice of 4 weeks of age were divided into 4 groups, which were a blank group, a model group and an experimental group, respectively, wherein the experimental group included a CCFM1299 group of Clostridium gastrinicum CCFM1299 and a NXYCHL3M3 group of Clostridium gastrinicum NXYCHL3M3. 8 animals are fed in each group at the center of experimental animals of university of Jiangnan, the constant temperature is 21-26 ℃, the humidity is 40-70%, the noise is less than or equal to 60dB, and the animal illuminance is 15-20LX (all animal experimental procedures are examined and approved by the animal welfare and ethics management committee of university of Jiangnan).
The experimental period totaled 13 weeks:
and the 1 st week is the adaptation period, and each cage of mice is free to drink water in the adaptation period, and common growth and propagation feed is given.
After the adaptation period is finished, the following weeks 2 to 13:
model group: 60% high-fat feed and 0.2mL physiological saline for gastric lavage are administered daily;
CCFM1299 group: the bacterial concentration of 0.2mL of 60% high-fat feed and gastric lavage is 5×10 8 CFU/mL of CCFM1299 bacterial suspension;
NXYCHL3M3 group: the bacterial concentration of 0.2mL of 60% high-fat feed and gastric lavage is 5×10 8 CFU/mL of NXYCHL3M3 bacterial suspension;
blank group: low fat control feed plus lavage 0.2mL physiological saline was administered daily.
The specific flow of the animal experiment is shown in figure 1.
After the completion of the experiment, the mice were subjected to eyeball blood collection, the blood was allowed to stand at room temperature for more than one hour, and serum was separated by centrifugation. The content of cholesterol TC in serum was measured by a biochemical analyzer, and the results are shown in fig. 9.
As can be seen from FIG. 9, the TC content in the serum of the blank group was 0.96.+ -. 0.12mmol/L, whereas the high-fat diet resulted in the TC content in the serum of the model group being raised to 1.94.+ -. 0.10mmol/L, which is significantly higher than that of the blank group. After the clostridium gastrectomy is filled with clostridium butyricum CCFM1299, the content of TC in serum (the content is 1.54+/-0.45 mmol/L) is obviously reduced compared with that of a model group, and after the clostridium gastrectomy is filled with clostridium gastrectomy NXYCHL3M3, the content of TC in serum is also reduced (the content is 1.66+/-0.18 mmol/L), but the TC is not obviously different from that of the model group. The above experimental results show that clostridium butyricum (Clostridium butyricum) CCFM1299 is more capable of reducing TC levels in serum of obese mice.
Example 10: effects of clostridium butyricum (Clostridium butyricum) CCFM1299 on liver lipid accumulation and non-alcoholic fatty liver disease scoring in obese mice
The method comprises the following specific steps:
SPF-class C57BL/6J male mice of 4 weeks of age were divided into 4 groups, which were a blank group, a model group and an experimental group, respectively, wherein the experimental group included a CCFM1299 group of Clostridium gastrinicum CCFM1299 and a NXYCHL3M3 group of Clostridium gastrinicum NXYCHL3M3. 8 animals are fed in each group at the center of experimental animals of university of Jiangnan, the constant temperature is 21-26 ℃, the humidity is 40-70%, the noise is less than or equal to 60dB, and the animal illuminance is 15-20LX (all animal experimental procedures are examined and approved by the animal welfare and ethics management committee of university of Jiangnan).
The experimental period totaled 13 weeks:
and the 1 st week is the adaptation period, and each cage of mice is free to drink water in the adaptation period, and common growth and propagation feed is given.
After the adaptation period is finished, the following weeks 2 to 13:
model group: 60% high-fat feed and 0.2mL physiological saline for gastric lavage are administered daily;
CCFM1299 group: the bacterial concentration of 0.2mL of 60% high-fat feed and gastric lavage is 5×10 8 CFU/mL of CCFM1299 bacterial suspension;
NXYCHL3M3 group: the bacterial concentration of 0.2mL of 60% high-fat feed and gastric lavage is 5×10 8 CFU/mL of NXYCHL3M3 bacteriumA suspension;
blank group: low fat control feed plus lavage 0.2mL physiological saline was administered daily.
The specific flow of the animal experiment is shown in figure 1.
After the experiment is finished, the mice are killed, the thoracic cavity is dissected, the liver is taken out and placed in 4% paraformaldehyde solution for fixing for 36 hours, and after ethanol gradient dehydration, xylene transparency, paraffin embedding and slice HE staining, pathological degree assessment is carried out on three layers of tissue from steatosis (steatosis < 5%,0 score, 5% -33%, 1 score, 34% -66%, 2 score, > 66%,3 score), liver lobular inflammation (the number of inflammation related focuses is 0, 0 score, less than 2, 1 score, 2-4, 2 score, more than 4, 3 score) and hepatocyte balloon transformation (without balloon transformation, 0 score, few balloon transformation, 1 score, most balloon transformation and 2 score). The liver staining results are shown in fig. 10. As can be seen from fig. 10, the high fat diet resulted in destruction of the hepatic lobular structure of mice, hepatomegaly and large lipid droplets were contained, and some hepatocytes were subjected to steatosis mainly due to macrovesicular nature and had inflammatory cell aggregation. Clostridium butyricum CCFM1299 has no large vesicular fat droplets in the liver after dry prognosis, and the fat droplets are small and evenly distributed, and the pathological characteristics are almost similar to those of a blank group. In the case of dry NXYCHL3M3, although the number and area of vacuoles in the liver of mice were significantly reduced, small amounts of macrovesicular lipid droplets were present.
As shown in fig. 11, the high fat diet resulted in an increase in the non-alcoholic fatty liver disease score of mice from 0.33±0.5 to 5.89±0.93 in the blank group, as seen in fig. 11. Clostridium butyricum CCFM1299 and NXYCHL3M3 interventions both promoted this score decrease, 1.11±0.60 (CCFM 1299) and 2.67±1.3 (NXYCHL 3M 3), respectively. Wherein the disease score was not significantly different from the blank under clostridium butyricum CCFM1299 intervention. Thus, clostridium butyricum CCFM1299 can also inhibit the onset of obesity-related non-alcoholic fatty liver disease.
Example 11: effect of Clostridium butyricum (Clostridium butyricum) CCFM1299 on TNF- α and IL-1β inflammatory factor levels in the liver of obese mice
The method comprises the following specific steps:
SPF-class C57BL/6J male mice of 4 weeks of age were divided into 4 groups, which were a blank group, a model group and an experimental group, respectively, wherein the experimental group included a CCFM1299 group of Clostridium gastrinicum CCFM1299 and a NXYCHL3M3 group of Clostridium gastrinicum NXYCHL3M3. 8 animals are fed in each group at the center of experimental animals of university of Jiangnan, the constant temperature is 21-26 ℃, the humidity is 40-70%, the noise is less than or equal to 60dB, and the animal illuminance is 15-20LX (all animal experimental procedures are examined and approved by the animal welfare and ethics management committee of university of Jiangnan).
The experimental period totaled 13 weeks:
and the 1 st week is the adaptation period, and each cage of mice is free to drink water in the adaptation period, and common growth and propagation feed is given.
After the adaptation period is finished, the following weeks 2 to 13:
model group: 60% high-fat feed and 0.2mL physiological saline for gastric lavage are administered daily;
CCFM1299 group: the bacterial concentration of 0.2mL of 60% high-fat feed and gastric lavage is 5×10 8 CFU/mL of CCFM1299 bacterial suspension;
NXYCHL3M3 group: the bacterial concentration of 0.2mL of 60% high-fat feed and gastric lavage is 5×10 8 CFU/mL of NXYCHL3M3 bacterial suspension;
blank group: low fat control feed plus lavage 0.2mL physiological saline was administered daily.
The specific flow of the animal experiment is shown in figure 1.
After the experiment is finished, the mice are killed, the thoracic cavity is dissected, the liver is removed, about 50mg of liver lobule tissue is sheared for total protein extraction, and then two inflammatory factors of TNF-alpha and IL-1 beta in the liver are detected by using an Elisa kit, and the results are shown in figures 12-13.
From FIGS. 12 and 13, it is evident that the high fat diet resulted in an increase in two inflammatory factors in the liver of mice, with TNF-. Alpha.increased from 2255.+ -. 214.5pg/mL in the blank to 3417.+ -. 189.4pg/mL and IL-1β increased from 2114.+ -. 146.4pg/mL to 3044.+ -. 425.7pg/mL.
Although Clostridium butyricum NXYCHL3M3 interventions each reduced the levels of TNF- α (3238.+ -. 135.4 pg/mL) and IL-1β (2820.+ -. 264.1 pg/mL) in the liver, there was no significant difference from the model group.
In contrast, clostridium butyricum CCFM1299 not only significantly reduced the levels of TNF- α (2514+ -381.3 pg/mL) and IL-1β (2033+ -232.1 pg/mL) in the liver compared to the model group, but also had no significant difference between the levels of both inflammatory factors and the blank group. Clostridium butyricum thus has the potential to inhibit liver inflammatory responses in high fat diets.
Example 12: preparation of clostridium butyricum (Lactobacillus plantarum) containing CCFM1189 bacterial powder
The method comprises the following specific steps:
(1) Preparation of clostridium butyricum CCFM1299 seed liquid
Inoculating clostridium butyricum CCFM1299 into RCM liquid culture medium, and culturing at 37 ℃ for 16h to obtain clostridium butyricum CCFM1299;
(2) Inoculating the prepared clostridium butyricum CCFM1299 seed solution into an RCM (RCM culture medium) according to the inoculum size accounting for 3% of the total mass of the culture medium, and culturing for 30 hours at 37 ℃ to obtain a culture solution;
centrifuging the culture solution, and collecting thalli; washing thalli for 3 times by using phosphate buffer solution with pH of 7.2, then re-suspending the thalli by using trehalose freeze-drying protective agent with the concentration of 100g/L, and controlling the mass ratio of the freeze-drying protective agent to the thalli to be 2:1 to obtain re-suspension;
and (3) immediately transferring the heavy suspension to a freeze dryer for drying for 24 hours after pre-cooling for 1.5 hours at the temperature of minus 80 ℃ to obtain clostridium butyricum CCFM1299 bacterial powder.
Example 13: preparation of CCFM1299 yogurt containing Clostridium butyricum (Clostridium butyricum)
The method comprises the following specific steps:
(1) Milk powder, inulin, stevioside and water are mixed according to the weight ratio of 20:5:5:75, mixing and homogenizing to prepare a fermentation raw material; sterilizing at 121deg.C for 300s, cooling to 42deg.C, inoculating mixed powder of Lactobacillus bulgaricus and Streptococcus thermophilus, fermenting at 42deg.C for 12 hr, and controlling the thallus concentration of Lactobacillus bulgaricus and Streptococcus thermophilus to 10 5 CFU/g and 10 7 CFU/g, and then blending; cooling the fermentation product to 37 ℃;
(2) Adding the fermentation product obtained by the method of example 11 to the cooled fermentation product obtained in the step (1)Clostridium butyricum CCFM1299 freeze-dried bacterial powder, and the feeding amount of the clostridium butyricum CCFM1299 freeze-dried bacterial powder is 10 9 CFU clostridium butyricum CCFM1299/mL yoghurt is stirred, canned, stored for 2 days at 4 ℃ and naturally finished after-ripening, and the probiotic yoghurt is prepared.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. Clostridium butyricum (Clostridium butyricum) CCFM1299, deposited at the cantonese province microorganism strain collection at 2023, month 01, 08, under the accession number GDMCC No:63125, the preservation address is Guangzhou Mr. first 100 college No. 59 building.
2. A microbial preparation comprising the clostridium butyricum CCFM1299 or a fermentation broth thereof according to claim 1 or the clostridium butyricum CCFM1299 lysate according to claim 1.
3. The microbial preparation according to claim 2, wherein the amount of clostridium butyricum CCFM1299 in the microbial preparation is not less than 1 x 10 6 CFU/mL or 1X 10 6 CFU/g。
4. A product comprising clostridium butyricum CCFM1299 according to claim 1 or comprising the microbial preparation according to claim 2 or 3.
5. The product according to claim 4, wherein the product includes, but is not limited to, general foods, special foods, medicines, feed additives.
6. The product according to claim 4 or 5, characterized in that in the product the live bacteria of clostridium butyricum CCFM1299The number is at least: 1X 10 6 CFU/mL or 1X 10 6 CFU/g。
7. Use of clostridium butyricum CCFM1299 of claim 1, or the microbial formulation of claim 2 or 3, for the preparation of a product for alleviating obesity.
8. The use according to claim 7, wherein the product comprises, but is not limited to, normal foods, special foods, medicines, feed additives.
9. The use according to claim 8, characterized in that in the product the viable count of clostridium butyricum CCFM1299 is at least: 1X 10 6 CFU/mL or 1X 10 6 CFU/g。
10. Use of clostridium butyricum according to claim 1, or of the microbial preparation according to claim 2 or 3 for the production of fermented food, feed additives.
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