CN114933994A - Composition with function of relieving ulcerative colitis and application thereof - Google Patents
Composition with function of relieving ulcerative colitis and application thereof Download PDFInfo
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- CN114933994A CN114933994A CN202210625876.XA CN202210625876A CN114933994A CN 114933994 A CN114933994 A CN 114933994A CN 202210625876 A CN202210625876 A CN 202210625876A CN 114933994 A CN114933994 A CN 114933994A
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Abstract
The invention discloses a composition with a function of relieving ulcerative colitis and application thereof, and belongs to the technical field of microorganisms. The composition is a composite microbial inoculum which contains bifidobacterium longum NSP002, bacteroides xylanisolvens NSP003 and enterococcus faecium NSP 004; experiments show that the composition provided by the invention can improve the disease activity index of an ulcerative colitis mouse, maintain the structural integrity of colon tissues, enhance intestinal barrier, reduce oxidative stress level and relieve the inflammation of the colon tissues. Moreover, the composition can restore the intestinal short-chain fatty acid level of mice with ulcerative colitis, thereby regulating the intestinal microecological balance. The composition provided by the invention is used for preparing foods, nutritional supplements, health products, pharmaceutical compositions and feed additives for relieving ulcerative colitis, and has a very wide application prospect.
Description
Technical Field
The invention relates to a composition with a function of relieving ulcerative colitis and application thereof, belonging to the technical field of microorganisms.
Background
Inflammatory Bowel Disease (IBD) is a chronic inflammatory bowel disease mainly affecting the digestive system, can be divided into two main types of crohn's disease and ulcerative colitis, has latent property, various symptoms and non-specificity, causes unclear until now, has complex pathogenesis and lacks of clear mechanism, and brings great challenge to clinical treatment. IBD was first discovered in western countries, however, in the last 20 years, the incidence and prevalence of IBD in western developed countries have tended to plateau, with a significant growth trend in south america and east asia. According to statistics, 150 ten thousand cases of Chinese IBD patients are expected to be reached in 2025, and the disability adjusting life years are far beyond the theoretical value, thereby causing huge economic burden of diseases.
The intestinal flora is closely related to the occurrence and development of intestinal inflammation, and is one of the hot spots of scientific research in recent years. Clinical studies indicate that patients with colitis have a common imbalance in intestinal mucosal flora. The immunodeficient mice cannot induce IBD under the intestinal sterile environment, and the intestinal tract returns to the normal flora state to show intestinal inflammation, so that the intestinal flora is a necessary condition for the formation of IBD. Therefore, modulation of the intestinal flora to alleviate the inflammatory response is an important research direction in the search for an effective treatment regimen for IBD. For example, Chinese patent application with publication number CN111424003A discloses a Lactobacillus casei zhang and its application in preventing and treating chronic gastroenteritis; the Chinese invention patent text with the publication number of CN105343133B discloses a composition containing lactobacillus acidophilus CGMCC10436 and lactobacillus plantarum CGMCC9961 and application thereof in treating ulcerative colitis; chinese invention patent application with publication number CN105434476A discloses a bacteroides fragilis ZY312 and application thereof in relieving ulcerative colitis.
Meanwhile, dietary fibers such as pectin, inulin, beta-glucan and the like can relieve intestinal inflammation by relieving colonic atrophy, enhancing intestinal barrier, regulating inflammatory factors, reducing oxidative stress level, maintaining intestinal microecological balance and the like (Fan L, et al, productive effects of peptides with varied degrees of freedom and ecological balance in microorganisms [ J ] Food & Function,2020,11, 2886-.
However, studies have shown that the use of single species of prebiotics, probiotics or synbiotics, while being able to regulate the abundance of some intestinal species, hardly causes significant changes in the overall intestinal flora, and thus has limited regulation of the physiological health of the host, and the diversification of gut bacteria and/or prebiotics as a stable micro-ecological community can promote the health and robustness of the microbial ecosystem more comprehensively (Tran TTT, et al. Therefore, a new idea can be provided for the development of pharmaceutical compositions or health care products for relieving ulcerative colitis by digging the intestinal bacteria and the composition of the intestinal bacteria and the dietary fibers.
Disclosure of Invention
The first purpose of the invention is to provide a composition, which comprises a composite microbial inoculum or the composite microbial inoculum and prebiotics. The complex microbial inoculum comprises Bifidobacterium longum (NSP 002), Bacteroides xylanisolvens (NSP 003) and Enterococcus faecium (NSP 004); the prebiotics are pectin.
The invention provides a composition, which comprises a complex microbial inoculum;
the complex microbial inoculum comprises Bifidobacterium longum (NSP 002), Bacteroides xylanisolvens (NSP 003) and Enterococcus faecium (NSP 004);
the bifidobacterium longum NSP002 has been preserved in the Guangdong province microorganism strain preservation center in 2021, 19.1, the preservation address is No. 59 building 5 of Michelia Tokyo No. 100 of Guangzhou city, and the preservation number is GDMCC No. 61440;
the bacteroides xylanisolvens NSP003 has been preserved in the Guangdong province microorganism culture preservation center at 19.1.1 in 2021, the preservation address is No. 59 floor 5 of Mirabilitum 100 of Guangzhou city, and the preservation number is GDMCC No. 61441;
the enterococcus faecium NSP004 strain is preserved in Guangdong province microorganism culture collection center at 19.1.1.2021, the preservation address is No. 59 floor 5 of Michelia Tokoro 100, Guangzhou city, and the preservation number is GDMCC No. 61442.
The Bifidobacterium longum (Bifidobacterium longum) NSP002, Bacteroides xylanisolvens (Bacteroides xylanisolvens) NSP003 and Enterococcus faecium (Enterococcus faecium) NSP004 are separated from healthy human fecal samples from south Chang area in Jiangxi, the strains are subjected to sequencing analysis, and the sequences obtained by sequencing are subjected to nucleic acid sequence alignment in NCBI, so that the three strains are respectively Bifidobacterium longum (Bifidobacterium longum), Bacteroides xylanisolvens (Bacteroides xylanisolvens) and Enterococcus faecium (Enterococcus faecium).
The bifidobacterium longum NSP002 has the following properties:
the characteristics of the thallus are as follows: gram-positive rod-shaped bacteria, sometimes Y-or V-shaped, are sporulate-free, flagellate-free, and have a width of about 0.5-1.5 μm and a length of about 1.3-8 μm. Colony characteristics: obvious colony is formed on the culture medium, the diameter is between 0.3 and 2mm, the front shape is circular, the middle part is convex, the edge is neat, the colony is slightly white and opaque, and the surface is moist and smooth. Growth characteristics: the strain is obligate anaerobe, the optimal growth temperature is 36-38 ℃, the optimal growth pH value is 6.6-7.0, the strain can grow well in a culture medium containing glucose, and the strain can enter the late logarithmic phase or the early stable phase within 16-24 hours.
The bacteroides xylanisolvens NSP003 has the following properties:
the characteristics of the thallus are as follows: gram-negative rod-shaped bacteria, spore-free, flagellum-free, about 0.9-1.2 μm wide and about 3-8 μm long. Colony characteristics: obvious colony is formed on the culture medium, the diameter is between 0.3 and 2mm, the front shape is circular, the middle part is convex, the edge is neat, the colony is slightly white and opaque, and the surface is moist and smooth. Growth characteristics: the strain is strict anaerobic bacteria, is sensitive to oxygen, optimally grows at the temperature of 35-38 ℃, has the optimal growth pH value of 6.6-7.0, well grows in a culture medium containing glucose, and can enter the logarithmic phase or the stable early phase within 16-24 hours.
The enterococcus faecium NSP004 has the following properties:
the characteristics of the thallus are as follows: gram-positive spherical bacteria, no spore, no flagellum, 0.8-1.0 μm in diameter. Colony characteristics: obvious colony is formed on the culture medium, the diameter is between 0.3 and 2mm, the front shape is circular, the middle part is convex, the edge is neat, the colony is slightly white and opaque, and the surface is moist and smooth. Growth characteristics: the strain is facultative anaerobe, the optimal growth temperature is 36-38 ℃, the optimal growth pH value is 6.6-7.0, the strain grows well in a culture medium containing glucose, and the strain can enter the late logarithmic phase or the early stable phase within 16-24 hours.
In one embodiment of the invention, the composition further contains prebiotics, and the mass ratio of the complex microbial inoculum to the prebiotics is as follows: (0.02-20): (0-1) in a proportion other than 0.
In one embodiment of the present invention, the number of viable bacteria in the complex microbial inoculum is at least: 1 x 10 6 CFU/mL or 1X 10 6 CFU/g。
In one embodiment of the invention, in the complex microbial inoculum, the bifidobacterium longum NSP002, the bacteroides xylanisolvens NSP003 and the enterococcus faecium NSP004 are as follows according to the viable bacteria ratio: (0.01-100): (0.01-100): (0.01-100) in proportion.
In one embodiment of the invention, the viable count of the bifidobacterium longum NSP002, the bacteroides xylanisolvens NSP003 and the enterococcus faecium NSP004 in the composite microbial inoculum is not less than 1 x 10 6 CFU/mL or 1X 10 6 CFU/g, and the addition ratio is 1:1:1 according to the volume ratio.
In one embodiment of the invention, the prebiotic is pectin having a degree of esterification of less than 50%.
The second object of the present invention is to provide a product containing the above composition.
In one embodiment of the invention, the product is a food product, a nutritional supplement, a nutraceutical, a pharmaceutical composition, a feed additive.
In one embodiment of the invention, the ratio of viable bacteria in the product is: (0.01-100): (0.01-100): (0.01-100) in proportion.
In one embodiment of the invention, the product further contains prebiotics, and the mass ratio of the complex microbial inoculum to the prebiotics is (0.02-20): (0-1) in the amount of the surfactant.
In one embodiment of the invention, the total viable count of bifidobacterium longum NSP002, bacteroides xylanisolvens NSP003, enterococcus faecium NSP004 is at least: 1X 10 6 CFU/mL or 1X 10 6 CFU/g。
In one embodiment of the invention, the prebiotic is pectin having a degree of esterification of less than 50%.
In one embodiment of the present invention, the product comprises Bifidobacterium longum (Bifidobacterium longum) NSP002, Bacteroides xylanisolvens (Bacteroides xylanisolvens) NSP003, Enterococcus faecium (Enterococcus faecium) NSP004, and further comprises acceptable carriers and/or adjuvants.
In one embodiment of the present invention, the product comprises Bifidobacterium longum (Bifidobacterium longum) NSP002, Bacteroides xylanisolvens (Bacteroides xylanisolvens) NSP003, Enterococcus faecium (Enterococcus faecalis) NSP004 and pectin, and further comprises acceptable carriers and/or adjuvants.
The third purpose of the invention is to provide the application of the composition in the preparation of products for preventing, assisting in treating, and/or repairing and improving the symptoms of the ulcerative colitis.
In one embodiment of the present invention, the composition is a complex microbial preparation, which contains Bifidobacterium longum (Bifidobacterium longum) NSP002, Bacteroides xylanisolvens (Bacteroides xylanisolvens) NSP003, Enterococcus faecium (Enterococcus faecium) NSP 004; in the composite microbial inoculum, bifidobacterium longum NSP002, bacteroides xylanisolvens NSP003 and enterococcus faecium NSP004 are as follows according to the viable count ratio: (0.01-100): (0.01-100): (0.01-100) in a proportion.
In one embodiment of the invention, the composition further contains prebiotics, and the mass ratio of the complex microbial inoculum to the prebiotics is (0.02-20): (0-1) in the amount of the surfactant.
In one embodiment of the present invention, the number of viable bacteria in the complex microbial inoculum is at least: 1 x 10 6 CFU/mL or 1X 10 6 CFU/g。
In one embodiment of the invention, the prebiotic is pectin having a degree of esterification of less than 50%.
In one embodiment of the invention, the product is a food product, a nutritional supplement, a nutraceutical, a pharmaceutical composition, a feed additive.
In one embodiment of the present invention, the dosage forms of the food, nutritional supplement, nutraceutical, pharmaceutical composition, feed additive include, but are not limited to, granules, capsules, tablets, pills, oral liquid.
In one embodiment of the invention, the food, nutritional supplement, health product, pharmaceutical composition, feed additive comprises Bifidobacterium longum (Bifidobacterium longum) NSP002, Bacteroides xylanisolvens (Bacteroides xylanisolvens) NSP003, Enterococcus faecium (Enterococcus faecium) NSP004, and further comprises acceptable carriers and/or adjuvants.
In one embodiment of the invention, the food, nutritional supplement, health product, pharmaceutical composition, feed additive comprises Bifidobacterium longum (Bifidobacterium longum) NSP002, Bacteroides xylanisolvens (Bacteroides xylanisolvens) NSP003, Enterococcus faecium (Enterococcus faecium) NSP004, pectin, and acceptable carriers and/or adjuvants.
In one embodiment of the present invention, the acceptable auxiliary materials include one or more of a thickening agent, an antioxidant, an acid-base regulator, an emulsifier, a preservative, a filler, a binder, a wetting agent, a disintegrating agent, a lubricant, and a flavoring agent.
In one embodiment of the invention, the filler is starch, sucrose, lactose, calcium sulfate and/or microcrystalline cellulose.
In one embodiment of the invention, the binder is a cellulose derivative, alginate, gelatin and/or polyvinylpyrrolidone.
In one embodiment of the invention, the wetting agent is water, ethanol, starch and/or syrup.
In one embodiment of the invention, the disintegrant is sodium carboxymethyl starch, carboxypropylcellulose, cross-linked carboxymethylcellulose, agar, calcium carbonate and/or sodium bicarbonate.
In one embodiment of the invention, the lubricant is talc, calcium stearate, magnesium stearate, aerosil and/or polyethylene glycol.
In one embodiment of the invention, the flavoring agent is simple syrup, sucrose, lecithin, orange peel syrup, cherry syrup, lemon, anise, peppermint oil, sodium alginate, gum arabic, gelatin, methyl cellulose, sodium carboxymethyl cellulose, citric acid, tartaric acid, and/or sodium bicarbonate.
The fourth purpose of the invention is to provide the application of the composition and the preparation containing the composition in preparing foods, nutritional supplements, health products, pharmaceutical compositions and feed additives for preventing, assisting in treating, repairing and improving the symptoms of ulcerative colitis.
Advantageous effects
The composition of the invention has the function of relieving ulcerative colitis, and is specifically characterized in that:
1) can improve the disease activity index of the mouse with the ulcerative colitis;
2) can reduce the intestinal permeability of the ulcerative colitis mouse, increase the level of tight junction protein in colon tissues and maintain the integrity of intestinal barriers;
3) can avoid the damage of the colon tissue structure of the ulcerative colitis mouse;
4) can reduce the level of Myeloperoxidase (MPO) in the colon tissue of the mouse with ulcerative colitis and relieve the oxidative damage of the colon tissue.
5) Can increase the level of tumor inhibiting factor TGF-beta in colon tissues of mice with ulcerative colitis and relieve inflammation.
Can increase the total level of short-chain fatty acid produced by the intestinal flora of mice with ulcerative colitis.
Biological material preservation
Bifidobacterium longum (Bifidobacterium longum) NSP002, taxonomic name: bifidobacterium longum has been deposited in the Guangdong province culture Collection at 19.1.2021 with GDMCC No. 61440, and has been deposited at Michelia Tokoro No. 59, 5 th of Dazhou institute of Michelia, Guangzhou province, institute of microbiology, Guangdong province.
A strain of Bacteroides xylanisolvens (Bacteroides xylanisolvens) NSP003 is taxonomically named as: bacteroides xylanisolvens has been deposited at the Guangdong province microorganism culture collection center at 19.1.2021 with the deposit number GDMCC No. 61441, and the deposit address is No. 59, 5, Guangdong province institute of microorganisms, Michelia Tokyo 100, Guangzhou, China.
An Enterococcus faecium (Enterococcus faecium) NSP004 strain which is taxonomically named as: the Enterococcus faecalium has been deposited at 19.1.2021 in the microbial cultures Collection of Guangdong province with the deposit number GDMCC No. 61442, and the deposit address is Michelia Tokyo No. 59, 5 th of Dazhou institute No. 100, Michelia Tokyo, Guangdong province microbial research institute.
Drawings
FIG. 1: the composition intervenes in the change of disease activity index of ulcerative colitis mouse, A is the change of disease activity index at different time, and B is the change of disease activity index after experiment.
FIG. 2: the composition intervenes in the changes in intestinal permeability (a) and in the level of claudin in the colon (B) following ulcerative colitis mice.
FIG. 3: the composition interferes with changes in colon tissue morphology following ulcerative colitis mice.
FIG. 4: the compositions interfere with changes in the level of myeloperoxidase MPO (B) in the colon following ulcerative colitis mice.
FIG. 5 is a schematic view of: the composition interferes with changes in the level of TGF- β in the colon following ulcerative colitis mice.
FIG. 6: the compositions intervene in the changes in the levels of total short chain fatty acids in the colon contents after ulcerative colitis mice.
The model group is a DSS colitis mouse group, NSP002 is a DSS + Bifidobacterium longum NSP002 group, NSP003 is a DSS + bacteroides xylanisolvens NSP003 group, NSP004 is a DSS + enterococcus faecium NSP004 group, the composite microbial inoculum is a DSS + Bifidobacterium longum NSP002+ bacteroides xylanisolvens NSP003+ enterococcus faecium NSP004 group, the composite microbial inoculum + prebiotics are a DSS + Bifidobacterium longum NSP002+ bacteroides xylanisolvens NSP003+ enterococcus faecium NSP004 group + pectin group, the pectin is a DSS + pectin group, and the mesalamine is a DSS + positive drug mesalamine group.
"" indicates a significant difference from the model group (.: p < 0.05;: p < 0.01;: p < 0.001); "#" indicates a significant difference from the normal group (#: p < 0.05; #: p < 0.01; #: p < 0.001).
Detailed Description
Dextran-FITC, referred to in the examples below, was purchased from Sigma-Aldrich, Dextran Sulfate Sodium (DSS) from MPBio, mesalazine granules from pharmaceutical company ltd, love, france.
The pectins referred to in the following examples are pectins with a degree of esterification of less than 50% and are available from CPKelco.
The media involved in the following examples:
preparation of a bifidobacterium liquid culture medium: preparing MRS culture medium (such as product of Qingdao Haibo biotechnology Co., Ltd.), dissolving in distilled water, adding 0.5-1g/L cysteine hydrochloride, mixing well, adjusting pH to 6.6-7.0, sterilizing at 121 deg.C for 15-20min to obtain the Bacillus bifidus liquid culture medium.
Preparation of a bifidobacterium solid culture medium: adding 1.5-2% of agar according to the formula of the bifidobacterium liquid culture medium, uniformly mixing, adjusting the pH value to 6.6-7.0, and sterilizing at the temperature of 115 ℃ and 121 ℃ for 15-20min to obtain the bifidobacterium solid culture medium.
Preparation of a bacteroides liquid culture medium: preparing a brain heart infusion BHI liquid culture medium (such as a product of Qingdao Haibo biotechnology, Inc.), dissolving in distilled water, adding 1g/L cysteine hydrochloride, 10.002g/L vitamin and 0.01g/L hemin, uniformly mixing, adjusting the pH value to 6.8-7.4, and sterilizing at 121 ℃ for 15-20min to obtain the bacteroides liquid culture medium.
Preparation of Bacteroides solid culture medium: adding agar of 1.5-2% according to the formula of Bacteroides liquid culture medium, mixing well, adjusting pH to 6.8-7.4, and sterilizing at 121 deg.C for 15-20 min. And (3) cooling to 50-60 ℃, and adding sterile vancomycin with the final concentration of 0.0075 per mill and kanamycin with the final concentration of 0.1 per mill into the culture medium to obtain the bacteroides solid culture medium.
Preparation of enterococcus liquid culture medium: preparing a brain heart infusion BHI liquid culture medium (such as a product of Qingdao Haibobo biotechnology limited), dissolving the mixture in distilled water, uniformly mixing, then adjusting the pH value to be 6.8-7.2, and sterilizing at the temperature of 121 ℃ of 115-.
Preparation of enterococcus solid medium: adding 1.5-2% of agar according to the formula of the enterococcus liquid culture medium, uniformly mixing, adjusting the pH value to 6.8-7.2, and sterilizing at the temperature of 115 ℃ for 15-20min to obtain the enterococcus solid culture medium.
The solutions referred to in the following examples were prepared as follows:
the preparation method of the 3% DSS sterilized distilled water comprises the following steps: weighing 30g of DSS, dissolving in distilled water, and sterilizing for 15min at 121 ℃ by adopting a high-pressure steam sterilization pot to obtain the 3% DSS sterilized distilled water.
The detection methods referred to in the following examples are as follows:
the criteria for the Disease Activity Index (DAI) are shown in table 1:
TABLE 1 Disease Activity Index (DAI) score
Note: normal feces: forming excrement; loosening feces: pasty, semi-formed feces that do not adhere to the anus; sparse feces: thin watery feces adhered to the anus.
Example 1: isolation screening of Bifidobacterium longum (Bifidobacterium longum) NSP002, Bacteroides xylanisolvens (Bacteroides xylosanisolvens) NSP003 and Enterococcus faecium (Enterococcus faecalis) NSP004
1. Sample collection
Collecting healthy human body feces samples in the south Chang district of Jiangxi, placing the samples in a storage tube, storing the samples in a heat preservation box with an ice bag, bringing the samples back to a laboratory, and rapidly placing the samples in a refrigerator at the temperature of-80 ℃ for separation and screening.
2. Separation and purification of bifidobacterium longum, bacteroides xylanisolvens and enterococcus faecium
(1) Gradient dilution of the fecal sample: taking about 1g of the feces sample collected in the step 1 in a sterile anaerobic environment, adding 9mL of normal saline to obtain a first gradient diluent, sucking 1mL of the first gradient diluent into 9mL of normal saline to obtain a second gradient diluent, and repeating the steps to prepare 9 gradient diluents;
(2) coating culture:
respectively sucking 100 μ L of the above gradient dilution solutions, respectively placing on Bacillus bifidus solid culture medium, coating, and culturing at 37 deg.C under anaerobic condition for 48 hr to obtain Bacillus bifidus dilution coating plate;
respectively sucking 100 mu L of all the gradient diluents, respectively placing the gradient diluents on a bacteroides solid culture medium, coating, and then culturing for 48h under an anaerobic condition at 37 ℃ to obtain a bacteroides dilution coating plate;
respectively sucking 100 mu L of all the gradient diluents, respectively placing the gradient diluents on an enterococcus solid culture medium, coating, and culturing at 37 ℃ for 48 hours under an anaerobic condition to obtain an enterococcus dilution coating plate;
(3) and (3) purification and culture:
selecting colonies with different forms on each solid culture medium, and streaking until pure single colonies with neat edges, slight whiteness, opacity, wet and smooth surfaces and consistent forms are obtained; respectively picking pure colonies on a bifidobacterium solid culture medium, a bacteroides solid culture medium and an enterococcus solid culture medium, inoculating the pure colonies on 5mL of corresponding liquid culture media (a bifidobacterium liquid culture medium, a bacteroides liquid culture medium and an enterococcus liquid culture medium), and culturing for 24h under an anaerobic condition at 37 ℃ to respectively obtain a bifidobacterium purified culture solution, a bacteroides purified culture solution and an enterococcus purified culture solution.
3. Strain preservation and identification
And (3) respectively centrifuging the purified culture solution of the bifidobacterium, the purified culture solution of the bacteroides and the purified culture solution of the enterococcus obtained in the step (2) for 10min at 8000rpm, and discarding the supernatant to respectively obtain bifidobacterium thalli, bacteroides thalli and enterococcus thalli which are respectively named as NSP002, NSP003 and NSP 004. PCR was performed with bacterial 16S rDNA primers (see Table 2).
TABLE 2 primer names
After the PCR product is confirmed by nucleic acid electrophoresis analysis, the amplified product is sent to a company for sequencing, 16S rDNA sequences of NSP002, NSP003 and NSP004 are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, and the sequencing result is compared with the sequences in an NCBI database for analysis; the comparison result shows that the strain with the number NSP002 is Bifidobacterium longum (Bifidobacterium longum) and is named as Bifidobacterium longum (Bifidobacterium longum) NSP002, the strain with the number NSP003 is Bacteroides xylanisolvens (Bacteroides xylanisolvens) and is named as Bacteroides xylanisolvens NSP003, and the strain with the number NSP004 is Enterococcus faecium (Enterococcus faecium) and is named as Enterococcus faecium NSP 004.
Example 2: influence of composition on pathological characterization indexes of ulcerative colitis mouse
The method comprises the following specific steps:
1. preparing a compound microbial inoculum cryopreservation agent:
(1) strain culture:
streaking the bifidobacterium longum NSP002 obtained in the embodiment 1 on a bifidobacterium solid culture medium, after a single colony grows out, selecting the single colony and inoculating the single colony to a bifidobacterium liquid culture medium, and carrying out anaerobic culture at 37 ℃ for 18-24 h to prepare a bifidobacterium longum NSP002 seed solution;
streaking the bacteroides xylanisolvens NSP003 obtained in the example 1 on a bacteroides solid culture medium, after a single colony grows out, selecting the single colony and a bacteroides liquid culture medium, and carrying out anaerobic culture at 37 ℃ for 18-24 h to prepare a bacteroides xylanisolvens NSP003 seed solution;
streaking the enterococcus faecium NSP004 obtained in the embodiment 1 on an enterococcus solid culture medium, after a single colony grows out, selecting the single colony and inoculating the single colony to an enterococcus liquid culture medium, and carrying out anaerobic culture at 37 ℃ for 18-24 h to prepare the enterococcus faecium NSP004 seed liquid.
(2) Preparing a protective agent:
preparing 20-25% of glycerol solution by using distilled water, adding 0.1% of cysteine hydrochloride by mass, and sterilizing at 121 ℃ for 15min to obtain the protective agent.
(3) Preparation of the cryopreservation agent:
respectively centrifuging the bifidobacterium longum NSP002 seed liquid, the bacteroides xylanisolvens NSP003 seed liquid and the enterococcus faecium NSP004 seed liquid prepared in the step (1) at 4 ℃ at 5000rpm for 10min, and taking precipitates to be bifidobacterium longum NSP002 thallus, bacteroides xylanisolvens NSP003 thallus and enterococcus faecium NSP004 thallus;
respectively washing the Bifidobacterium longum NSP002, Bacteroides xylanolyticus NSP003 and enterococcus faecium NSP004 with sterile PBS (pH 7.2) for 1-2 times, respectively re-suspending with the protective agent prepared in step (2) until the bacterial liquid concentration is 5.0 × 10 8 CFU/mL, and storing at-80 deg.C to obtain bacterial concentrations of 5.0 × 10 8 CFU/mL Bifidobacterium longum NSP002 cryopreservation agent with bacterial concentration of 5.0 × 10 8 CFU/mL bacteriodes xylanisolvens NSP003 cryopreservation agent with bacterial concentration of 5.0 × 10 8 CFU/mL enterococcus faecium NSP004 cryopreservation agent.
Preparing a compound microbial inoculum group cryopreservation agent:
respectively washing the Bifidobacterium longum NSP002, Bacteroides xylanolyticus NSP003 and enterococcus faecium NSP004 with sterile PBS (pH 7.2) for 1-2 times, respectively re-suspending with the protective agent prepared in step (2) until the bacterial liquid concentration is 5.0 × 10 8 CFU/mL, mixing according to the volume ratio of 1:1:1, and storing at-80 ℃ for later use; the concentrations of Bifidobacterium longum NSP002, Bacteroides xylanisolvens NSP003 and enterococcus faecium NSP004 in the mixed bacterial liquid reach 5.0 × 10 8 And (5) CFU/mL to obtain the cryopreservation agent of the composite microbial inoculum.
2. Preparation of pectin solution
Weighing pectin (such as CPKelco) with esterification degree lower than 50% according to mouse weight, and dissolving in water to obtain pectin solution. The gavage dose is 300mg pectin per kilogram mouse body weight, the gavage volume is 0.2mL, and the gavage is prepared for use.
3. Preparation of mesalazine solution
And weighing mesalazine granules according to the weight of the mouse, and dissolving the mesalazine granules in a sodium carboxymethylcellulose aqueous solution with the mass fraction of 5% to obtain the mesalazine solution. The intragastric dose is 300mg mesalazine per kilogram of mouse body weight, and the intragastric volume is 0.2 mL.
4. Animal experiment method
90 healthy male C57BL/6 mice at 5 weeks of age were randomly assigned to nine groups: the composition comprises a normal group, an ulcerative colitis group (a model group), a DSS + bifidobacterium longum NSP002 group (an NSP002 group), a DSS + bacteroides xylanisolvens NSP003 group (an NSP003 group), a DSS + enterococcus faecium NSP004 group (an NSP004 group), a DSS + bifidobacterium longum NSP002+ bacteroides xylanisolvens NSP003+ enterococcus faecium NSP004 group (a composite microbial agent group), a DSS + bifidobacterium longum NSP002+ bacteroides xylanisolvens NSP003+ enterococcus faecium NSP004 group + a pectin group (a composite microbial agent + a probiotic group), a DSS + pectin group (a pectin group), and a DSS + a positive drug mesalazine group (a mesalazine group). Each group contained 10 mice.
The mice are raised in a standardized laboratory with the temperature of 25 +/-2 ℃, the relative humidity of 50 +/-5 percent and the illumination of 12h for darkness, the experiment begins for 14 days after the mice are fed normally in an adaptive manner, and the mice are freely fed with standard feed in the whole experiment.
The specific process is as follows:
normal group: on day 1-14, freely drinking sterilized distilled water;
intervention treatment experimental process:
ulcerative colitis group (model group): freely drinking sterilized distilled water on days 1-7, and freely drinking sterilized distilled water containing 3% DSS on days 8-14;
DSS + bifidobacterium longum NSP002(NSP002 group): freely drinking sterilized distilled water on days 1-7, and freely drinking sterilized distilled water containing 3% DSS on days 8-14; and, gavage once daily on days 1-14 for 0.2mL of Bifidobacterium longum NSP002 cryopreservation agent (5.0 × 10) prepared in step 1 8 CFU/mL);
DSS + bacteroides xylanolyticus NSP003(NSP003 group): freely drinking sterilized distilled water on days 1-7, and freely drinking sterilized distilled water containing 3% DSS on days 8-14; and, gavage once daily on days 1-14 0.2mL of Bacteroides xylanisolvens NSP003 cryopreservative agent (5.0 × 10) prepared in step 1 8 CFU/mL);
DSS + enterococcus faecium NSP004(NSP004 group): freely drinking sterilized distilled water on days 1-7, and freely drinking sterilized distilled water containing 3% DSS on days 8-14; and, gavage once daily on days 1-14 with 0.2mL of enterococcus faecium NSP004 cryopreservation agent prepared in step 1 (5.0 × 10) 8 CFU/mL);
DSS + Bifidobacterium longum NSP002+ xyloglucanBacteroides NSP003+ enterococcus faecium NSP004 group (composite microbial inoculum group): freely drinking sterilized distilled water on days 1-7, freely drinking sterilized distilled water containing 3% DSS on days 8-14, and intragastrically administering once a day on days 1-14 for 0.2mL of the composite microbial inoculum cryopreservation agent (5.0 × 10) prepared in step 1 8 CFU/mL Bifidobacterium longum NSP002, 5.0 × 10 8 CFU/mL Bacteroides xylanolyticus NSP003 and 5.0 × 10 8 CFU/mL enterococcus faecium NSP 004);
DSS + bifidobacterium longum NSP002+ bacteroides xylanolyticus NSP003+ enterococcus faecium NSP004 group + pectin group (complex bacterial agent + prebiotic group): freely drinking sterilized distilled water on days 1-7, freely drinking sterilized distilled water containing 3% DSS on days 8-14, and intragastrically administering once a day on days 1-14 for 0.2mL of the composite microbial inoculum cryopreservation agent (5.0 × 10) prepared in step 1 8 CFU/mL Bifidobacterium longum NSP002, 5.0 × 10 8 CFU/mL Bacteroides xylanolyticus NSP003 and 5.0X 10 8 CFU/mL enterococcus faecium NSP004) and 0.2mL of the pectin solution prepared in step 2 (300mg/kg bw);
DSS + pectin group (pectin group): freely drinking sterilized distilled water on days 1-7, freely drinking sterilized distilled water containing 3% DSS on days 8-14, and intragastrically administering 0.2mL of the pectin solution prepared in step 2 (300mg/kg bw) once a day on days 1-14;
DSS + mesalazine (positive drug control, mesalazine group): and (3) freely drinking sterilized distilled water on days 1-7, freely drinking sterilized distilled water containing 3% DSS on days 8-14, and performing intragastric administration once a day on days 1-14 to obtain 0.2mL of the mesalamine solution (300mg/kg bw) prepared in the step 3.
Mice were sacrificed after the end of the experiment, the status of each group of mice was monitored and recorded daily during the experiment, the body weight of the mice was recorded, and Disease Activity Index (DAI) was calculated according to the criteria in table 1.
4. Results of the experiment
As shown in fig. 1A to 1B, the DAI value of the model group mice rose from 0 (normal group) to 2.76(p <0.001) after 7 consecutive days of drinking of sterilized distilled water containing 3% DSS. After the complex microbial inoculum is dried, the DAI value is reduced to 2.30(p is less than 0.05). After combined dry prediction of the complex microbial inoculum and the pectin prebiotics, the DAI value is remarkably reduced to 2.10(p is less than 0.001), and is lower than that of an NSP002 group (2.39), an NSP003 group (2.40), an NSP004 group (2.44), an isolated pectin dry prediction group (2.21) and a positive drug mesalazine group (2.38) of an isolated strain dry prediction group. The results show that the composition can reduce the disease activity index, improve the pathological characterization index of the ulcerative colitis, and has better effect than the gavage prebiotics, the intestinal bacteria and the clinical drug group.
Example 3: effect of the composition on the intestinal Barrier in ulcerative colitis mice
Experimental methods As in example 2, all groups of mice were fasted starting 6h before sacrifice, and after fasting by gastric lavage of 600mg/kg mouse body weight Dextran-FITC 4h before sacrifice, all mice were sacrificed and serum and colon tissue samples were collected. The FITC content in serum was measured with a fluorescence spectrophotometer with excitation light 485nm and emission light 535 nm. The content of claudin (Occludin) in the colon tissue was measured by using an ELISA kit (product of Nanjing Senega Biotech Co., Ltd.).
As shown in FIG. 2A, continuous drinking of 3% DSS-containing sterilized distilled water for 7 days significantly increased the Dextran-FITC content in mouse serum from 0.06mg/L (normal group) to 1.09mg/L (model group, p < 0.001). After the combined dry pretreatment of the complex microbial inoculum and the pectin prebiotics, the content of Dextran-FITC in serum is remarkably reduced to 0.53mg/L (p <0.05), and after the combined dry pretreatment of the complex microbial inoculum, the content of Dextran-FITC in serum is reduced to 0.35mg/L (p <0.01), which is lower than that of an NSP002 group (0.49mg/L), an NSP003 group (0.63mg/L), an NSP004 group (0.57mg/L), an individual pectin dry pretreatment group (0.48mg/L) and a positive drug mesalazine group (0.78mg/L) of an individual strain dry pretreatment group.
Meanwhile, as shown in fig. 2B, the content of claudin (Occludin) in the colon of the mice in the model group was significantly reduced from 110.64pg/mL (normal group) to 75.14pg/mL (p < 0.001). After the dry prediction of the complex microbial inoculum, the increase of the Occludin content in the colon is 90.14pg/mL (p <0.05), and after the combined dry prediction of the complex microbial inoculum and the pectin prebiotics, the increase of the Occludin content in the colon is 103.75pg/mL (p <0.001), which is higher than that of an NSP002 group (102.25pg/mL), an NSP003 group (93.80pg/mL), an NSP004 group (87.16pg/mL), an individual pectin dry prediction group (101.11pg/mL) and a positive drug mesalamine group (87.38pg/mL) in an individual strain intervention group.
The results show that the composition can reduce the intestinal permeability of the ulcerative colitis mouse, adjust the level of tight connexin upwards, maintain the integrity of the intestinal barrier, and has better effect than the gavage prebiotics, the intestinal bacteria and the clinical medicine group.
Example 4: effect of the composition on colon histopathology in ulcerative colitis mice.
In the same manner as in example 2, all mice were sacrificed after the experiment, colon tissue samples were collected, and the pathological structure changes of the colon tissue of the mice were observed by hematoxylin-eosin staining method in combination with microscope, and the experimental results are shown in fig. 3.
The results show that the epithelial cell structure of the colon mucosa of the normal mice is intact, the colon villus and crypt structure are intact, and no ulcer or damage exists. The mice in the model group can show obvious damage of intestinal mucosa, cytoplasma looseness, infiltration of a large number of inflammatory cells, damage of colon villi and disappearance of intestinal crypts. Intervention of the compound microbial inoculum can effectively reduce inflammatory cell infiltration and maintain the integrity of intestinal mucosa epithelial cells. Through the combined dry prognosis of the compound microbial inoculum and the pectin prebiotics, inflammatory cell infiltration can be effectively reduced, the arrangement of colon tissue cells is more compact, the colon tissue morphology is close to that of a normal group, and the improvement effect is obviously superior to that of an NSP002 group, an NSP003 group, an NSP004 group, an independent pectin dry-prognosis group and a positive drug mesalazine group of an independent strain dry-prognosis group.
Example 5: effect of the composition on the level of oxidative stress in mice with ulcerative colitis.
The experimental procedure was the same as in experimental example 2, all mice were sacrificed after the experiment, and colon tissue samples were collected. The myeloperoxidase MPO content in colon was measured by ELISA kit (product of Nanjing Senega Biotech Co., Ltd.).
As shown in FIG. 4, the MPO content in the colon of the model mouse significantly increased from 8.91ng/L (normal group) to 13.82ng/L (p < 0.001). After the drying of the complex microbial inoculum, the MPO content in the colon is reduced to 10.03ng/L (p is less than 0.05). Through the combined dry prognosis of the complex microbial inoculum and the pectin prebiotics, the MPO content in the colon is remarkably reduced to 7.62ng/L (p is less than 0.001), which is lower than that of an NSP002 group (8.64ng/L), an NSP003 group (9.12ng/L), an NSP004 group (12.39ng/L), an individual pectin dry-predicted group (8.09ng/L) and a positive drug mesalazine group (9.74ng/L) of an individual strain dry-predicted group.
The results show that the composition can reduce the oxidative stress level of the ulcerative colitis mouse, so that systemic and tissue and organ oxidative damage related to colitis is relieved, and the effect is superior to that of gavage prebiotics, intestinal bacteria and clinical medicine groups.
Example 6: effect of the composition on the level of inflammatory factors in ulcerative colitis mice.
The experimental procedure was the same as in experimental example 2, all mice were sacrificed after the experiment, and colon tissue samples were collected. The TGF-. beta.content of the colon was determined using an ELISA kit (product of Nanjing Senega Biotech Co., Ltd.).
As shown in FIG. 5, the TGF-beta content in the colon of the mouse in the model group is remarkably reduced from 47.81ng/L (normal group) to 21.79ng/L (p < 0.001). After the dry prognosis of the complex microbial inoculum, the content of TGF-beta in the colon is increased to 44.11ng/L (p is less than 0.001), and after the combined dry prognosis of the complex microbial inoculum and pectin prebiotics, the content of TGF-beta in the colon is obviously increased to 38.40ng/L (p is less than 0.01), which is higher than that of an NSP002 group (30.29ng/L), an NSP003 group (29.72ng/L) and an NSP004 group (31.78ng/L) of a single strain intervention group. And the TGF-beta content of the compound microbial inoculum group is higher than that of a single pectin intervention group (40.29ng/L) and a positive drug mesalazine group (40.58 ng/L).
The results show that the composition can obviously increase the level of tumor inhibition factors in colon tissues of ulcerative colitis mice so as to relieve colonic inflammation, and the effect is superior to that of gastric perfusion prebiotics, intestinal bacteria and clinical drug groups.
Example 7: the composition has effect on short chain fatty acid production of mouse intestinal flora with ulcerative colitis.
The experimental procedure was the same as in experimental example 2, and all mice were sacrificed after the experiment. A100 mg sample of colon contents was collected, homogenized by adding 0.8mL of saturated physiological saline, centrifuged at 14000rpm at 4 ℃ for 15min, and the supernatant was filtered through a 0.22 μm aqueous filter head. Taking 200 mu L of supernatant after passing through the membrane, adding 0.1mL of 50% (v/v) sulfuric acid and 400 mu L of anhydrous ether, standing for 2min after vortexing, centrifuging for 1min at 14000rpm at 4 ℃, taking the supernatant, and detecting the content of total short chain fatty acids including acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid and isovaleric acid by using an Agilent 7890B gas chromatograph.
As shown in FIG. 6, the total short-chain fatty acid content of colon contents of model group mice was significantly reduced from 0.5944mmol/g (normal group) to 0.3992mmol/g (p < 0.01). After the drying of the composite microbial inoculum, the content of the total short-chain fatty acid is increased to 0.5176 mmol/g. After the combined dry pretreatment of the compound microbial inoculum and the pectin prebiotics, the content of total short-chain fatty acid is remarkably increased to 0.5692 mmol/g (p is less than 0.05), which is higher than that of an NSP002 group (0.5429mmol/g), an NSP003 group (0.4746 mmol/g), an NSP004 group (0.5406mmol/g), an individual pectin dry pretreatment group (0.5588mmol/g) and a positive drug mesalazine group (0.5390 mmol/g).
The results show that the composition can remarkably increase the content of short-chain fatty acid in the intestinal contents of mice with ulcerative colitis, so as to adjust the intestinal microecological health, and the effect is superior to that of the gavage prebiotics, the intestinal bacteria and the clinical medicine group.
Example 8: use of a composition
The composition can be used for preparing capsule products, and the capsule products are prepared by the following specific steps:
(1) respectively inoculating the purified culture solution of the bifidobacterium longum NSP002, the purified culture solution of the bacteroides xylanisolvens NSP003 and the purified culture solution of the enterococcus faecium NSP004 obtained in the example 1 into corresponding culture media in the inoculation amount of 3 percent (v/v), culturing for 36h at 37 ℃, centrifuging at 6000 rpm for 20min, and collecting bacterial sludge; after the bacterial sludge is cleaned by using sterile water, the bacterial sludge is resuspended by using skim milk and the three bacterial liquids are mixed until the concentrations of the bifidobacterium longum NSP002, the bacteroides xylanisolvens NSP003 and the enterococcus faecium NSP004 are all 2 multiplied by 10 10 CFU/mL to obtain a suspension;
(2) adding the suspension prepared in the step (1) into a sodium alginate solution with the concentration of 3 percent to the concentration of 2 multiplied by 10 9 After CFU/mL, fully stirring to uniformly disperse the cells of the bifidobacterium longum NSP002, the bacteroides xylanisolvens NSP003 and the enterococcus faecium NSP004 in the sodium alginateObtaining a mixed solution in the solution; extruding the mixed solution into a calcium chloride solution with the concentration of 2% to form colloidal particles; standing and solidifying the formed colloidal particles for 30min, and filtering and collecting the colloidal particles; freeze-drying the collected colloidal particles for 48 hours to obtain powder;
(3) and (3) putting the powder prepared in the step (2) and pectin powder into a medicinal capsule together to obtain a capsule product.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that 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.
SEQUENCE LISTING
<110> university of Nanchang
<120> composition with function of relieving ulcerative colitis and application thereof
<130> BAA220394A
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<170> PatentIn version 3.3
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gcggtagggg agactggaat tcccggtgta acggtggaat gtgtagatat cgggaagaac 660
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Claims (10)
1. The composition is a complex microbial inoculum, and the complex microbial inoculum contains Bifidobacterium longum (Bifidobacterium longum) NSP002, Bacteroides xylanisolvens (Bacteroides xylosans) NSP003 and Enterococcus faecium (Enterococcus faecium) NSP 004; in the composite microbial inoculum, bifidobacterium longum NSP002, bacteroides xylanisolvens NSP003 and enterococcus faecium NSP004 are as follows according to the viable count ratio: (0.01-100): (0.01-100): (0.01-100) in proportion;
the bifidobacterium longum NSP002 has been preserved in the microbial strain preservation center of Guangdong province in 2021, 19 months, the preservation address is No. 59 floor 5 of Michelia Tokyo No. 100 of Guangzhou city, and the preservation number is GDMCC No. 61440;
the bacteroides xylanisolvens NSP003 has been preserved in the Guangdong province microorganism culture preservation center at 19.1.1 in 2021, the preservation address is No. 59 floor 5 of Mirabilitum 100 of Guangzhou city, and the preservation number is GDMCC No. 61441;
the enterococcus faecium NSP004 strain is preserved in Guangdong province microorganism culture collection center at 23.1.1.2021, the preservation address is No. 59 floor 5 of Michelia Tokoro 100, Guangzhou city, and the preservation number is GDMCC No. 61442.
2. The composition according to claim 1, further comprising prebiotics, wherein the mass ratio of the complex microbial inoculum to the prebiotics is (0.02-20): (0-1) in the amount of the surfactant.
3. The composition of claim 2, wherein the number of viable bacteria in the complex microbial inoculum is at least: 1X 10 6 CFU/mL or 1X 10 6 CFU/g。
4. The composition of claim 3, wherein the prebiotic is pectin having a degree of esterification of less than 50%.
5. A product comprising the composition of any one of claims 1 to 4.
6. The product of claim 5, wherein the product is any one of a food product, a nutritional supplement, a nutraceutical, a pharmaceutical composition, a feed additive.
7. Use of the composition of any one of claims 1 to 4 in the preparation of a product for preventing, adjunctively treating, and/or repairing and ameliorating symptoms of ulcerative colitis.
8. Use according to claim 7, wherein the product is any of a food product, a nutritional supplement, a nutraceutical, a pharmaceutical composition, a feed additive.
9. The use of claim 8, wherein the food, nutritional supplement, nutraceutical, pharmaceutical composition, feed additive is in a dosage form including, but not limited to, granules, capsules, tablets, pills, oral liquid.
10. Use according to claim 9, wherein the product comprises a composition according to any one of claims 1 to 4, together with acceptable carriers and/or adjuvants.
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CN113797232A (en) * | 2021-10-28 | 2021-12-17 | 南昌大学 | Composition with function of relieving insulin resistance and application thereof |
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CN114164134A (en) * | 2021-09-30 | 2022-03-11 | 东北农业大学 | Bifidobacterium longum subspecies longum with functions of preventing and relieving colitis symptoms and application thereof |
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CN113897302A (en) * | 2021-08-06 | 2022-01-07 | 东北农业大学 | Bifidobacterium capable of relieving colitis and application thereof |
CN114164134A (en) * | 2021-09-30 | 2022-03-11 | 东北农业大学 | Bifidobacterium longum subspecies longum with functions of preventing and relieving colitis symptoms and application thereof |
CN113755409A (en) * | 2021-10-28 | 2021-12-07 | 南昌大学 | Bifidobacterium longum for relieving insulin resistance and application thereof |
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