CN117618472A - Clostridium praecox capable of reducing intestinal gas generation and application thereof - Google Patents
Clostridium praecox capable of reducing intestinal gas generation and application thereof Download PDFInfo
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- CN117618472A CN117618472A CN202311564770.4A CN202311564770A CN117618472A CN 117618472 A CN117618472 A CN 117618472A CN 202311564770 A CN202311564770 A CN 202311564770A CN 117618472 A CN117618472 A CN 117618472A
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- clostridium
- ccfm1204
- intestinal
- prasugrel
- fermentation
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Abstract
The invention discloses clostridium prasugrel capable of reducing intestinal gas generation and application thereof, and belongs to the technical field of microorganisms and medicines. The invention provides an application of clostridium prasugrel CCFM1204 in reducing intestinal gas generation, which is specifically expressed in the following steps: reducing the generation of fermentation by-product gas in the intestinal tract of an adult; lowering the pH of the intestinal tract; improving the content of short chain fatty acids such as acetic acid, propionic acid, butyric acid and the like in the intestinal tract; and (3) regulating the intestinal flora structure of intestinal flatulence people. The clostridium prasugrel CCFM1204 and the active ingredients thereof have good safety and therapeutic application, and can be used for developing probiotic products. Therefore, the clostridium prasugrel CCFM1204 has great application prospect in the aspect of preventing and/or treating intestinal flatulence.
Description
Technical Field
The invention relates to clostridium prasugrel capable of reducing intestinal gas generation and application thereof, and belongs to the technical field of microorganisms and medicines.
Background
In a typical diet and metabolic process of endogenous ingredients in the large intestine, the intestinal microbiota of most healthy people can produce 0.2L-1.5L of gas per day. However, excessive intestinal gas can negatively affect individuals and may also be a symptom of chronic diseases, such as irritable bowel syndrome. The gas exiting the anus is mainly from the colon, and unabsorbed food residues are fermented by colonic bacteria. But even in the same diet, gas production shows a large individual difference. Thus, the amount of gas produced and expelled by the anus has a close relationship with the composition and metabolic activity of the colonic microbiota. It has been pointed out that intestinal flatulence is often accompanied by increased intestinal inflammation and an imbalance in intestinal micro-ecology, as increased by harmful microorganisms, such as Proteus, enterobacteriaceae, clostridium perfringens, etc.
Currently, some patents relate to reducing intestinal gas or ameliorating flatulence, mainly focused on drug therapy, assisted relief using probiotics, etc. Reported medicines comprise Chinese herbal medicine formulas, medicines for improving intestinal peristalsis, enzyme supplements or antibiotics and the like, and for example, patent CN101785845A provides a traditional Chinese medicine composition which can be used for treating intestinal flatulence; patent CN110787155a provides an itopride hydrochloride pharmaceutical composition that can eliminate flatulence by enhancing gastrointestinal motility; patent CN114246833a discloses a lactase composition which can effectively relieve intestinal flatulence caused by lactose intolerance; patent CN106999477a proposes repeated treatment of patients with irritable bowel syndrome with rifaximin, which can sufficiently relieve abdominal distention symptoms. In addition, some patents are directed to certain probiotic species/strains, including typical probiotic strains of lactobacillus, bifidobacterium, and the like. For example, patent CN112075637a provides a probiotic composition comprising bifidobacterium longum, bifidobacterium breve, bifidobacterium infantis, bifidobacterium bifidum and lactobacillus helveticus that reduces the production of intestinal gas in infants. For example, the patent CN116746683A provides a bifidobacterium animalis subspecies of the BAL-28 which can promote digestion and absorption functions, relieve dyspepsia and effectively improve intestinal flatulence symptoms. However, no patent is reported for treatment based on the characteristic flora structure of specific flatulence, and no patent exists for the action of clostridium praecox in relieving flatulence.
Clostridium prasugrel is a gram-negative bacterium belonging to the genus faecium, which is one of the important producers of butyric acid in the intestinal tract, and studies have shown that its abundance is significantly reduced in patients suffering from constipation, celiac disease, inflammatory bowel disease and irritable bowel syndrome. As a next generation probiotic, its related probiotic function has also been reported in patents, for example, patent CN115279383a reports that a combination of clostridium prasugrel and mesalamine or its derivatives can be used for treating gastrointestinal inflammation. Patent CN115093999A provides a clostridium prasugrel CCFM1205 strain which can effectively improve blood lipid disorders. Also, studies have shown that Clostridium praecox can be effective in alleviating nonalcoholic fatty liver disease by reducing glycolysis, pyruvate metabolism, etc. (Nutrients, 2022, 19;14 (14): 2945). However, there is no patent yet related to a clostridium praecox capable of reducing intestinal gas generation, and therefore, it is necessary to provide a clostridium praecox capable of reducing intestinal gas generation, so as to provide an effective target for treating intestinal flatulence.
Disclosure of Invention
The invention provides an application of clostridium prasugrel (Faecalibacterium prausnitzii) CCFM1204 or fermentation broth thereof in preparing medicines, enteral nutrition or probiotic preparations for preventing, relieving, improving and/or treating intestinal flatulence, wherein the clostridium prasugrel CCFM1204 is preserved in the microorganism strain preservation center of Guangdong province at the month 27 of 2022, and the preservation number is GDMCC No:62239, the preservation address is 5 buildings of Guangzhou Mr. Xian Zhonglu 100 college 59; the fermentation broth contains clostridium prasugrel CCFM1204.
In one embodiment of the invention, the amount of clostridium prasugrel CCFM1204 in the pharmaceutical, enteral nutritional or probiotic preparation is not less than 1X 10 9 CFU/mL or 1X 10 9 CFU/g。
In one embodiment of the invention, the probiotic preparation comprises wet cells or lyophilized cells of the clostridium prasugrel CCFM1204.
In one embodiment of the invention, the probiotic preparation contains adjuvants in addition to clostridium prasugrel CCFM1204, including but not limited to excipients or food additives.
In one embodiment of the present invention, the content of the clostridium prasugrel CCFM1204 in the probiotic preparation is not less than 1×10 6 CFU/mL or 1X 10 6 CFU/g。
In one embodiment of the present invention, the preparation method of the microbial preparation comprises: and mixing the cultured clostridium prasugrel CCFM1204 cells with a protective agent.
In one embodiment of the present invention, the protective agent comprises one or more of skimmed milk powder, glycerol, maltodextrin, trehalose, and sodium L-glutamate.
In one embodiment of the invention, the medicament contains the clostridium prasugrel CCFM1204, a medicament carrier and/or a pharmaceutical adjuvant.
In one embodiment of the invention, the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles and/or liposomes.
In one embodiment of the invention, the pharmaceutical excipients comprise one or more of 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 medicament is granules, capsules, tablets, pills or oral liquid.
In one embodiment of the present invention, the effect of reducing intestinal gas production comprises at least one of the following:
(1) Reducing the generation of fermentation by-product gas in the intestinal tract of an adult;
(2) Lowering the pH of the intestinal tract;
(3) Improving the content of short chain fatty acids such as acetic acid, propionic acid, butyric acid and the like in the intestinal tract;
(4) Regulating intestinal flora structure of intestinal flatulence crowd;
the invention also provides an application of the clostridium prasugrel CCFM1204 or the fermentation liquid thereof in reducing the gas content in a culture medium containing fecal suspension; the fermentation broth contains clostridium prasugrel CCFM1204.
In one embodiment of the invention, the application is that clostridium prasugrel CCFM1204 or a fermentation broth thereof is added into a culture medium containing faecal suspension of a person suffering from flatulence for fermentation culture.
In one embodiment of the invention, the preparation method of the faecal suspension for the flatulence population comprises the following steps: fecal samples from the bloated population were collected and Phosphate Buffered Saline (PBS) at pH 7.4 was mixed with fresh fecal samples at a ratio of 1:7-1:10 (w/v) to prepare fecal inoculum.
In one embodiment of the present invention, the clostridium prasugrel CCFM1204 is added in an amount of at least: 10 9 CFU/mL。
In one embodiment of the invention, the fecal suspension is added to the culture medium in an amount of: 10% -15% (v/v)
The invention also provides an application of the clostridium prasugrel CCFM1204 or a fermentation broth thereof in reducing the pH value of a culture medium containing fecal suspension; the fermentation broth contains clostridium prasugrel CCFM1204.
In one embodiment of the invention, the application is that clostridium prasugrel CCFM1204 or a fermentation broth thereof is added into a culture medium containing faecal suspension of a person suffering from flatulence for fermentation culture.
In one embodiment of the invention, the preparation method of the faecal suspension for the flatulence population comprises the following steps: fecal samples from the bloated population were collected and Phosphate Buffered Saline (PBS) at pH 7.4 was mixed with fresh fecal samples at a ratio of 1:7 to 1:10 (w/v) to prepare fecal inoculum.
In one embodiment of the present invention, the clostridium prasugrel CCFM1204 is added in an amount of at least: 10 9 CFU/mL。
In one embodiment of the invention, the fecal suspension is added to the culture medium in an amount of: 10% -15% (v/v)
The invention also provides an application of the clostridium prasugrel CCFM1204 or a fermentation broth thereof in promoting the yield of short-chain fatty acids of intestinal flora, wherein the application is for the purpose of non-disease diagnosis and treatment; the fermentation broth contains clostridium prasugrel CCFM1204.
In one embodiment of the invention, the application is that clostridium prasugrel CCFM1204 or a fermentation broth thereof is added into a culture medium containing faecal suspension of a person suffering from flatulence for fermentation culture.
In one embodiment of the invention, the preparation method of the faecal suspension for the flatulence population comprises the following steps: fecal samples from the bloated population were collected and Phosphate Buffered Saline (PBS) at pH 7.4 was mixed with fresh fecal samples at a ratio of 1:7-1:10 (w/v) to prepare fecal inoculum.
In one embodiment of the present invention, the clostridium prasugrel CCFM1204 is added in an amount of at least: 10 9 CFU/mL。
In one embodiment of the invention, the fecal suspension is added to the culture medium in an amount of: 10% -15% (v/v).
Advantageous effects
1. The invention provides an application of clostridium prasugrel (Faecalibacterium prausnitzii) CCFM1204 in reducing intestinal gas generation, which is specifically expressed in the following steps:
(1) Reducing the generation of fermentation by-product gas in the intestinal tract of an adult;
(2) Lowering the pH of the intestinal tract;
(3) Improving the content of short chain fatty acids such as acetic acid, propionic acid, butyric acid and the like in the intestinal tract;
(4) And (3) regulating the intestinal flora structure of intestinal flatulence people.
2. The clostridium prasugrel CCFM1204 and the active ingredients thereof have good safety and therapeutic application, and can be used for developing probiotic products. Therefore, the clostridium prasugrel CCFM1204 has great application prospect in the aspect of preventing and/or treating intestinal flatulence.
Drawings
Fig. 1: intestinal flatulence population and healthy control group intestinal flora phylum, family and genus horizontal stacking bar chart; wherein: a is a horizontal stacking bar graph of intestinal flora phylum of two groups of people; b is a horizontal stacking bar graph of intestinal flora of two groups of people; c is a horizontal stacking bar graph of intestinal flora of two groups of people.
Fig. 2: intestinal flora difference between intestinal flatulence population and healthy control group belongs to a relative abundance diagram; wherein: a is the relative abundance of bifidobacteria in the intestinal flora of two groups of people; b is the relative abundance of lactobacillus of intestinal flora of two groups of people; c is the relative abundance of the intestinal flora and the faeces bacillus of two groups of people; d is the relative abundance of the intestinal flora of two groups of people, namely the shigella; e is the relative abundance of the intestinal flora of two groups of people, namely the shigella; f is the relative abundance of intestinal flora Ruminococcaceae UCG-010 in two groups of people.
Fig. 3: in vitro fermentation and gas production difference of feces of intestinal tract distention people and healthy control people; wherein: a is intestinal microbial culture medium (GMM); b is simulated chyme medium (SM).
Fig. 4: effect of different groups of strains on the yield of fermentation gas.
Fig. 5: effects of different groups of strains on pH of fermentation broth.
Fig. 6: effects of different groups of strains on short chain fatty acid content.
Fig. 7: influence of different groups of strains on intestinal flora diversity; wherein: a is the influence of different strains on the diversity of flora alpha; b is the effect of different groups of strains on the beta diversity of the flora.
Fig. 8: and (3) analyzing the LefSe of the intestinal flora composition by different strains.
Fig. 9: and (5) flora analysis.
Detailed Description
Chemicals such as tryptone and yeast powder, which are referred to in the following examples, were purchased from the national drug group; the Fast DNA Spin Kit for Feces kit referred to in the examples below was purchased from MP Biomedicals company.
The bifidobacterium breve FFJND2DLM11, bifidobacterium adolescentis FHNFQ48M5, bifidobacterium longum subspecies FJSWXI4MIM1 referred to in the following examples were all deposited in the university of south of the river food institute biotechnology center strain resource library; the bacillus coagulans CCFM1041 and clostridium praecox CCFM1204 are respectively recorded in Chinese patent application texts with publication numbers of CN114908023A and CN 115074276A.
The following examples relate to the following media:
GMM liquid Medium (g/L): 2g/L of peptone, 1g/L of yeast extract, 0.4g/L of glucose, 0.5g/L of cysteine, 1g/L of cellobiose, 1g/L of maltose, 1g/L of fructose, 5g/L of beef extract, 100mL of potassium dihydrogen phosphate (100 mM), 0.00 g/L of magnesium sulfate heptahydrate, 0.4g/L of sodium bicarbonate, 0.08g/L of sodium chloride, 1mL of calcium chloride (0.80%), 11mL of Vitamin K, 1mL of ferrous sulfate (1.44 mM), 1mL of histidine heme solution (0.1%), 80 of Tween, 10mL of ATCC Vitamin Mix (0.1%), ATCC Trace Mineral Mix mL (0.1%).
SM liquid medium (g/L): 2g/L of citrus pectin, 2g/L of xylan, 1g/L of arabinogalactan, 1g/L of guar gum, 1g/L of inulin, 5g/L of soluble potato starch, 4g/L of mucin, 3g/L of acid hydrolyzed casein, 5g/L of peptone water, 5g/L of tryptone, 4.5g/L of yeast extract, 0.8g/L of cysteine, 0.4g/L of bile salt, 0.5g/L of potassium dihydrogen phosphate, 1.5g/L of sodium bicarbonate, 4.5g/L of sodium chloride, 4.5g/L of potassium chloride, 0.6g/L of anhydrous magnesium sulfate, 0.1g/L of calcium chloride dihydrate, 0.2g/L of manganous chloride tetrahydrate, 0.005g/L of ferrous sulfate heptahydrate, 0.05g/L of heme, 80 g/L of tween, and 11 mL/L of vitamin K.
M2GSC liquid medium (g/L): 5g/L of yeast powder, 10g/L of casein peptone, 5g/L of glucose, 2g/L of cellobiose, 2g/L of fructose, 4g/L of sodium bicarbonate, 0.9g/L of sodium chloride, 0.45g/L of monopotassium phosphate, 0.45g/L of dipotassium phosphate, 0.09g/L of magnesium sulfate, 0.09g/L of calcium chloride, 0.5g/L of cysteine, 1.0mg/L of resazurin and 10mL/L of clarified tumor gastric juice.
The screening criteria for intestinal flatulence population and healthy control population as referred to in the examples below were as follows:
before the study started, participants were asked to fill out a questionnaire and the following parameters were evaluated: (a) The number of bowel movements and stool morphology were assessed using a bristol scale; (b) Subjective sensations of bloating (defined as anal venting), abdominal distension (pressure/filling), abdominal distension (increased waist circumference), abdominal distension and abdominal discomfort/pain were assessed using a corresponding 0-10 analog scale; (c) Digestive tract comfort was assessed using a 10-level scale from +5 (satisfactory) to-5 (unsatisfactory).
The exclusion criteria included: major intestinal diseases (ileus, intussusception, colon cancer, colitis), respiratory diseases or infections; antibiotics have been used in the past 2 months; the habit of using probiotics is presented; probiotic products or foods containing probiotics have been used in the past 1 month. Only patients with anus displacement of more than or equal to 5 (10 minutes) and meeting the exclusion standard can participate in the study. Healthy people are gender-matched volunteers without gastrointestinal symptoms.
The specific clinical parameters of the two groups of people are as follows: flatulence (defined as anal exhaust) (7.159 ±0.272vs.2.219±0.209, p < 0.0001); abdominal distension (pressure/filling) (3.125±0.264 vs.6.409±0.316, p < 0.0001); abdominal distension (increased waist circumference) (5.273 ±0.459vs.2.125±0.347, p < 0.0001); subjective feeling of abdominal distension and abdominal discomfort/pain (5.932 ±0.327vs.2.281±0.346, p < 0.0001); digestive tract comfort (-1.364+ -0.361 vs.3.350+ -0.406, p < 0.0001).
Example 1: intestinal flora difference between intestinal flatulence population and healthy control population
The method comprises the following specific steps:
collecting 44 fecal samples from the flatulence population and 32 fecal samples from the healthy control population in total, preserving at-80 ℃, extracting bacterial metagenome of the two groups of fecal samples by using a Fast DNA Spin Kit for Feces kit, carrying out PCR amplification on the 16s V3-V4 region sequence, and carrying out 1.5% agarose gel electrophoresis on all the obtained PCR products. The target gene was recovered and purified from agarose gel containing the target band using a gel recovery kit, tested on a Illumina Miseq PE300 platform, and the next data was analyzed by QIIMEII software.
The results show that there is a significant difference between intestinal flora composition in the flatulence population and healthy control population as seen in figures 1 and 2. Specifically, at the door level, it is mainly composed of the phylum firmicutes and bacteroides; at the department level, peptostococcuae, egberthellac ae, christensenelaceae and clostridium in the flatus group increased over the healthy control group; at the genus level, the faecal rod genus was significantly reduced compared to the healthy control group; the escherichia-shigella, enterobacter and ruminococcus UCG-010 are significantly increased; typical probiotic bifidobacteria have no significant differences in the two groups of people, whereas lactobacillus is significantly increased in the flatulence group.
Example 2: intestinal tract flatulence crowd and healthy control crowd excrement in-vitro fermentation and gas production difference
The method comprises the following specific steps:
1. in-vitro fermentation and gas production of faeces of intestinal tract distention people and healthy control people
Collecting 20 fecal samples from the flatulence population and the healthy control population respectively, mixing Phosphate Buffer Solution (PBS) with pH of 7.4 with fresh fecal samples according to the ratio of 1:7 (w/v), and preparing fecal inoculation liquid; a20 mL sealed anaerobic fermentation tube with a rubber plug is adopted, 13.5mL intestinal microorganism culture medium (GMM) or simulated colon chyme culture medium (SM) is added into each tube, feces are inoculated according to the concentration of 10% (v/v), the inoculation amount is 1.5mL, and the fermentation is carried out by shaking for 48 hours at 37 ℃. The experiment adopts two culture mediums to carry out in vitro verification experiments of gas production, and aims to verify the gas production of intestinal microorganisms of two groups of people under different nutrient conditions. All of the above operations are performed at an anaerobic workstation.
After 48h fermentation, the fermentation gas and fermentation broth were collected.
The detection method of the gas volume comprises the following steps: measured by inserting a sterile needle into the rubber cap of each container. Pressure builds up in the headspace pushing the syringe barrel to record the volume. After each measurement, the headspace of each container was allowed to equilibrate with the atmosphere in the anaerobic tank. The gas volumes at each time point were measured using the same container. 3 gas production experiments were performed for each sample.
2. Experimental results
The results are shown in table 1 below:
table 1: in-vitro fermentation gas production rate of stool samples of healthy people and intestinal tract flatulence people
Data: mean ± standard deviation
As can be seen from table 1 and fig. 3, in vitro fermentation experiments were performed to further verify whether the flatulence population produced more gas. The results show that fecal samples from both groups of people produced higher amounts of gas in the simulated chyme medium (SM), consistent with the rich nutrition in this medium and the rich fermentable carbon source. Importantly, in both media, the stool samples of the flatulence population fermented to significantly higher amounts of gas than the healthy control group, indicating that the intestinal flora of the flatulence population will produce more gas under the same substrate conditions.
3. The gas production of each sample was analyzed for Spearman correlation with the flora results in example 1.
The results show that: as can be seen from fig. 4, the faecalcibacterium is significantly inversely correlated with the gas yield (p=0.022), indicating that the higher the faecalcibacterium abundance, the lower the gas yield.
Example 3: effect of Clostridium praecox CCFM1204 on gas yield
The method comprises the following specific steps:
1. culture of bifidobacterium breve FFJND2DLM11, bifidobacterium adolescentis FHNFQ48M5, bifidobacterium longum subspecies infantis FJSWXI4MIM1, bacillus coagulans CCFM1041, clostridium praecox CCFM1204 strain:
inoculating Bifidobacterium breve FFJND2DLM11, bifidobacterium adolescentis FHNFQ48M5, bifidobacterium longum subspecies FJSWXI4MIM1, and Bacillus coagulans CCFM1041 in an inoculum size of 2% (v/v) into MRS liquid culture medium, respectively, and culturing in an anaerobic tank (80% N) 2 ,10％CO 2 ,10％H 2 ) Culturing at 37deg.C for 24 hr, sequentially transferring to 3 rd generation according to 2% (v/v) inoculum size, centrifuging at 6000g for 3min to collect bacterial mud, washing with 0.1M PBS (pH 7.2, containing 0.05% cysteine) and re-suspending to obtain bacterial concentration of 1×10 9 CFU/mL bacterial suspension, immediately on the day.
Inoculating Clostridium praecox CCFM1204 preserved in-80deg.C environment into M2GSC liquid medium at an inoculum size of 2% (v/v), and culturing in an anaerobic tank (80% N) 2 ,10％CO 2 ,10％H 2 ) Culturing at 37deg.C for 24 hr, sequentially transferring to 3 rd generation according to 2% (v/v) inoculum size, vacuum filtering in anaerobic workstation to obtain bacterial mud, washing with 0.1M PBS (pH 7.2, containing 0.05% cysteine), and re-suspending to obtain bacterial concentration of 1×10 9 CFU/mL bacterial suspension, immediately on the day.
2. Bifidobacterium breve FFJND2DLM11, bifidobacterium adolescentis FHNFQ48M5, bifidobacterium longum subspecies infantis FJSWXI4MIM1, bacillus coagulans CCFM1041, clostridium praecox CCFM1204 co-cultured with intestinal flora, respectively
(1) Collecting 6 faecal samples from the flatulence population, mixing Phosphate Buffer Solution (PBS) with pH of 7.4 with fresh faecal sample according to the ratio of 1:7 (w/v), preparing faecal inoculation liquid, adding 12.15mL of simulated colon chyme culture medium (SM) into each tube by adopting a 20mL sealed anaerobic fermentation tube with a rubber plug, inoculating faeces and bacterial liquid according to the concentration of 10% (v/v), wherein the inoculation amount of faecal inoculation liquid is 1.35mL, and the bacterial suspension (10 9 CFU/mL viable count) was 1.5mL;
in addition, 1.5mL of Phosphate Buffer Solution (PBS) with pH of 7.4 is added to replace the bacterial suspension;
(2) The above-mentioned fermentation tubes were each subjected to fermentation by shaking at 37℃for 48 hours.
All of the above operations are performed at an anaerobic workstation. After 48h fermentation, the fermentation gas was collected.
The gas volume was determined by inserting a sterile needle into the rubber cap of each container. Pressure builds up in the headspace pushing the syringe barrel to record the volume. After each measurement, the headspace of each container was allowed to equilibrate with the atmosphere in the anaerobic tank. The gas volumes at each time point were measured using the same vessel and 4 parallel experiments were performed for each group.
3. Experimental results
The specific results are shown in table 2 below:
table 2: gas production after co-cultivation of different strains with fecal suspension
Data: mean ± standard deviation
As can be seen from table 2 and fig. 5, the addition of the clostridium prasugrel CCFM1204 to the fermentation system reduced the gas production of the flatulence sample (2.65±0.20) compared to the blank (3.83±0.21), and was statistically significant (p < 0.05). Clostridium praecox is used as an important beneficial bacterium in the intestinal tract, has cross-breeding relationship with other beneficial intestinal bacteria, and promotes the proliferation of the beneficial bacterium in the intestinal tract. Metabolites produced by clostridium praecox have anti-inflammatory and pathogenic bacteria inhibiting effects, which inhibit the growth of harmful gas-producing bacteria in the intestinal tract, thereby reducing gas production.
Example 4: effect of Clostridium praecox CCFM1204 on broth pH
The method comprises the following specific steps:
1. culture of bifidobacterium breve FFJND2DLM11, bifidobacterium adolescentis FHNFQ48M5, bifidobacterium longum subspecies infantis FJSWXI4MIM1, bacillus coagulans CCFM1041, clostridium praecox CCFM1204 strain:
bifidobacterium breve FFJND2DLM11 and Bifidobacterium adolescentis preserved in-80deg.C environment respectivelyBacillus bifidus FHNFQ48M5, bifidobacterium longum subspecies infantis FJSWXI4MIM1, bacillus coagulans CCFM1041 was inoculated into MRS liquid medium at an inoculum size of 2% (v/v), in an anaerobic tank (80% N) 2 ,10％CO 2 ,10％H 2 ) Culturing at 37deg.C for 24 hr, sequentially transferring to 3 rd generation according to 2% (v/v) inoculum size, centrifuging at 6000g for 3min to collect bacterial mud, washing with 0.1M PBS (pH 7.2, containing 0.05% cysteine) and re-suspending to obtain bacterial concentration of 1×10 9 CFU/mL bacterial suspension, immediately on the day.
Inoculating Clostridium praecox CCFM1204 preserved in-80deg.C environment into M2GSC liquid medium at an inoculum size of 2% (v/v), and culturing in an anaerobic tank (80% N) 2 ,10％CO 2 ,10％H 2 ) Culturing at 37deg.C for 24 hr, sequentially transferring to 3 rd generation according to 2% (v/v) inoculum size, vacuum filtering in anaerobic workstation to obtain bacterial mud, washing with 0.1M PBS (pH 7.2, containing 0.05% cysteine), and re-suspending to obtain bacterial concentration of 1×10 9 CFU/mL bacterial suspension, immediately on the day.
2. Bifidobacterium breve FFJND2DLM11, bifidobacterium adolescentis FHNFQ48M5, bifidobacterium longum subspecies infantis FJSWXI4MIM1, bacillus coagulans CCFM1041, clostridium praecox CCFM1204 co-cultured with intestinal flora, respectively
(1) Collecting 6 faecal samples from the flatulence population, mixing Phosphate Buffer Solution (PBS) with pH of 7.4 with fresh faecal sample according to the ratio of 1:7 (w/v), preparing faecal inoculation liquid, adding 12.15mL of simulated colon chyme culture medium (SM) into each tube by adopting a 20mL sealed anaerobic fermentation tube with a rubber plug, inoculating faeces and bacterial liquid according to the concentration of 10% (v/v), wherein the inoculation amount of faecal inoculation liquid is 1.35mL, and the bacterial suspension (10 9 CFU/mL viable count) was 1.5mL;
in addition, 1.5mL of Phosphate Buffer Solution (PBS) with pH of 7.4 is added to replace the bacterial suspension;
(2) The above-mentioned fermentation tubes were each subjected to fermentation by shaking at 37℃for 48 hours.
All of the above operations are performed at an anaerobic workstation. After 48h of fermentation, the anaerobic fermentation tubes were immediately placed on ice for 15min, respectively, and fermentation broth was collected and centrifuged at 8000g at 4℃for 10min to obtain the supernatant. Each group was subjected to 4 replicates. And detecting the pH value of the fermentation liquor.
3. Experimental results
The specific results are shown in table 3 below:
table 3: pH value of fermentation liquor after co-culture of different strains and fecal suspension
Data: mean ± standard deviation
As can be seen from Table 3 and FIG. 6, the addition of Clostridium praecox CCFM1204 to the fermentation system can reduce the pH of the fermentation broth. Studies show that reducing the pH value in the intestinal lumen can inhibit the proliferation of harmful bacteria, and protect the intestinal tract from colonization by sensitive pathogens, such as harmful gas-producing bacteria, such as escherichia coli, clostridium perfringens, klebsiella and the like. In contrast, the beneficial bacteria in the intestinal tract can tolerate lower pH values, such as probiotics like bifidobacteria, lactobacilli, etc., which provide a favorable condition for their survival.
Example 5: effect of Clostridium praecox CCFM1204 on short chain fatty acid content
The method comprises the following specific steps:
1. culture of bifidobacterium breve FFJND2DLM11, bifidobacterium adolescentis FHNFQ48M5, bifidobacterium longum subspecies infantis FJSWXI4MIM1, bacillus coagulans CCFM1041, clostridium praecox CCFM1204 strain:
inoculating Bifidobacterium breve FFJND2DLM11, bifidobacterium adolescentis FHNFQ48M5, bifidobacterium longum subspecies FJSWXI4MIM1, and Bacillus coagulans CCFM1041 in an inoculum size of 2% (v/v) into MRS liquid culture medium, respectively, and culturing in an anaerobic tank (80% N) 2 ,10％CO 2 ,10％H 2 ) Culturing at 37deg.C for 24 hr, sequentially transferring to 3 rd generation according to 2% (v/v) inoculum size, centrifuging at 6000g for 3min to collect bacterial mud, washing with 0.1M PBS (pH 7.2, containing 0.05% cysteine) and re-suspending to obtain bacterial concentration of 1×10 9 CFU/mL bacterial suspension, immediately on the day.
Will be preserved in an environment of-80 DEG CClostridium praecox CCFM1204 was inoculated into M2GSC broth at an inoculum size of 2% (v/v) in an anaerobic tank (80% N 2 ,10％CO 2 ,10％H 2 ) Culturing at 37deg.C for 24 hr, sequentially transferring to 3 rd generation according to 2% (v/v) inoculum size, vacuum filtering in anaerobic workstation to obtain bacterial mud, washing with 0.1M PBS (pH 7.2, containing 0.05% cysteine), and re-suspending to obtain bacterial concentration of 1×10 9 CFU/mL bacterial suspension, immediately on the day.
2. Bifidobacterium breve FFJND2DLM11, bifidobacterium adolescentis FHNFQ48M5, bifidobacterium longum subspecies infantis FJSWXI4MIM1, bacillus coagulans CCFM1041, clostridium praecox CCFM1204 co-cultured with intestinal flora, respectively
(1) Collecting 6 faecal samples from the flatulence population, mixing Phosphate Buffer Solution (PBS) with pH of 7.4 with fresh faecal sample according to the ratio of 1:7 (w/v), preparing faecal inoculation liquid, adding 12.15mL of simulated colon chyme culture medium (SM) into each tube by adopting a 20mL sealed anaerobic fermentation tube with a rubber plug, inoculating faeces and bacterial liquid according to the concentration of 10% (v/v), wherein the inoculation amount of faecal inoculation liquid is 1.35mL, and the bacterial suspension (10 9 CFU/mL viable count) was 1.5mL;
in addition, in the control group, 1.5ml of Phosphate Buffer Solution (PBS) with pH of 7.4 is added to replace the bacterial suspension;
(2) The above-mentioned fermentation tubes were each subjected to fermentation by shaking at 37℃for 48 hours.
All of the above operations are performed at an anaerobic workstation. After 48h fermentation, the anaerobic fermentation tube is immediately placed on ice for 15min to stop fermentation, then fermentation liquid is collected, the fermentation liquid is respectively centrifuged for 10min at 4 ℃ and 8000g, and the supernatant is taken. 0.5mL of the supernatant was taken, 500. Mu.L of saturated NaCl solution was added thereto, and 20. Mu.L of 10% H was added thereto 2 SO 4 Acidifying; adding 1000 mu L of anhydrous diethyl ether, shaking uniformly, extracting fatty acid, and centrifuging for 15min at 18000 g; taking the upper diethyl ether phase, adding 0.25g of anhydrous Na 2 SO 4 Drying; standing for 30min, centrifuging for 10min at 18000g, collecting upper diethyl ether phase, and determining short chain fatty acid content in fermentation broth by GC-MS. Rtx-Wax column (column length 30m, inner diameter 25 μm) was used; the carrier gas is He, and the flow rate is 2mL/min;the sample injection volume is 1 mu L, the temperature is increased to 140 ℃ according to 7.5 ℃/min, then the temperature is increased to 200 ℃ according to 60 ℃/min, and the ionization temperature is 20 ℃ after 3 min; the analysis adopts a full scanning mode, and standard curves are prepared by an external standard method, so that the concentration of each short chain fatty acid is calculated. Each group was subjected to 4 replicates.
3. Experimental results
The specific results are shown in table 4 below:
table 4: concentration value of each short chain fatty acid after co-culture of different strains and fecal suspension
Data: mean ± standard deviation
As can be seen from table 4 and fig. 7, the addition of clostridium prasugrel CCFM1204 to the fermentation system can significantly increase the yield of short chain fatty acids, including acetic acid, propionic acid, butyric acid, isobutyric acid and valeric acid, compared to the control group. Since clostridium prasugrel is the main butyrate producing strain in the intestinal tract, NADH is continuously consumed in the pathway of producing butyrate by microbial metabolism in the intestinal tract, and NADH is required as a reducing agent for synthesizing free H in the intestinal tract, and hydrogen is consumed in the pathway of synthesizing butyrate from acetyl-coa. Thus, an increase in the yield of butyric acid will reduce the formation of hydrogen.
Example 6: modulation of intestinal flora structure by clostridium praecox CCFM1204
The method comprises the following specific steps:
1. culture of bifidobacterium breve FFJND2DLM11, bifidobacterium adolescentis FHNFQ48M5, bifidobacterium longum subspecies infantis FJSWXI4MIM1, bacillus coagulans CCFM1041, clostridium praecox CCFM1204 strain:
inoculating Bifidobacterium breve FFJND2DLM11, bifidobacterium adolescentis FHNFQ48M5, bifidobacterium longum subspecies FJSWXI4MIM1, and Bacillus coagulans CCFM1041 in an inoculum size of 2% (v/v) into MRS liquid culture medium, respectively, and culturing in an anaerobic tank (80% N) 2 ,10％CO 2 ,10％H 2 ) Culturing at 37deg.C for 24 hr, sequentially transferring to the first stage according to 2% (v/v) of inoculation amount3 rd generation, centrifuging 6000g for 3min to collect bacterial sludge, washing with 0.1M PBS (pH 7.2, containing 0.05% cysteine), and re-suspending to obtain bacterial concentration of 1×10 9 CFU/mL bacterial suspension, immediately on the day.
Inoculating Clostridium praecox CCFM1204 preserved in-80deg.C environment into M2GSC liquid medium at an inoculum size of 2% (v/v), and culturing in an anaerobic tank (80% N) 2 ,10％CO 2 ,10％H 2 ) Culturing at 37deg.C for 24 hr, sequentially transferring to 3 rd generation according to 2% (v/v) inoculum size, vacuum filtering in anaerobic workstation to obtain bacterial mud, washing with 0.1M PBS (pH 7.2, containing 0.05% cysteine), and re-suspending to obtain bacterial concentration of 1×10 9 CFU/mL bacterial suspension, immediately on the day.
2. Bifidobacterium breve FFJND2DLM11, bifidobacterium adolescentis FHNFQ48M5, bifidobacterium longum subspecies infantis FJSWXI4MIM1, bacillus coagulans CCFM1041, clostridium praecox CCFM1204 were co-cultured with the intestinal flora, respectively.
(1) Collecting 6 faecal samples from the flatulence population, mixing Phosphate Buffer Solution (PBS) with pH of 7.4 with fresh faecal sample according to the ratio of 1:7 (w/v), preparing faecal inoculation liquid, adding 12.15mL of simulated colon chyme culture medium (SM) into each tube by adopting a 20mL sealed anaerobic fermentation tube with a rubber plug, inoculating faeces and bacterial liquid according to the concentration of 10% (v/v), wherein the inoculation amount of faecal inoculation liquid is 1.35mL, and the bacterial suspension (10 9 CFU/mL viable count) was 1.5mL;
in addition, 1.5mL of Phosphate Buffer Solution (PBS) with pH of 7.4 is added to replace the bacterial suspension;
(2) The above-mentioned fermentation tubes were each subjected to fermentation by shaking at 37℃for 48 hours.
All of the above operations are performed at an anaerobic workstation. After 48h fermentation, the anaerobic fermentation tube was immediately placed on ice for 15min to stop fermentation, and the fermentation broth was then collected. After extracting bacterial genome of each group of fermentation liquor by adopting a Fast DNA Spin Kit for Feces kit, carrying out PCR amplification on the 16s V3-V4 region sequence, and carrying out 1.5% agarose gel electrophoresis on all obtained PCR products. The target gene was recovered and purified from agarose gel containing the target band using a gel recovery kit, tested on a Illumina Miseq PE300 platform, and the next data was analyzed by QIIMEII software.
As can be seen from fig. 8 and 9, the addition of the clostridium prasuum CCFM1204 to the fermentation system can maintain the intestinal flora diversity, and the ACE index, the Chao1 index, the Shannon index and the Simpson index are all significantly improved (p < 0.05) compared with the control group, the bifidobacterium adolescentis, the bifidobacterium longum and the bacillus coagulans group. More and more studies have shown that higher intestinal flora diversity represents a healthier flora characteristic, whereas a decrease in flora diversity is often accompanied by the occurrence of diseases such as Irritable Bowel Syndrome (IBS), inflammatory Bowel Disease (IBD), colon cancer (CRC) etc. In addition, clostridium prasugrel CCFM1204 can significantly reduce the abundance of intestinal harmful bacteria, such as Proteus, enterobacteriaceae and the like, and can also reduce the abundance of gas-producing bacteria in the intestinal tract, such as Ruminococcus. Supplementation with clostridium prasugrel CCFM1204 significantly increases the abundance of butyric acid-producing bacteria in the system, such as genus Eubacterium halliigroup, genus Coprococcus1 and genus Butyricicoccus. Butyric acid is one of important short chain fatty acids in intestinal tract, has antibacterial and anti-inflammatory effects, is a main energy supply substance of intestinal epithelial cells, and has important effect on maintaining intestinal barrier. Thus, clostridium praecox CCFM1204 helps to maintain intestinal health, provides a lower pH intestinal lumen environment, inhibits harmful bacteria proliferation, thereby reducing over-fermentation gas production thereof.
Example 7: preparation of solid beverage containing clostridium prasugrel CCFM1204
The method comprises the following specific steps:
inoculating clostridium praecox CCFM1204 into a culture medium according to an inoculum size accounting for 2% of the total mass of the culture medium M2GSC, and culturing for 18 hours at 37 ℃ to obtain a culture solution; centrifuging the culture solution to obtain thalli; the thalli is washed 3 times by phosphate buffer solution with pH of 7.2 and then resuspended by trehalose freeze-drying protective agent with the trehalose concentration of 100g/L (the mass ratio of the freeze-drying protective agent to the thalli is 2:1), and the concentration of the thalli is 5 multiplied by 10 8 CFU/mL of resuspension; and freeze-drying the heavy suspension by adopting a vacuum freezing method to obtain the clostridium prasugrel CCFM1204 bacteria powder.
Example 8: clostridium praecox CCFM1204 for preparing capsule product
The method comprises the following specific steps:
inoculating clostridium praecox CCFM1204 into a culture medium according to an inoculum size accounting for 2% of the total mass of the culture medium M2GSC, and performing anaerobic culture for 18 hours at 37 ℃ to obtain bacterial liquid; vacuum filtering in anaerobic environment to obtain bacterial sludge; the thalli is washed 3 times by phosphate buffer solution with pH of 7.2 and then resuspended by trehalose freeze-drying protective agent with the trehalose concentration of 100g/L (the mass ratio of the freeze-drying protective agent to the thalli is 2:1), and the concentration of the thalli is 5 multiplied by 10 8 CFU/mL of resuspension; extruding the mixed solution into a calcium chloride solution with the concentration of 20g/L to form colloidal particles; after the formed colloidal particles are stationary and solidified for 30min, filtering and collecting the colloidal particles; freeze-drying the collected colloidal particles for 48 hours to obtain powder; filling the powder into a medicinal capsule to obtain a capsule product;
the preparation method of the culture medium comprises the following steps: the medium was obtained by dissolving 10% enzyme hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract with 87.7% water based on the total weight of the medium, and then adjusting the pH to 6.8.
Example 9: clostridium praecox CCFM1204 for preparing tablets
The method comprises the following specific steps:
inoculating clostridium praecox CCFM1204 into a culture medium according to an inoculum size accounting for 2% of the total mass of the culture medium M2GSC, and performing anaerobic culture for 18 hours at 37 ℃ to obtain bacterial liquid; vacuum filtering in anaerobic environment to obtain bacterial sludge; washing the bacterial mud with physiological saline for 3 times, and re-suspending with protective agent to a concentration of 1×10 10 CFU/mL, obtaining bacterial suspension; pre-culturing the bacterial suspension at 37 ℃ for 60min, and freeze-drying to obtain clostridium prasuum CCFM1204 bacterial powder;
the preparation method of the culture medium comprises the following steps: dissolving 10% enzyme hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract with 87.7% water based on the total weight of the culture medium, and adjusting pH to 6.8 to obtain culture medium;
the components of the protective agent comprise: 100g/L skimmed milk powder, 30mL/L glycerol, 100g/L maltodextrin, 150g/L trehalose and 10g/L L-sodium glutamate.
Weighing 25.7 parts by weight of clostridium praecox CCFM1204 bacteria powder, 55.0 parts by weight of starch, 4.5 parts by weight of cellulose derivative, 12.0 parts by weight of carboxymethyl starch sodium, 0.8 part by weight of talcum powder, 1.0 part by weight of sucrose and 1.0 part by weight of water to obtain raw materials; mixing the raw materials to obtain wet particles; tabletting the wet granules by a tablet press of a pharmaceutical machinery factory in the middle south, and drying by a small-sized drug dryer of Yikang traditional Chinese medicine machinery Co., ltd.
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. Use of clostridium prasugrel (Faecalibacterium prausnitzii) CCFM1204 or a fermentation broth thereof in the manufacture of a medicament, enteral nutritional or probiotic preparation for preventing, alleviating, ameliorating and/or treating intestinal flatulence, wherein clostridium prasugrel CCFM1204 has a deposit number of GDMCC No:62239 the fermentation broth contains clostridium prasugrel CCFM1204.
2. The use according to claim 1, wherein the amount of clostridium prasugrel CCFM1204 in the pharmaceutical, enteral nutritional or probiotic preparation is not less than 1 x10 6 CFU/mL or 1X 10 6 CFU/g。
3. The use according to claim 1 or 2, characterized in that the probiotic preparation comprises wet cells or lyophilized cells of clostridium prasugrel CCFM1204.
4. The use according to claim 3, wherein the probiotic preparation is prepared by: and mixing the cultured clostridium prasugrel CCFM1204 cells with a protective agent.
5. The use according to claim 4, wherein the protective agent comprises one or more of skimmed milk powder, glycerol, maltodextrin, trehalose, sodium L-glutamate.
6. The use according to claim 1 or 2, wherein the medicament comprises the clostridium prasugrel CCFM1204, a pharmaceutical carrier and/or a pharmaceutical adjuvant.
7. The use according to claim 6, wherein the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles and/or liposomes.
8. The use according to claim 7, wherein the pharmaceutical excipients comprise one or more of anti-adhesive agents, 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, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, foaming agents, suspending agents, coating materials, fragrances, diluents, flocculants and deflocculants, filter aids, and release retarders.
9. The use according to any one of claims 1 to 8, wherein the medicament is in the form of granules, capsules, tablets, pills or oral liquids.
10. The use according to any one of claims 1 to 9, wherein the medicament comprises at least one of the following actions:
(1) Reducing the generation of fermentation by-product gas in the intestinal tract of an adult;
(2) Lowering the pH of the intestinal tract;
(3) Improving the content of short chain fatty acids such as acetic acid, propionic acid, butyric acid and the like in the intestinal tract;
(4) And (3) regulating the intestinal flora structure of intestinal flatulence people.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311564770.4A CN117618472A (en) | 2023-11-22 | 2023-11-22 | Clostridium praecox capable of reducing intestinal gas generation and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311564770.4A CN117618472A (en) | 2023-11-22 | 2023-11-22 | Clostridium praecox capable of reducing intestinal gas generation and application thereof |
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| Publication Number | Publication Date |
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| CN117618472A true CN117618472A (en) | 2024-03-01 |
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| CN202311564770.4A Pending CN117618472A (en) | 2023-11-22 | 2023-11-22 | Clostridium praecox capable of reducing intestinal gas generation and application thereof |
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| Country | Link |
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| CN (1) | CN117618472A (en) |
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2023
- 2023-11-22 CN CN202311564770.4A patent/CN117618472A/en active Pending
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