CN113234619B - Bifidobacterium bifidum and application thereof in relieving intestinal injury - Google Patents
Bifidobacterium bifidum and application thereof in relieving intestinal injury Download PDFInfo
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- CN113234619B CN113234619B CN202110388079.XA CN202110388079A CN113234619B CN 113234619 B CN113234619 B CN 113234619B CN 202110388079 A CN202110388079 A CN 202110388079A CN 113234619 B CN113234619 B CN 113234619B
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Abstract
The invention discloses bifidobacterium bifidum and application thereof in relieving intestinal injury, belonging to the field of microorganisms. The bifidobacterium bifidum CCFM1172 can obviously relieve or prevent acute colon tissue injury, improve the disease states of weight loss, mouse laziness, chilliness, reduced eating, dull and uneven hair color, diarrhea and the like, and reduce the death rate from 53 percent to 0 percent. Therefore, the bifidobacterium bifidum can be used for preparing food, medicines or health products for relieving or preventing acute colon injury, and has wide application prospect.
Description
Technical Field
The invention relates to bifidobacterium bifidum and application thereof in relieving intestinal injury, belonging to the field of microorganisms.
Background
Bifidobacterium bifidum, a probiotic, has received some attention because of its potential benefits to human health. Bifidobacterium bifidum is the first colonizer in the gastrointestinal tract of newborns because of its ability to metabolize human milk oligosaccharides. One study by Elahe et al showed that specific bifidobacterium bifidum was effective in inducing anti-tumor immune responses and inhibiting the growth of Human Papillomavirus (HPV) -associated TC-1 tumor cells in C57BL/6 mice. Results of clinical trials in Viola et al indicate that certain bifidobacterium bifidum is able to alleviate the symptoms of Irritable Bowel Syndrome (IBS). One study by Ahamd et al showed that specific bifidobacterium bifidum is able to slow down colitis caused by DSS and partially restore malnutrition caused by enteritis. Verma et al have shown that specific Bifidobacterium bifidum and its polysaccharides can promote the formation of Treg cells in the intestinal tract and show inhibitory activity on T cell metastatic colitis. As can be seen from these studies, Bifidobacterium bifidum has great potential in the aspects of metabolism, tumor resistance, influence on immune regulation and further relief of intestinal inflammation, and the like. Patent CN107629988B provides a Bifidobacterium bifidum which can reduce the tumor number of the node and rectum parts of a model mouse by regulating the expression of Notch1, Notch2 signal paths and VEGFR2 molecules in colorectal tissues. Patent CN111450260A provides a method for preparing an anti-tumor preparation of bifidobacterium bifidum. Patent CN110331119A provides a strain of bifidobacterium bifidum, which can relieve the symptoms caused by type ii diabetes. Patent CN106834187A provides a strain of bifidobacterium bifidum, which can relieve constipation.
Intestinal injury is a common syndrome in clinic, and has both acute lesion and chronic lesion. Trinitrobenzene sulfonic acid (2,4,6-trinitrobenzene sulfonic acid, TNBS) is dissolved in ethanol (30% -50%) with a certain concentration, and rectal administration of trinitrobenzene sulfonic acid to mice or rats of specific strains causes intestinal injury, which is a commonly applied method in experimental research. Differences in the dose administered and the concentration of ethanol cause varying degrees of intestinal injury, both with and without an inflammatory response. When the administration dosage is larger, the early-stage intestinal injury is serious and is not accompanied with inflammatory reaction; after 5-7 days, the intestinal injury is relieved, and then the conversion to chronic inflammation is started. Therefore, TNBS modeling can cause two disease models due to differences in the dose and observation period: one is TNBS-induced acute intestinal injury; the second is TNBS-induced colitis. Patent CN107485625A provides a strain of bifidobacterium bifidum for relieving colitis induced by TNBS. However, to date, no patent report has been made on the ability of bifidobacterium bifidum to alleviate acute intestinal injury.
Disclosure of Invention
The invention provides a strain of bifidobacterium bifidumBifidobacterium bifidum) CCFM1172 with the deposit number GDMCC No: 61550, deposited at the Guangdong province Collection of microorganisms at 2021, 3, 9 days.
The invention also provides a microbial preparation containing the bifidobacterium bifidum CCFM 1172.
In one embodiment of the present invention, the viable count of Bifidobacterium bifidum CCFM1172 in the microbial preparation is not less than 1X 106CFU/mL or 1X 106 CFU/g。
The invention also provides application of the bifidobacterium bifidum CCFM1172 or the microbial preparation in preparing a medicament for preventing or relieving acute colon injury.
In one embodiment of the present invention, the medicament contains the bifidobacterium bifidum CCFM1172 or the microbial preparation.
In one embodiment of the invention, the number of viable bacteria of Bifidobacterium bifidum CCFM1172 in the medicament is not less than 1 x 106CFU/mL or 1X 106 CFU/g。
In one embodiment of the invention, the medicament comprises bifidobacterium bifidum CCFM1172, a pharmaceutical carrier and/or a pharmaceutical excipient.
In one embodiment of the invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and/or liposomes.
In one embodiment of the invention, the pharmaceutical excipient comprises a filler, a binder, a wetting agent, a disintegrant, a lubricant and/or 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.
In one embodiment of the invention, the dosage form of the medicament is powder, granules, capsules, tablets, pills or oral liquid.
The invention has the beneficial effects that:
the bifidobacterium bifidum CCFM1172 can obviously relieve or prevent acute colon tissue injury, improve disease states such as weight loss, mouse laziness, intolerance of cold, reduced eating, dull and uneven hair color, diarrhea and the like, and obviously reduce death rate. Therefore, the bifidobacterium bifidum with the function of relieving the colon injury has wide application prospect in the directions of food and microbial preparations.
Biological material preservation
The invention provides bifidobacterium bifidum (B)Bifidobacterium bifidum) CCFM1172, which is preserved in Guangdong province microorganism culture collection center at 2021, 3 and 9 days, wherein the preservation address is the microbial research institute of Guangdong province, No. 59 building, No. 5 building, No. 59 Ministry of Middleway, Guangzhou city, and the preservation number is GDMCC No: 61550.
drawings
FIG. 1 shows the body weight changes of mice gavaged with CCFM1172 before and after TNBS-induced colon injury.
FIG. 2 mortality in mice gavaged CCFM1172 before and after TNBS-induced colonic injury.
FIG. 3 Colon images of CCFM1172 mice gazed before and after TNBS-induced colon injury.
FIG. 4 TNBS induced colon injury before and after perfusion of CCFM1172 mouse colon cytokine.
FIG. 5 weight change following TNBS-induced colonic injury treated by gastric gavage CCFM 1172.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
The media involved in the following examples are as follows:
MRS solid medium (g/L): 10g/L of peptone, 10g/L of beef extract, 20 g/L of glucose, 2 g/L of sodium acetate, 5 g/L of yeast powder and 2 g/L, K of diammonium hydrogen citrate2HPO4·3H2O 2.6 g/L、MgSO4·7 H2O 0.1 g/L、MnSO4·H2O0.05 g/L, Tween 801 mL/L, agar 20 g/L and cysteine hydrochloride 1 g/L.
MRS liquid medium (g/L): 10g/L of peptone, 10g/L of beef extract, 20 g/L of glucose, 2 g/L of sodium acetate, 5 g/L of yeast powder and 2 g/L, K of diammonium hydrogen citrate2HPO4·3H2O 2.6 g/L、MgSO4·7 H2O 0.1 g/L、MnSO4·H2O0.05 g/L, Tween 801 mL/L and cysteine hydrochloride 1 g/L.
Bifidobacterium bifidum (b)Bifidobacterium bifidum) CCFM16 is a published strain: the preservation number is CGMCC NO.13632, which is preserved in the China general microbiological culture Collection center on 17.01.2017, and the preservation address is the microbiological research institute of China academy of sciences, No. 3 of Xilu No.1 Hospital, Beijing, Chaoyang. Is disclosed in patent CN 110720512A.
Example 1: isolation and screening of Bifidobacterium bifidum CCFM1172
1. Sample collection
Collecting healthy infant feces samples in Jiangning district of Nanjing city, Jiangsu province, placing the samples in a storage tube filled with 30% glycerol, storing in a heat preservation box filled with an ice bag, taking the samples back to a laboratory, and rapidly placing the samples in a refrigerator at-80 ℃ for separation and screening.
2. Separation and purification of bifidobacteria
(1) Dilution coating: in a sterile environment, about 0.5 g of a 30% glycerol/feces mixture was added to a 10 mL centrifuge tube containing 4.5 mL of physiological saline to give 10-1Diluting solution, repeating the above dilution steps to obtain 10-2、10-3、10-4、10-5、10-6Diluting the solution;
(2) coating culture: sucking 100 μ L of the above 10-4、10-5、10-6Three gradient sample dilutions were plated on MRS fixed media containing 0.1% cysteine hydrochloride and mupirocin (100. mu.g/mL) at pH 6.8 and incubated at 37 ℃ for 48h under anaerobic conditions to obtain diluted plating plates;
(3) and (3) purification and culture: taking a diluted coating flat plate with the colony number within the range of 30-300, selecting colonies with different forms on a solid culture medium, and carrying out streaking separation until pure single colonies which are milky white or white, smooth in surface, neat in edge and consistent in form are obtained; and (3) selecting the pure colonies on the solid culture medium, inoculating the pure colonies in 5mL of liquid MRS culture medium, and culturing for 24-36 h under an anaerobic condition at 37 ℃ to obtain a purified culture solution.
3. Strain preservation and identification
(1) And (3) strain preservation:
uniformly mixing the purified culture solution obtained in the step 2, sucking 500 mu L of the purified culture solution into a sterilized strain storage tube, adding 500 mu L of 60% glycerol, uniformly mixing, and storing in a refrigerator at the temperature of-80 ℃; and (3) putting another 1 mL of bacterial liquid into a preservation tube for strain identification, centrifuging at 8000r/min for 10min, and removing the supernatant to obtain the thalli.
(2) And (3) strain identification:
carrying out PCR by using a bacteria 16S rDNA PCR specific primer, and carrying out sequencing on a PCR product after nucleic acid electrophoresis analysis and confirmation; the 27F and 1492R splice sequences returned by sequencing were uploaded to BLAST (http:// www.ncbi.nlm.nih.gov/BLAST) at NCBI for species validation; the strain with the number of CCFM1172 is bifidobacterium bifidum according to the comparison result.
TABLE 1 identification of bacterial species 25 muL 16S rDNA PCR reaction system
| Components | Adding amount (mu L) |
| 27F | 0.25 |
| 1492R | 0.25 |
| Taq enzyme Mix | 12.5 |
| |
1 |
| Double distilled water | 11 |
TABLE 2 primer names
| 16S rDNA PCR primer name | Sequence of |
| 27F | 5’-AGAGTTTGATCCTGGCCTCA-3’（SEQ ID NO.1） |
| 1492R | 5’-GGTTACCTTGTTACGACTT-3’（SEQ ID NO.2） |
Example 2: the TNBS induced colon injury before and after intragastric administration CCFM1172 has obvious protective effect
1. Preparation of the bacterial suspension
The bifidobacterium bifidum CCFM1172 in the glycerol storage tube obtained in the example 1 is coated on an MRS solid plate and cultured for 48 hours at 37 ℃ in an anaerobic workstation; selecting a single colony, inoculating the single colony in a 5mLMRS culture medium, and culturing for 36h at 37 ℃ in an anaerobic workstation; the activated bacteria liquid is enlarged and cultured into 1L MRS culture medium, cultured in an anaerobic workstation at 37 ℃ for 2 days, centrifuged at 8000r/min for 20min, the bacterial sludge is collected, and the PBS is used for resuspension to adjust the viable count to 5 multiplied by 109CFU/mL, portioned, placed in a 4 ℃ freezer for use, and used up within 5 days. A Bifidobacterium bifidum CCFM16 and a Bifidobacterium bifidum FHNXY17M1 bacterial suspension screened in the same batch were prepared by the same method.
2. Laboratory animal
SPF grade 8 week old male Balb/c mice, purchased from Witongli Limited; culturing in dark environment with relative humidity of 50 + -5% at 25 + -2 deg.C for 12 h.
3. Experimental methods
The drug preparation method comprises the following steps: a5% aqueous TNBS solution was prepared into a 35% aqueous ethanol solution (35% ethanol concentration, 25ug/uL TNBS concentration) at 25mg/uL using a 75% ethanol solution and sterile water.
Male Balb/c mice of 8 weeks of age were randomly divided into 5 groups, including a model group, a blank group, and an experimental group of bifidobacterium bifidum CCFM1172, CCFM16, or FHNXY17M1, with 13 model groups, and 10 in the remaining groups. The experimental period is 10 days, PBS is continuously infused once a day in a molding group and a blank group, and 200 mu L/bacterial suspension obtained in the step 1 is continuously infused once a day in a Bifidobacterium bifidum CCFM1172 group, a CCFM16 group and a FHNXY17M1 group respectively. Fasting is started after the gastric lavage is finished on the 4 th day, the molding is started on the 5 th day after 24 hours, the molding group and each experimental group except a blank group are connected with a plastic hose by a syringe, and 50 uL/piece of TNBS ethanol solution is rectally filled in the blank group, and 50 uL/piece of 35% ethanol solution is filled in the blank group.
On the day of administration to the end of the experiment, the building group and the blank group were continuously subjected to intragastric gavage once a day by PBS, and the bifidobacterium bifidum CCFM1172 group, the CCFM16 group and the FHNXY17M1 group were respectively subjected to intragastric gavage once a day by 200 μ L/piece of the bacterial suspension obtained in the step 1.
The weight of the mice is recorded before modeling, the weight, mental state, hair color, food intake and excrement conditions of the mice are recorded every day after modeling, the death condition of the mice is recorded, and all the mice are euthanized at the 5 th day after modeling.
4. General Condition Change in mice
The body weight change of the mice is shown in fig. 1. After TNBS induces acute colon injury, the weight of a mouse of the modeling group is obviously reduced, and the mouse cannot recover by 5 days after modeling; and the bifidobacterium bifidum CCFM1172 can obviously relieve the weight loss caused by acute colon tissue injury, and the weight of half of mice is recovered to the weight before modeling on the 5 th day after modeling, and the average weight change fluctuation is between 20 and 25 g. As shown in FIG. 1, the mice in the CCFM1172 group had a smaller weight change, a weight loss of at most 0.5 g and a rapid weight recovery, while the mice in the control CCFM16 group had a weight loss of at most 1.5 g and the mice in the FHNXY17M1 group had a weight loss of at most 2 g.
On the 1 st day after the model building, the mice of the model building group have the defects of laziness, intolerance of cold, reduced food intake, dark and uneven hair color, and the conditions of most of the mice can not be obviously improved before the experiment is finished; some mice have black feces adhered to the perianal area from day 2 to day 3; the hair color of mice in the CCFM1172 group is normal, and the mice do not have the conditions of chilly and the like, and only do not move easily when observed on the 1 st day after the molding, and return to normal on the 2 nd day.
Mortality of mice as shown in figure 2, the mortality of mice in CCFM1172 group was zero, significantly lower than the mortality of 50% in the building and FHNXY17M1 groups and 20% in CCFM16 group.
5. Gross pathological observation
The colon image of the mouse is shown in fig. 3, the colon wall of the mouse with the model is remarkably thickened, the intestinal cavity is remarkably expanded, the intestinal mucosa is congested, ulcer and fibrosis are serious, the colonic enteric nerve of the mouse is damaged, so that the normal peristalsis can not be realized, and the colon cavity is completely thin and convenient and cannot be discharged; the colon of mice in the CCFM16 group and the FHNXY17M1 group has serious pathological conditions; the colons of the CCFM1172 mice are close to the normal state on the 5 th day after model building, the congestion, the thickening of the intestinal wall and the ulcer are not seen, and the colons have good peristalsis capability and the stool form is close to normal.
6. Mouse colon tissue cytokine levels
After the mice die, colon tissues are taken, intestinal contents are emptied, the colon tissues are washed clean in ice PBS, water is drained on a water wiping paper, the colon tissues are weighed, magnetic beads and PBS 9 times the weight of the tissues are added to prepare 10% tissue homogenate, and the contents of cytokines (IFN-gamma, IL-17 and IL-10) in the colon tissues are measured by an ELISA method, wherein the result is shown in figure 4. The detection results of the CCFM1172 group cytokines are all close to the level of normal mice; the acute injury condition of the mouse colon induced by the high-dose TNBS is serious, and the generation of cytokines cannot be induced all the time from the model building to the end of the experiment without inflammatory reaction; the levels of the three cytokines in the CCFM16 group and the FHNXY17M1 group as the control experimental group are obviously higher than those in the blank group, and it can be seen that the continuous supply of the bifidobacterium bifidum CCFM16 and the FHNXY17M1 can also enhance the resistance of the mice and help the body to initiate immune response compared with the model group, but the death rate of the mice cannot be obviously improved.
Example 3: therapeutic effects of CCFM1172 following TNBS-induced colonic injury
1. Experimental methods
Preparation of bacterial suspension and experimental animals the same as in example 2.
The drug preparation method comprises the following steps: a5% aqueous TNBS solution was prepared into a 35% aqueous ethanol solution (ethanol concentration 35%, TNBS concentration 20ug/uL) using a 75% ethanol solution and sterile water.
Male Balb/c mice of 8 weeks of age were randomly divided into 3 groups, including a molding group, a therapeutic group of bifidobacterium bifidum by gavage CCFM1172, and a blank group, each group containing 10 mice. After one week acclimation of the mice in the SPF grade barrier environment, fasting was performed for 24h, and then the plastic hoses were connected using syringes to rectally perfuse the mice of the manufacturing and treatment groups with 50 uL/mouse of TNBS in ethanol solution, and the blank group was perfused with 50 uL/mouse of 35% ethanol solution in the same manner.
Gavage once 1X 10 on the day of administration9CFU/mL Bifidobacterium bifidum CCFM1172, the gastric lavage with PBS once for the model group and the blank group, and the continuous gavage is kept for 5 days. The weight of the mice is recorded before modeling, the weight, mental state, hair color, food intake and excrement conditions of the mice are recorded every day after modeling, scoring is carried out according to disease activity indexes, the death condition of the mice is recorded, and all the mice are euthanized at the 5 th day after modeling.
2. General conditions of mice
The body weight change of the mice is shown in figure 5, the body weight of the mice in the bifidobacterium bifidum CCFM1172 treatment group is recovered to the body weight before the model building on the 5 th day, and the body weight change fluctuates between 20 and 25 g. The conditions of obvious reduction of the whole body weight, laziness in movement, intolerance of cold, reduction of food intake, dull and uneven hair color and diarrhea appear after the model building of the model group mice, and the symptoms of most of the mice are not improved before the experiment is finished; and the bifidobacterium bifidum CCFM1172 after the model building is filled can obviously relieve the weight loss caused by acute colon tissue injury, and the whole mouse has the conditions of no moving and reduced food intake, but the degree is lighter than that of the model building group.
Example 4: prevention of CCFM1172 before TNBS-induced colonic injury
1. Experimental methods
Preparation of bacterial suspension and experimental animals the same as in example 2.
The drug preparation method comprises the following steps: a5% aqueous TNBS solution was prepared into a 35% aqueous ethanol solution (ethanol concentration 35%, TNBS concentration 20ug/uL) using a 75% ethanol solution and sterile water.
Male Balb/c mice of 6 weeks old were randomly divided into 3 groups, including a group of mice, a prophylactic group of Bifidobacterium bifidum by gavage CCFM1172, and a blank group, each group containing 10 mice. CCFM1172 group mice were gavaged once a day by 1X 109CFU/mL Bifidobacterium bifidum CCFM1172, the building block and the blank group were continuously gavaged once daily with PBS for 7 days, fasting was started after the completion of gavage on day 7, and then the mice in the building block and the treatment group were rectally perfused with 50 uL/L TNBS ethanol solution using a syringe connected to a plastic hose, and the blank group was perfused with 50 uL/L35% ethanol solution in the same manner. The stomach is not filled any more after the model is made.
The weight of the mice is recorded before modeling, the weight, mental state, hair color, food intake and excrement conditions of the mice are recorded every day after modeling, the death condition of the mice is recorded, and all the mice are euthanized at the 5 th day after modeling.
2. General condition and mortality of mice
The conditions of obvious reduction of the whole body weight, laziness in movement, intolerance of cold, reduction of food intake, dull and uneven hair color and diarrhea of the model-making mice occur after model making, and the symptoms of most of the mice are not improved before the experiment is finished; compared with the model building group, the administration of the bifidobacterium bifidum CCFM1172 is continued for 7 days before the model building, so that the mouse is in a normal state on the 1 st to 2 th days after the model building, and symptoms such as chilliness, laziness, weight reduction and the like do not appear. Compared with enteric nerve disorder caused by intestinal injury of model group and incapability of defecation of mice, the CCFM1172 prevention group recovers the normal intestinal motility on day 3 and begins to defecate.
Example 5 application of Bifidobacterium bifidum CCFM1172
Bifidobacterium bifidum CCFM1172 can be used for preparing cow milk, and the specific preparation process of cow milk is as follows:
(1) will carry outInoculating the purified culture solution of bifidobacterium bifidum CCFM1172 obtained in example 1 into a culture medium with the inoculation amount of 3% (v/v), culturing for 36h at 37 ℃ in an anaerobic workstation, centrifuging for 20min at 6000r/min, and collecting bacterial sludge; after the bacterial sludge was washed with PBS, it was resuspended to a concentration of 1' 10 with a protective agent10CFU/mL to obtain a suspension; pre-culturing the suspension at 37 deg.C for 60min, and lyophilizing to obtain starter;
wherein the culture medium is MRS liquid culture medium;
the components of the protective agent comprise: 100g/L of skimmed milk powder, 30mL/L of glycerol, 100g/L of maltodextrin, 150g/L of trehalose and 10g/L L-sodium glutamate;
(2) sterilizing skimmed milk at 95 deg.C for 20min, and cooling to 4 deg.C to obtain raw material; adding the starter culture obtained in step (1) to the raw material to a concentration of not less than 1' 106CFU/mL to obtain cow milk (the cow milk needs to be refrigerated at 4 ℃).
Example 6 application of Bifidobacterium bifidum CCFM1172
Bifidobacterium bifidum CCFM1172 can be used for preparing soybean milk, and the specific preparation process of the soybean milk is as follows:
(1) inoculating the culture solution obtained in example 1 after the purification of Bifidobacterium bifidum CCFM1172 into a culture medium with the inoculation amount of 3% (v/v), culturing for 36h at 37 ℃ in an anaerobic workstation, centrifuging for 20min at 6000r/min, and collecting bacterial sludge; after the bacterial sludge was washed with PBS, it was resuspended to a concentration of 1' 10 with a protective agent10CFU/mL to obtain a suspension; pre-culturing the suspension at 37 deg.C for 60min, and lyophilizing to obtain starter;
wherein the culture medium is MRS liquid culture medium;
the components of the protective agent comprise: 100g/L of skimmed milk powder, 30mL/L of glycerol, 100g/L of maltodextrin, 150g/L of trehalose and 10g/L L-sodium glutamate;
(2) soaking soybean at 80 deg.C for 2 hr, removing soybean hull to obtain peeled soybean; draining the peeled soybeans from the soaking water, adding boiling water, and grinding into soybean milk to obtain soybean milk; keeping the temperature of the soybean milk at a temperature higher than 80 ℃ for 12 min to obtain cooked soybean milk; filtering the cooked soybean milk with a 150-mesh screen, centrifuging,obtaining coarse soybean milk; heating the coarse soybean milk to 140-150 ℃, and then quickly introducing the coarse soybean milk into a vacuum cooling chamber for vacuumizing, so that peculiar smell substances in the coarse soybean milk are quickly discharged along with water vapor to obtain cooked soybean milk; cooling cooked soybean milk to about 37 deg.C, and adding the starter obtained in step (1) to the cooked soybean milk to a concentration of not less than 1' 10 ″6CFU/mL to obtain soybean milk (the soybean milk needs to be stored at 4 deg.C under refrigeration).
Example 7 application of Bifidobacterium bifidum CCFM1172
Bifidobacterium bifidum CCFM1172 can be used for preparing vegetable beverage, and the vegetable beverage is prepared by the following steps:
(1) inoculating the culture solution obtained by purifying the bifidobacterium bifidum CCFM1172 obtained in the example 1 into a culture medium by the inoculation amount of 3% (v/v), culturing for 36h at 37 ℃ in an anaerobic workstation, centrifuging for 20min at 6000r/min, and collecting bacterial sludge; after the bacterial sludge was washed with PBS, it was resuspended to a concentration of 1' 10 with a protective agent10CFU/mL to obtain a suspension; pre-culturing the suspension at 37 deg.C for 60min, and lyophilizing to obtain starter;
wherein the culture medium is MRS liquid culture medium;
the components of the protective agent comprise: 100g/L of skimmed milk powder, 30mL/L of glycerol, 100g/L of maltodextrin, 150g/L of trehalose and 10g/L L-sodium glutamate;
(2) cleaning fresh vegetables, and squeezing to obtain vegetable juice; thermally sterilizing the vegetable juice at 140 deg.C for 2 s to obtain sterilized vegetable juice; cooling the sterilized vegetable juice to about 37 deg.C, and adding the starter culture obtained in step (1) to the sterilized vegetable juice to a concentration of not less than 1' 10 ℃6CFU/mL to obtain vegetable beverage (the vegetable beverage needs to be stored at 4 deg.C under refrigeration).
Example 8 use of Bifidobacterium bifidum CCFM1172
The bifidobacterium bifidum CCFM1172 can be used for preparing a capsule product, and the specific preparation process of the capsule product is as follows:
(1) the purified culture solution of Bifidobacterium bifidum CCFM1172 obtained in example 1 was inoculated into a culture medium at an inoculum size of 3% (v/v), and cultured at 37 ℃ for 36 hours in an anaerobic workstationCentrifuging at 6000r/min for 20min, and collecting bacterial sludge; after the bacterial sludge was washed with PBS, it was resuspended to a concentration of 2' 10 with skim milk10CFU/mL to obtain a suspension;
(2) adding the suspension prepared in the step (1) into a sodium alginate solution with the concentration of 3% to the concentration of 2' 109Fully stirring after CFU/mL to uniformly disperse cells of bifidobacterium bifidum CCFM1172 in the sodium alginate solution to obtain a mixed 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 30 min, and filtering and collecting the colloidal particles; freeze-drying the collected colloidal particles for 48 hours to obtain powder; and filling the powder into a medicinal capsule to obtain a capsule product.
Example 9 use of Bifidobacterium bifidum CCFM1172
Bifidobacterium bifidum CCFM1172 can be used for preparing fermented milk, and the specific preparation process of the fermented milk is as follows:
(1) inoculating the culture solution obtained in example 1 after the purification of Bifidobacterium bifidum CCFM1172 into a culture medium with the inoculation amount of 3% (v/v), culturing for 36h at 37 ℃ in an anaerobic workstation, centrifuging for 20min at 6000r/min, and collecting bacterial sludge; after the bacterial sludge was washed with PBS, it was resuspended to a concentration of 1' 10 with a protective agent10CFU/mL to obtain a suspension; pre-culturing the suspension at 37 deg.C for 60min, and lyophilizing to obtain lyophilized powder;
wherein the culture medium is MRS liquid culture medium;
the components of the protective agent comprise: 100g/L of skimmed milk powder, 30mL/L of glycerol, 100g/L of maltodextrin, 150g/L of trehalose and 10g/L L-sodium glutamate;
(2) mixing the freeze-dried powder with commercial dry powder starter lactobacillus bulgaricus and commercial dry powder starter streptococcus thermophilus according to the mass ratio of 1:1:1 to obtain starter;
(3) adding sugar into fresh milk to reach a concentration of 5% to obtain a mixed solution; homogenizing the mixed solution at 65 deg.C and 20MPa, and sterilizing at 95 deg.C for 5 min to obtain fermentation raw material; cooling the fermentation raw material to 35 ℃, inoculating the starter prepared in the step (2) into the fermentation raw material in an inoculation amount of 0.03% (v/v), and fermenting at the temperature of 35 ℃ for 16h to obtain fermented milk; after curdling the fermented milk at 42 ℃, refrigerating the fermented milk at 4 ℃ for 24h for after-ripening to obtain a fermented milk finished product.
Example 10 use of Bifidobacterium bifidum CCFM1172
Bifidobacterium bifidum CCFM1172 can be used for preparing tablets, and the specific preparation process of the tablets is as follows:
(1) inoculating the culture solution obtained in example 1 after the purification of Bifidobacterium bifidum CCFM1172 into a culture medium with the inoculation amount of 3% (v/v), culturing for 36h at 37 ℃ in an anaerobic workstation, centrifuging for 20min at 6000r/min, and collecting bacterial sludge; after the bacterial sludge was washed with PBS, it was resuspended to a concentration of 1' 10 with a protective agent10CFU/mL to obtain a suspension; pre-culturing the suspension at 37 deg.C for 60min, and lyophilizing to obtain bacterial powder;
wherein the culture medium is MRS liquid culture medium;
the components of the protective agent comprise: 100g/L of skimmed milk powder, 30mL/L of glycerol, 100g/L of maltodextrin, 150g/L of trehalose and 10g/L L-sodium glutamate;
(2) weighing 25.7 parts by weight of the fungus powder prepared in the step (1), 55.0 parts by weight of starch, 4.5 parts by weight of cellulose derivative, 12.0 parts by weight of sodium carboxymethyl starch, 0.8 part by weight of talcum powder, 1.0 part by weight of cane sugar and 1.0 part by weight of water to obtain a raw material; mixing the raw materials to obtain wet granules; the wet granules were tableted with a tablet press of pharmaceutical machinery of south-central institute, and then dried with a small-sized drug dryer of Yikang Chinese medicine machinery, Inc., of Qingzhou city, to obtain tablets.
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 one skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> bifidobacterium bifidum and application thereof in relieving intestinal injury
<130> BAA210321A
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence
<400> 1
agagtttgat cctggcctca 20
<210> 2
<211> 19
<212> DNA
<213> Artificial sequence
<400> 2
ggttaccttg ttacgactt 19
Claims (8)
1. Bifidobacterium bifidumBifidobacterium bifidum) CCFM1172, deposited at the Guangdong province culture Collection center at 2021, 3/9, with the deposit number GDMCC No: 61550.
2. a microbial agent comprising Bifidobacterium bifidum CCFM1172 according to claim 1.
3. The microbial agent according to claim 2, wherein the viable count of Bifidobacterium bifidum CCFM1172 is not less than 1 x 106CFU/mL or 1X 106 CFU/g。
4. Use of bifidobacterium bifidum CCFM1172 of claim 1 or a microbial agent of claim 2 in the manufacture of a medicament for preventing or ameliorating acute colon injury.
5. The use according to claim 4, wherein the medicament contains Bifidobacterium bifidum CCFM1172 of no less than 1 x 10 viable count6CFU/mL or 1X 106 CFU/g。
6. Use according to claim 4, characterized in that the medicament comprises Bifidobacterium bifidum CCFM1172 according to claim 1, a pharmaceutical carrier and/or a pharmaceutical excipient.
7. Use according to claim 6, wherein the drug carrier is a microcapsule, microsphere, nanoparticle and/or liposome.
8. The use of claim 6, wherein the medicament is in the form of a powder, granules, capsules, tablets, pills or oral liquid.
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