CN114561325B - Bifidobacterium longum capable of changing bile acid content in simulated gastrointestinal tract environment and having constipation relieving effect and application thereof - Google Patents
Bifidobacterium longum capable of changing bile acid content in simulated gastrointestinal tract environment and having constipation relieving effect and application thereof Download PDFInfo
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- CN114561325B CN114561325B CN202210236496.7A CN202210236496A CN114561325B CN 114561325 B CN114561325 B CN 114561325B CN 202210236496 A CN202210236496 A CN 202210236496A CN 114561325 B CN114561325 B CN 114561325B
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Abstract
The invention discloses bifidobacterium longum capable of changing bile acid content in simulated gastrointestinal tract environment and having constipation relieving effect and application thereof, and belongs to the technical field of microorganisms. The bifidobacterium longum CCFM1077 provided by the invention can obviously reduce the content of glycine and bile acid, further plays a role in relieving hyperlipidemia, and simultaneously has the effects of improving intestinal power and relieving constipation. The capacity of bifidobacterium longum CCFM1077 for reducing the content of glycine bile acid is obviously better than that of other three bifidobacterium longum I3, J3 and B3. Therefore, the bifidobacterium longum CCFM1077 with the function of reducing the content of the glycine bile acid has wide application prospect in the directions of foods and microecologics.
Description
Technical Field
The invention relates to bifidobacterium longum capable of changing bile acid content in simulated gastrointestinal tract environment and having constipation relieving effect and application thereof, and belongs to the technical field of microorganisms.
Background
Bile acid is an important component of human bile, and plays roles of digesting fat and absorbing in human body. More and more reports now show that bile acids are important signaling molecules in the human body and play an important role in various organs. The bile acid pool of normal people is about 3-5 g, and the liver and intestine circulation is carried out for 8-12 times every day, wherein 95% of bile acid participates in circulation metabolism, and the rest 5% can be discharged out of the body through feces and urine. The liver is responsible for synthesizing this 5% of bile acids daily to maintain the stability of the bile acid pool. Cholesterol is taken as a raw material in the liver, and is metabolized step by liver enzymes, so that the combined primary bile acid is finally synthesized and discharged into the intestinal tract. Bile acids in the intestinal tract are metabolized through multiple steps of intestinal flora, and the abundant diversity of bile acid spectrums is formed. After the conjugated bile acid enters the intestinal tract, the conjugated bile acid is substituted by Xie Chengfei conjugated bile acid from intestinal bacteria rich in bile salt hydrolase, and the bacterial group rich in bile salt hydrolase comprises bacteroides, clostridium, lactobacillus, bifidobacterium, listeria and the like. Further, intestinal flora modifies hydroxyl groups on the skeleton structure of bile acid, mainly at 7 position, and also at 3 position, 6 position and 12 position, and performs dehydroxylation, dehydrogenation, isomerization and other functions, thereby finally forming secondary bile acid. The bacterial groups involved in these metabolism include Bacteroides, clostridium, escherichia, ruminococcus, and the like. Glycine bile acid is an important bile acid, and can cause liver diseases, metabolic diseases and other diseases under the condition of abnormal metabolism. In general, glycocholic acid is elevated only when liver excretion of cholic acid is impaired when hepatocytes are severely damaged or bile is stagnated. However, there are many causes of liver cell damage, and various viral hepatitis diseases are the main causes of liver function index glycocholic acid to be high, so that it is critical that patients be alerted to viral hepatitis. In addition, according to the statistics of the related data, the liver function inspection index glycocholic acid of patients with acute hepatitis, chronic active hepatitis, primary liver cancer and liver cirrhosis is obviously higher than that of normal people, and the presentability is increased. Moreover, it is verified that the increase in the content of glycocholate in these patients is caused by pathological changes of the liver, impaired liver function and reduced capacity of hepatic cells to take up glycocholate, which results in an increase in the content of glycocholate in serum. In addition, if cholestasis occurs, liver excretion of glycocholic acid is also hindered, and reflux in blood promotes increase of glycocholic acid content. Therefore, the regulation of bile acid metabolism, especially the metabolism of glycine bile acid, can play a certain role in promoting the regulation of the actions of the glycine bile acid on the aspects of body health and diseases.
In recent years, as research methods are being advanced, a great deal of research has found that the regulation of bile acids is associated with lipid metabolism diseases or liver diseases. For example, a literature published by Degirolamo et al in 2014 at Cell Reports demonstrates that supplementing the probiotic product VSL#3 can significantly alter the hepatointestinal circulation of bile acids, mainly the TCA/TβMCA and total bile acid content. Susan A. Joyce et al published in 2014 in PNAS a document that demonstrated that bile salt hydrolase produced by probiotics was able to significantly alter the composition of bile acids, mainly by altering the content of taurine bile acids.
Constipation is receiving increasing attention as a disease that severely affects quality of life. Epidemiological investigation showed that constipation has a global incidence of 0.7% -79% (on average up to 16%); there is a 3% -17% incidence in China alone. There are many causes of constipation, such as imbalance of diet, microbial disorder, side effects of drugs, complications caused by metabolic diseases such as diabetes, genetic diseases, mechanical ileus, and neurological diseases. The treatment means are different for different pathogenesis reasons. For lighter patients, people are often encouraged to eat foods with rich dietary fibers such as fresh vegetables, fruits, beans and the like, but some patients with serious diseases can use permeable or irritant laxatives such as polyethylene glycol, lactulose or anthraquinone derivatives and the like, but the laxatives often cause dependency, even side effects such as nausea, abdominal pain, diarrhea and the like, and if the patients are serious enough to cause obvious lesions on intestinal tissues, the patients need to be treated by surgery. Therefore, probiotics are widely accepted at present as a treatment means with less side effects, obvious alleviation effect and weak dependence.
At present, the physiological functions related to bifidobacteria are studied by scholars at home and abroad, and the application of the bifidobacteria to constipation relieving effect is further explored. The microbial inoculum prepared by the microbial inoculum or the functional food prepared by adding the microbial inoculum into the food has great application prospect, and can prevent constipation and even treat constipation. By studying bifidobacteria for relieving constipation, there will be a great influence on various aspects of food science, microbiology, preventive medicine and the like, and therefore, studies on bifidobacteria for relieving constipation are necessary.
Related patent: the Mark G-Corii et al patent (CN 111050755A) reports that colesevelam or colesevelam hydrochloride bile acid sequestrants can reduce the bile acid level in the digestive tract and thereby alleviate the symptoms of gastroesophageal reflux disease patients. These data indicate that intake of some drugs has a significant effect on regulating bile acid metabolism. However, regulation of glycyrhizinate metabolism for probiotics remains blank.
Disclosure of Invention
In order to solve the above problems, the present invention provides a bifidobacterium longum (Bifidobacterium longum subsp. Longum) CCFM1077 deposited at the microorganism strain deposit center in guangdong province at 9/5/2019 under the deposit number GDMCC No:60769, the preservation address is building 5 of road 100 university, no. 59 in Guangzhou city martyr.
The invention also provides a microbial preparation containing the bifidobacterium longum CCFM 1077.
In one embodiment, the viable count of bifidobacterium longum CCFM1077 in the microbial preparation is not less than 1X 10 6 CFU/mL or 1X 10 6 CFU/g.
The invention also provides a product for regulating bile acid metabolism, which contains the bifidobacterium longum CCFM1077.
In one embodiment, the viable count of bifidobacterium longum CCFM1077 in the product is no less than 1X 10 6 CFU/mL or 1X 10 6 CFU/g.
In one embodiment, the product is a food, pharmaceutical or nutraceutical product that is ingestible into the gastrointestinal tract.
In one embodiment, the food product comprises fermented fruit and vegetable, fermented milk, cheese, milk-containing beverages, milk powder or other food products comprising bifidobacterium longum as described above.
The invention also provides application of the bifidobacterium longum CCFM1077 or the microbial preparation in preparing products for reducing the content of glycine and bile acid, improving the content of dehydrobile acid and/or relieving constipation.
In one embodiment, the glycocholate includes, but is not limited to, at least one of glycopig bile acid, glycohyodeoxycholic acid, glycobear deoxybile acid.
In one embodiment, the dehydrobile acid includes, but is not limited to, at least one of 3-dehydrocholic acid, 7-dehydrocholic acid, 12-dehydrocholic acid.
In one embodiment, the relief of constipation includes, but is not limited to, increasing fecal moisture content, decreasing intestinal transit time.
The beneficial effects are that:
The bifidobacterium longum CCFM1077 provided by the invention has good tolerance performance in simulated gastric and intestinal fluids, and can obviously change the composition of bile acid composition (compared with a control strain, the bifidobacterium longum CCFM1077 reduces the content of glycine by nearly 8 times), thereby relieving the occurrence of hyperlipidemia. And bifidobacterium longum CCFM1077 was significantly better in reducing glycobile acid content than the control strain bifidobacterium longum strains I3, J3, B3 (disclosed in paper "Strain-Specific Effects of Bifidobacterium longum on Hypercholesterolemic Rats and Potential Mechanisms", and selected from the same sample as bifidobacterium longum CCFM1077 screen). Bifidobacterium longum CCFM1077 also has the effect of relieving loperamide-induced mouse constipation. The bifidobacterium longum strain with the function of changing the composition of bile acid has wide application prospect in the directions of foods and microecologics.
Preservation of biological materials
Bifidobacterium longum (Bifidobacterium longum subsp. Longum) CCFM1077, taxonomically designated Bifidobacterium longum subsp. Longum, deposited at the center of collection of microorganisms in the cantonese province at 5/9/2019 under accession number GDMCC No:60769, the preservation address is building 5 of road 100 university, no. 59 in Guangzhou city martyr.
Drawings
FIG. 1 is a graph of colonies of bifidobacterium longum CCFM1077 grown on mMRS medium.
Figure 2 is the effect of bifidobacterium longum CCFM1077 on the concentration of glycine bile acid in a simulated gastrointestinal environment.
FIG. 3 is the effect of bifidobacterium longum CCFM1077 on glycohyocholic acid content in simulated gastrointestinal environments.
FIG. 4 is the effect of bifidobacterium longum CCFM1077 on the content of glycohyodeoxycholic acid in simulated gastrointestinal environments.
FIG. 5 is the effect of bifidobacterium longum CCFM1077 on the content of glycoursodeoxycholic acid in simulated gastrointestinal environment.
FIG. 6 is the effect of Bifidobacterium longum CCFM1077 on the 3-dehydrocholic acid content in a simulated gastrointestinal environment.
FIG. 7 is the effect of Bifidobacterium longum CCFM1077 on the 7-dehydrocholic acid content in a simulated gastrointestinal environment.
FIG. 8 is the effect of Bifidobacterium longum CCFM1077 on 12-dehydrocholic acid content in simulated gastrointestinal environment.
Fig. 9 is the effect of bifidobacterium longum CCFM1077 on loperamide-induced reduction in the frequency of bowel movements in mice.
Fig. 10 is the effect of bifidobacterium longum CCFM1077 on loperamide-induced reduction in intestinal transit time in mice.
Fig. 11 is the effect of bifidobacterium longum CCFM1077 on loperamide-induced reduction of fecal moisture in mice.
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 with reference to the accompanying drawings.
The following examples relate to the following media:
MRS liquid medium: 10g/L of peptone, 10g/L of beef extract, 5g/L of yeast extract, 20g/L of glucose, 2g/L of anhydrous sodium acetate, 1g/L of citric acid diamine 2g/L,K2HPO4·3H2O 2.6g/L,MgSO4·7H2O 0.5g/L,MnSO4·H2O 0.25g/L, cysteine hydrochloride, 1g/L of tween-80 and 1000g/L of distilled water.
MRS solid medium: 10g/L of peptone, 10g/L of beef extract, 5g/L of yeast extract, 20g/L of glucose, 2g/L of anhydrous sodium acetate, 1g/L of citric acid diamine 2g/L,K2HPO4·3H2O 2.6g/L,MgSO4·7H2O 0.5g/L,MnSO4·H2O 0.25g/L, cysteine hydrochloride, 1g/L of tween-80, 20g/L of agar and 1000g/L of distilled water.
The preparation method of the general bacteroides bacterial suspension related in the following examples comprises the following steps:
Scribing on MRS solid culture medium, anaerobic culturing at 37deg.C for 48 hr to obtain single colony; picking single colony, inoculating in MRS liquid culture medium, culturing at 37deg.C for 18 hr for activation, and activating for two generations to obtain activating solution; inoculating the activating solution into MRS liquid culture medium according to the inoculum size of 2% (v/v), and culturing for 18h at 37 ℃ to obtain bacterial solution; centrifuging the bacterial liquid for 10min by 8000g to obtain bifidobacterium longum bacterial cells; the bifidobacterium longum thalli is washed by normal saline and then resuspended in a glycerol solution with the concentration of 200g/L (containing 1g/L cysteine hydrochloride) until the bacterial concentration is 1 multiplied by 10 10 CFU/mL, so as to obtain bacterial suspension, and the bacterial suspension is preserved at the temperature of minus 80 ℃ for standby.
Example 1: separation and screening of bifidobacterium longum CCFM111077
(L) 1g of fresh feces of healthy people is taken. Coating the diluted solution on mMRS solid culture medium, and culturing at 37 ℃ for 72 hours in anaerobic environment;
(2) Observing and recording the morphology of the bacterial colony, picking the bacterial colony, and streaking and purifying;
(3) The colonies obtained were gram stained in MRS liquid medium at 37℃for 48 hours, and colony morphology was recorded.
(4) Removing gram-negative bacterial strains and gram-positive cocci in the bacterial colonies, and selecting to obtain the gram-positive bacilli.
(5) After the catalase analysis, the catalase positive strain was discarded, and the catalase negative strain was retained.
(II) preliminary identification of bifidobacteria: fructose-6-phosphate phosphoketolase assay
(1) Culturing the lactobacillus obtained by screening in the step (one) in a liquid mMRS culture solution for 24 hours, and centrifuging the lmL culture at 8000rpm for 2 minutes;
(2) Washing twice with a 0.05M KH 2PO4 solution of pH6.5 containing 0.05% by mass of cysteine hydrochloride;
(3) Resuspended in 200. Mu.L of the above phosphate buffer with the addition of 0.25% (mass percent) Triton X-100;
(4) 50. Mu.L of a mixture of sodium fluoride at a concentration of 6mg/mL and sodium iodoacetate at a concentration of 10mg/mL was added, and 50. Mu.L of fructose-6-phosphate at a concentration of 80mg/mL was incubated at 37℃for 1 hour;
(5) 300. Mu.L of hydroxylamine hydrochloride having a concentration of 0.139g/mL and a pH of 6.5 was added and left at room temperature for 10min;
(6) 200. Mu.L of 15% (mass percent) trichloroacetic acid and 4M HCI were added, respectively;
(7) 200 mu L of 0.1M HCI containing 5 percent (mass percent) of ferric trichloride is added, the system rapidly turns red, namely F6PPK positive is obtained, and the bifidobacterium is preliminarily judged.
(III) molecular biological identification of bifidobacteria:
(l) Single bacterial genome extraction: culturing the bifidobacterium obtained by screening in the step (II) overnight, taking bacterial suspension lmL cultured overnight, centrifuging at 10000rpm for 2min in a 1.5mL centrifuge tube, and discarding the supernatant to obtain thalli; washing the thalli with lmL sterile water, centrifuging at 10000rpm for 2min, and discarding the supernatant to obtain thalli; 200 mu L of SDS lysate is added, and water bath is carried out for 30min at 80 ℃; adding 200 mu L of phenol-chloroform solution into a thallus lysate, wherein the phenol-chloroform solution comprises the components of Tris saturated phenol and chloroform isoamyl alcohol=25:24:1 in volume ratio, mixing the components in a reversed way, centrifuging at 12000rpm for 5-10min, and taking 200 mu L of supernatant; adding 400 μl of ice ethanol or ice isopropanol to 200uL of supernatant, standing at-20deg.C for 1h, centrifuging at 12000rpm for 5-10min, and discarding supernatant; adding 500 μL70% (volume percent) of ice ethanol to resuspend the precipitate, centrifuging at 12000rpm for 1-3min, and discarding the supernatant; oven drying at 60deg.C, or naturally air drying; 50 μl ddH 2 O was redissolved for PCR;
(2)16S rDNA PCR：
A. Bacterial 16S rDNA 50. Mu. LPCR reaction System:
10×Taq buffer, 5. Mu.L; dNTP, 5. Mu.L; 27F, 0.5. Mu.L; 1492R, 0.5. Mu.L; taq enzyme, 0.5. Mu.L; template, 0.5 μl; ddH 2 O, 38. Mu.L.
PCR conditions:
95℃5min；95℃10s；55℃30s；72℃30s；step2-4 30×；72℃5min；12℃2min；
C. Preparing 1% agarose gel, mixing the PCR product with 10000×loading buffer, loading 2 μl, running at 120V for 30min, and performing gel imaging;
D. The obtained PCR product was sent to a professional sequencing company, and the obtained sequencing result was compared with the search and similarity in GeneBank using BLAST, and identified as Bifidobacterium longum.
(3) Whole genome sequencing
The extracted whole genome is sent to a professional sequencing company, a second generation sequencer is used for sequencing the whole genome of the strain, the obtained sequence result is searched in a GeneBank by using BLAST and is subjected to similarity comparison, the identification result shows that the Average Nucleotide Identity (ANI) between B.longum CCFM 1077 and a standard strain Bifidobacterium longum NCC2705 genome is 98.3 percent and is higher than a strain identification level threshold (more than or equal to 95 percent), and the result shows that the strain is a new bifidobacterium longum strain. Strains I3, B3, J3, s7 were screened from healthy adult human fecal samples, identified as bifidobacterium longum, and subsequent experiments were used as control strains (strains I3, B3, J3 have been disclosed in paper "Strain-Specific Effects of Bifidobacterium longum on Hypercholesterolemic Rats and Potential Mechanisms"). The strain is preserved at-80 ℃ for standby.
Example 2: bifidobacterium longum subspecies longum CCFM1077 has good tolerance to simulated gastrointestinal fluids
The frozen and preserved bifidobacterium longum subspecies CCFM1077 is inoculated in mMRS culture medium (MRS culture medium plus 0.05 percent cysteine hydrochloride), anaerobic culture is carried out for 48 hours at the temperature of 37 ℃, and after 2-3 times of subculture by mMRS culture solution, 1mL of culture solution of the bifidobacterium subspecies CCFM1077 is taken, mixed with 9.0mL of artificial simulated gastric fluid (mMRS culture medium containing 1 percent gastric protease and pH=2.5) with the pH of 2.5, anaerobic culture is carried out at the temperature of 37 ℃, sampling is carried out at the time of 0 hour, 0.5 hour, 1 hour and 2 hours respectively, and the colony count of the flat plate is carried out by casting culture with mMRS agar culture medium, and the viable count is measured and the survival rate is calculated.
The survival rate is the ratio of the number of viable bacteria to the number of viable bacteria at the time of sampling in the culture solution to the number of viable bacteria at the time of 0h, expressed in%. 1mL of a culture solution of Bifidobacterium longum subspecies longum CCFM1077 was added to 9mL of an artificial simulated intestinal fluid (mMRS medium containing 0.3% bovine bile salt, 1% trypsin and pH=8.0), anaerobic culture was performed at 37℃and samples were taken at 0h, 0.5h, 1 h and 2h, respectively, plate colony counts were performed by casting culture with mMRS agar medium, the viable count was determined and the survival rate was calculated. The survival rate is the ratio of the number of viable bacteria to the number of viable bacteria at the time of sampling in the culture solution to the number of viable bacteria at the time of 0h, expressed in%. The experimental results are shown in tables 1 and 2. The result shows that the bifidobacterium longum subspecies longum CCFM1077 has better tolerance to artificial gastrointestinal fluids.
TABLE 1 tolerance of Bifidobacterium longum subspecies longum CCFM1077 in artificial simulated gastric fluid
TABLE 2 tolerance of Bifidobacterium longum subspecies longum CCFM1077 in artificial simulated intestinal juice
Example 3: effect of bifidobacterium longum CCFM1077 in reducing content of glycine and bile acid in simulated gastrointestinal fluid environment
Single cells from the freshly streaked and grown solid media were inoculated into 10mL MRS broth and placed in an anaerobic incubator at 37℃for 18 hours. Each broth was normalized to OD 600 of 1 and then the cells were prepared and washed twice with PBS. The washed cells were resuspended, and 1ml of the cells were added to LB broth containing 0.5% of bile derived from porcine gall bladder. Adding bile acid in human body which is deleted into LB culture medium which is added with pig bile acid, comprising: cholic acid (cholic acid), α -muricholic cholic acid, β -muricholic cholic acid, ω -muricholic cholic acid). 1mL of the bacterial suspension with the bacterial concentration of 1X 10 7 CFU/mL is incubated with bile for 1.5 hours. Deuterated internal standards of cholic acid and chenodeoxycholic acid are added simultaneously. Followed by centrifugation in a centrifuge, centrifugation conditions: 10000g,10min. The supernatant was extracted with 1 volume of methanol (divided into 2 tubes) to a final concentration of 50% methanol. The mixture was allowed to stand at-20℃for 30 minutes and subjected to vortex treatment. Then dried under vacuum under nitrogen and re-extracted with acetonitrile and formic acid (1 ml). In this step, the samples are pooled together. The extract was centrifuged and dried and resuspended in 150. Mu.l of 50% methanol on the day of MS detection or stored at-20℃until the day of UPLC-MS detection. The results show that CCFM1077 can significantly reduce the content of glycobile acid (1858.4 ng/mL), 87.7 percent (figure 2) compared with a blank control group (15156.4 ng/mL), the content of glycopig bile acid is reduced from 440317.95ng/mL to 20112.2ng/mL, 95.4 percent (figure 3), the content of glycopig deoxycholic acid is reduced from 348530.8ng/mL to 3350.6 ng/mL, 99.1 percent (figure 4), and the content of glycobear deoxybile acid is reduced from 5061.4ng/mL to 928.0ng/mL, and 81.7 percent (figure 5).
Example 4: function of bifidobacterium longum CCFM1077 in improving content of dehydrobile acid in simulated gastrointestinal fluid environment
Single cells from the freshly streaked and grown solid media were inoculated into 10mL MRS broth and placed in an anaerobic incubator at 37℃for 18 hours. Each broth was normalized to OD 600 of 1 and then the cells were prepared and washed twice with PBS. The final concrete was resuspended in LB broth medium containing 0.5% of bile of porcine cholecystokinis. The corresponding missing bile acid-bile acid, cholic acid (alpha, beta, omega) and conjugated forms thereof are added into the main mixed bile acid LB. The cells were incubated with bile for 1.5 hours. Deuterated internal standards of cholic acid and chenodeoxycholic acid are added simultaneously. Followed by centrifugation in a centrifuge, centrifugation conditions: 10000g, 10min. The supernatant was extracted with 1 volume of methanol (divided into 2 tubes) to a final concentration of 50% methanol. The mixture was allowed to stand at-20℃for 30 minutes and subjected to vortex treatment. Then dried under vacuum under nitrogen and re-extracted with acetonitrile and formic acid (1 ml). In this step, the samples are pooled together. The extract was centrifuged and dried and resuspended in 150. Mu.l of 50% methanol on the day of MS detection or stored at-20℃until the day of UPLC-MS detection. The results showed that bifidobacterium longum CCFM1077 increased 3-dehydrocholate from 8.8 to 1331.5ng/mL by 150-fold (FIG. 6), 7-dehydrocholate from 16.6 to 180.7ng/mL by 9.9-fold (FIG. 7), and 12-dehydrocholate from 120.0 to 408.1ng/mL by 2.4-fold (FIG. 8) compared to the blank.
Example 5: bifidobacterium longum CCFM1077 for preparing fermented cow milk
The bifidobacterium longum CCFM1077 can be used for preparing fermented cow milk, and the specific preparation process of the cow milk is as follows:
(1) Inoculating the secondary purified culture solution of the bifidobacterium longum CCFM1077 obtained in the example 1 into a culture medium with an inoculum size of 3% (v/v), and culturing for 18 hours at 37 ℃ to obtain a bacterial solution; centrifuging the bacterial liquid to obtain bacterial mud; washing the bacterial sludge with phosphate buffer solution with pH of 7.2 for 3 times, and then re-suspending the bacterial sludge with a protective agent until the concentration is 1X 10 10 CFU/mL to obtain suspension; pre-culturing the suspension at 37 ℃ for 60min, and freeze-drying to obtain a starter;
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, 10g/L L-sodium glutamate;
(2) Sterilizing the skimmed milk at 95deg.C for 20min, and cooling to 4deg.C to obtain raw materials; adding the starter obtained in the step (1) into raw materials until the concentration is not lower than 1× 6 CFU/mL to obtain cow milk (cow milk is required to be refrigerated and stored at 4deg.C).
Example 6: bifidobacterium longum CCFM1077 for preparing fermented soybean milk
Bifidobacterium longum CCFM1077 can be used for preparing soymilk, and the specific preparation process of the soymilk is as follows:
(1) Inoculating the secondary purified culture solution of the bifidobacterium longum CCFM1077 obtained in the example 1 into a culture medium with an inoculum size of 3% (v/v), and culturing for 18 hours at 37 ℃ to obtain a bacterial solution; centrifuging the bacterial liquid to obtain bacterial mud; washing the bacterial sludge with phosphate buffer solution with pH of 7.2 for 3 times, and then re-suspending the bacterial sludge with a protective agent until the concentration is 1X 10 10 CFU/mL to obtain suspension; pre-culturing the suspension at 37 ℃ for 60min, and freeze-drying to obtain a starter;
The preparation method of the culture medium comprises the following steps: dissolving 10% of skim milk, 0.5% of glucose, 1.5% of tryptone and 0.3% of yeast extract by using 87.7% of water based on the total weight of the culture medium, and then adjusting the pH value to 6.8 to obtain the culture medium;
the components of the protective agent comprise: 100g/L skimmed milk powder, 30mL/L glycerol, 100g/L maltodextrin, 150g/L trehalose, 10g/L L-sodium glutamate;
(2) Soaking soybean at 80deg.C for 2 hr, removing soybean hull to obtain peeled soybean; removing the soaked water from peeled soybean, adding boiling water, and grinding to obtain soybean milk; maintaining the temperature of the soybean milk at a temperature higher than 80 ℃ for 12min to obtain cooked soybean milk; filtering cooked soybean milk with 150 mesh sieve, and centrifuging to obtain coarse soybean milk; heating the crude soymilk to 140-150 ℃, then rapidly introducing the heated crude soymilk into a vacuum cooling chamber for vacuumizing, so that the peculiar smell substances in the crude soymilk are rapidly discharged along with water vapor to obtain cooked soymilk; cooling cooked soybean milk to 37deg.C, adding the starter prepared in step (1) to the cooked soybean milk until the concentration is not lower than 1× 6 CFU/mL, and obtaining soybean milk (the soybean milk is stored at 4deg.C).
Example 7: bifidobacterium longum CCFM1077 for preparing fruit and vegetable beverage
The bifidobacterium longum CCFM1077 can be used for preparing vegetable beverages, and the specific preparation process of the vegetable beverages is as follows:
(1) Inoculating the secondary purified culture solution of the bifidobacterium longum CCFM1077 obtained in the example 1 into a culture medium with an inoculum size of 3% (v/v), and culturing for 18 hours at 37 ℃ to obtain a bacterial solution; centrifuging the bacterial liquid to obtain bacterial mud; washing the bacterial sludge with phosphate buffer solution with pH of 7.2 for 3 times, and then re-suspending the bacterial sludge with a protective agent until the concentration is 1X 10 10 CFU/mL to obtain suspension; pre-culturing the suspension at 37 ℃ for 60min, and freeze-drying to obtain a starter;
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, 10g/L L-sodium glutamate;
(2) Cleaning fresh vegetables, and squeezing to obtain vegetable juice; sterilizing the vegetable juice at 140 ℃ for 2 seconds to obtain sterilized vegetable juice; cooling the sterilized vegetable juice to about 37 ℃, and adding the starter prepared in the step (1) into the sterilized vegetable juice until the concentration is not lower than 1X 10 6 CFU/mL, thereby obtaining the vegetable beverage (the vegetable beverage is required to be refrigerated and stored at 4 ℃).
Example 8: bifidobacterium longum CCFM1077 for preparing capsule product
The bifidobacterium longum CCFM1077 can be used for preparing capsule products, and the specific preparation process of the capsule products is as follows:
(1) Inoculating the secondary purified culture solution of the bifidobacterium longum CCFM1077 obtained in the example 1 into a culture medium with an inoculum size of 3% (v/v), and culturing for 18 hours at 37 ℃ to obtain a bacterial solution; centrifuging the bacterial liquid to obtain bacterial mud; washing the bacterial sludge with phosphate buffer solution with pH of 7.2 for 2 times, and then re-suspending the bacterial sludge with skimmed milk until the concentration is 2X 10 10 CFU/mL to obtain suspension;
(2) Adding the suspension prepared in the step (1) into a sodium alginate solution with the concentration of 3% until the concentration is 2 multiplied by 10 9 CFU/mL, and fully stirring to uniformly disperse cells of bifidobacterium longum CCFM1077 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; 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; and (5) filling the powder into a medicinal capsule to obtain a capsule product.
Example 9: bifidobacterium longum CCFM1077 for preparing fermented dairy products
The bifidobacterium longum CCFM1077 can be used for preparing fermented milk, and the specific preparation process of the fermented milk is as follows:
(1) Inoculating the secondary purified culture solution of the bifidobacterium longum CCFM1077 obtained in the example 1 into a culture medium with an inoculum size of 3% (v/v), and culturing for 18 hours at 37 ℃ to obtain a bacterial solution; centrifuging the bacterial liquid to obtain bacterial mud; washing the bacterial sludge with phosphate buffer solution with pH of 7.2 for 3 times, and then re-suspending the bacterial sludge with a protective agent until the concentration is 1X 10 10 CFU/mL to obtain suspension; pre-culturing the suspension at 37 ℃ for 60min, and freeze-drying to obtain freeze-dried powder, wherein the concentration of thalli in the powder is 1 multiplied by 10 9 CFU/g;
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, 10g/L L-sodium glutamate;
(2) Mixing the freeze-dried powder with commercial dry powder starter lactobacillus bulgaricus (the thallus concentration is 1×10 9 CFU/g) and commercial dry powder starter streptococcus thermophilus (the thallus concentration is 1×10 9 CFU/g) according to the mass ratio of 1:1:1 to obtain starter, wherein the bacterial concentration of the lactobacillus bulgaricus and the streptococcus thermophilus in the starter is 2%;
(3) Adding sugar into fresh milk until the concentration is 5% to obtain a mixed solution; homogenizing the mixed solution at 65deg.C and 20MPa, and sterilizing at 95deg.C for 5min 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 with an inoculum size of 0.03% (v/v), and fermenting at 35 ℃ for 16 hours to obtain fermented milk; and (3) after the fermented milk is curdled at 42 ℃, refrigerating at 4 ℃ for 24 hours for after-ripening, thus obtaining a fermented milk finished product.
Example 10: application of bifidobacterium longum CCFM1077
Bifidobacterium longum CCFM1077 can be used for preparing tablets, and the specific preparation process of the tablets is as follows:
(1) Inoculating the secondary purified culture solution of the bifidobacterium longum CCFM1077 obtained in the example 1 into a culture medium with an inoculum size of 3% (v/v), and culturing for 18 hours at 37 ℃ to obtain a bacterial solution; centrifuging the bacterial liquid to obtain bacterial mud; washing the bacterial sludge with phosphate buffer solution with pH of 7.2 for 3 times, and then re-suspending the bacterial sludge with a protective agent until the concentration is 1X 10 10 CFU/mL to obtain suspension; pre-culturing the suspension at 37 ℃ for 60min, and freeze-drying to obtain 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, 10g/L L-sodium glutamate;
(2) Weighing 25.7 parts by weight of the bacterial 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 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 a raw material; 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.
The products prepared in examples 5-10 were added to simulated gastrointestinal fluid environment, and the results showed that the products containing bifidobacterium longum CCFM1077 all had the effect of regulating the content of glycine bile acid and/or the content of dehydrobile acid.
Example 11: bifidobacterium longum CCFM1077 induces relief of constipation related symptoms to loperamide
After 50 male BALB/c mice of 8 weeks of age were acclimatized for 1 week, 10 mice per group were randomly divided into 5 groups including a blank control (gavage normal saline), a constipation model group (gavage loperamide and normal saline), a positive drug (phenolphthalein) control group (gavage loperamide and 7mg/mL phenolphthalein solution), a bifidobacterium longum S7 group and a bifidobacterium longum CCFM1077 group (gavage loperamide and bacterial suspension of bifidobacterium longum with bacterial concentration of 1X 10 9 CFU/mL), and gavage was performed for 14 days. Mice were kept in IVC cages in animal houses at constant temperature and humidity (temperature: 25±2 ℃; humidity: 55% ±5%) for 12h darkness and 12h light cycles, and mice were given quasi-commercial mice with food and free water during the experiment. Animal protocols were approved by the university of south China ethical committee (JN.No20180615b0800804 [159 ]), and experimental procedures were performed according to the European Union established guidelines for laboratory animals (direct 2010/63/EU).
And collecting the mouse feces before the experiment is finished, injecting 1% pentobarbital sodium solution into the abdominal cavity of the mouse for anesthesia after the experiment is finished, collecting blood and colon contents, measuring the pushing length of the ink, and freezing the sample in a refrigerator at the temperature of minus 80 ℃ for measuring other indexes. Collecting the faeces of mice on 8-10 early days 0, 7 and 14, placing each mouse in a clean IVC cage box independently, collecting the faeces of the mice in time, recording the particle number of the faeces, storing in an ice box, recording the dry weight of the faeces after freeze drying, and calculating the water content of the faeces by adopting the following formula: fecal moisture (%): (wet fecal weight-dry fecal weight)/(wet fecal weight x 100%). The initial black stool time of mice was measured on day 14, and the mice of the remaining groups were filled with 0.2mL loperamide in addition to the blank group mice, and after 1h, the blank group mice and the constipation group mice were filled with 0.25mL ink, and the bifidobacterium longum CCFM1077 group, bifidobacterium longum S7 group and the positive drug control group were filled with 0.25mL ink containing the respective gastric lavage contents, and the gastric lavage mice were placed in a clean IVC cage with white filter paper laid thereon, and the time for discharging the first black stool after each of the mice was filled with the gastric ink was recorded.
As can be seen from fig. 9-11, the bifidobacterium longum CCFM1077 group can significantly increase the fecal water content, the defecation frequency and the intestinal transit time compared with the constipation model group, wherein the fecal water content after the bifidobacterium longum CCFM1077 is irrigated can reach 62.8%, which is 67% higher than the constipation model group (P < 0.0001); after the bifidobacterium longum CCFM1077 is infused, the intestinal transit time (167 min) is shortened to a certain extent, and is shortened by 36.7% compared with a constipation model group; the frequency of bowel movement was significantly reduced (P < 0.001) after lavage with bifidobacterium longum CCFM 1077. Meanwhile, compared with a control group (the water content of the excrement is 50.8%, the intestinal transit time is 178 min), the water content of the excrement is increased by 23.7%, and the intestinal transit time is reduced by 6.3%. And on the three apparent indexes, the action effect of the bifidobacterium longum CCFM1077 is better than that of the bifidobacterium longum S7. In conclusion, bifidobacterium longum CCFM1077 has a good constipation relieving effect from the apparent index.
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 (2)
1. Use of bifidobacterium longum (Bifidobacterium longum subsp. Longum) CCFM1077 or a microbial preparation containing bifidobacterium longum CCFM1077 in the production of dehydrobile acid; the bifidobacterium longum CCFM1077 has been deposited at the microorganism strain collection in Guangdong province on 9/5 th 2019 under accession number GDMCC No:60769.
2. The use according to claim 1, wherein the dehydrobile acid is at least one of 3-dehydrocholic acid, 7-dehydrocholic acid, 12-dehydrocholic acid.
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| CN111073828A (en) * | 2019-11-19 | 2020-04-28 | 江南大学 | Bifidobacterium longum subspecies longum and application thereof |
| CN113068837A (en) * | 2021-04-06 | 2021-07-06 | 江南大学 | Bifidobacterium longum capable of eliminating nonyl phenol and relieving poisoning symptoms caused by nonyl phenol |
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| CN111073828A (en) * | 2019-11-19 | 2020-04-28 | 江南大学 | Bifidobacterium longum subspecies longum and application thereof |
| CN113068837A (en) * | 2021-04-06 | 2021-07-06 | 江南大学 | Bifidobacterium longum capable of eliminating nonyl phenol and relieving poisoning symptoms caused by nonyl phenol |
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| Jinchi Jiang等.Strain-Specific Effects of Bifidobacterium longum on Hypercholesterolemic Rats and Potential Mechanisms.Int J Mol Sci.2021,第22卷(第3期),参见摘要. * |
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