Bifidobacterium longum capable of changing bile acid content in simulated gastrointestinal tract environment and relieving constipation and application of bifidobacterium longum
A technology of bifidobacteria longum and bile acid, applied in the field of microorganisms, can solve the problem of glycine bile acid being in a blank state and the like
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Embodiment 1
[0039] Example 1: Isolation and screening of Bifidobacterium longum CCFM111077
[0040] (l) Take 1g of fresh feces from a healthy person. After gradient dilution, spread on mMRS solid medium and culture in an anaerobic environment at 37°C for 72 hours;
[0041] (2) Observe and record the colony morphology, pick and purify the colony by streaking;
[0042] (3) Cultivate in MRS liquid medium at 37° C. for 48 hours, perform Gram staining on the obtained colonies, and record the colony morphology.
[0043] (4) Discard the Gram-negative bacterial strains and Gram-positive cocci in the colonies, and select the Gram-positive bacilli.
[0044] (5) After the catalase analysis, discard the catalase-positive strains and keep the catalase-negative strains.
[0045] (2) Preliminary identification of bifidobacteria: fructose-6-phosphate phosphoketolase assay
[0046] (1) the lactic acid bacteria that step (1) is screened is cultivated in liquid mMRS nutrient solution for 24h, then get 1...
Embodiment 2
[0064] Example 2: Bifidobacterium longum subspecies CCFM1077 has good tolerance to simulated gastrointestinal fluid
[0065] The cryopreserved Bifidobacterium longum subsp. longum CCFM1077 was inoculated in mMRS medium (MRS medium + 0.05% cysteine hydrochloride), cultured anaerobically at 37°C for 48 hours, and then subcultured in mMRS medium After 2 to 3 times, take 1 mL of the culture solution of Bifidobacterium longum subsp. longum CCFM1077, mix it with 9.0 mL of pH 2.5 artificial simulated gastric juice (mMRS medium containing 1% pepsin, pH=2.5), and incubate at 37°C. For anaerobic culture, samples were taken at 0h, 0.5h, 1h and 2h respectively, and mMRS agar medium was poured for culture to count plate colonies, determine the number of viable bacteria and calculate their survival rate.
[0066] The survival rate is the ratio of the logarithmic value of the number of viable bacteria at the time of sampling to the logarithmic value of the number of viable bacteria at 0h i...
Embodiment 3
[0071] Example 3: Effect of Bifidobacterium longum CCFM1077 on reducing glycine bile acid content in simulated gastrointestinal fluid environment
[0072] Inoculate 10 mL of MRS liquid medium with a single bacterium derived from the newly streaked and grown solid medium, and place it in an anaerobic incubator at 37°C for 18 hours. Each medium was normalized to OD 600The value is 1, and then the cells are prepared and washed twice with PBS. The washed bacterium was resuspended, and 1 ml of thalline was added to the LB broth medium containing 0.5% porcine gallbladder-derived bile. In the LB medium to which porcine bile acids have been added, the missing bile acids in the human body are added, including: cholic acid, α-muricholic cholic acid, β-muricholic cholic acid, ω-muricholic cholic acid). 1mL bacterial concentration is 1×10 7 CFU / mL bacterial suspension was co-incubated with bile for 1.5 hours. Deuterated internal standards of cholic acid and chenodeoxycholic acid were ...
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