Lactobacillus reuteri composition capable of relieving rheumatoid arthritis
A Lactobacillus reuteri, rheumatoid technology, applied in the field of Lactobacillus reuteri composition, to achieve the effect of inhibiting level, relieving rheumatoid arthritis, and improving the composition of intestinal flora
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Embodiment 1
[0038] Example 1: Adhesion performance of Lactobacillus reuteri to human colon adenocarcinoma cell Caco2 in vitro
[0039] Caco2 cells were cultured in DEME (5% fetal bovine serum, 1% antibiotics) medium, and the medium was changed every day until the degree of cell aggregation was 80-90%. After washing three times with PBS, the cells were digested with pre-warmed 0.25% trypsin, and the cell concentration was adjusted to 5×10 5 cells / mL, and inoculated into a six-well culture dish, and placed the culture dish at 37°C, 95% air / 5% CO 2 Cultivate in an incubator, replace the medium once a week or two, and change the medium every day after one week until the growth becomes a dense monolayer.
[0040] Wells with a single cell layer were used for the adhesion experiment. After washing three times with 2 mL of pre-warmed PBS, 1 mL of DEME medium containing 5% fetal bovine serum without antibiotics and 1 mL of DEME culture medium resuspended with end-logarithmic bacteria were added t...
Embodiment 2
[0045] Example 2: Determination of Lactobacillus reuteri tolerance to simulated gastrointestinal fluid in vitro
[0046] Prepare simulated gastric juice: weigh a certain amount of pepsin, dissolve it in 2 / 3 of the required volume of 0.5% (w / v) normal saline, adjust the pH to 3.0 with 6M hydrochloric acid, and make the final concentration of pepsin 3g / L.
[0047] Preparation of simulated intestinal juice: Weigh a certain amount of trypsin and bile salts, dissolve in 2 / 3 of the required volume of 0.5% (w / v) normal saline, adjust to pH 8.0 with 0.1mol / L NaOH, constant volume, make The final concentrations were 1g / L and 0.3g / L, respectively.
[0048] The simulated gastric juice and simulated intestinal juice are prepared and used immediately, and the simulated liquid after constant volume is filtered and sterilized by a 0.22 μm sterile filter membrane before use.
[0049] The activated two-generation Lactobacillus reuteri continued to be subcultured, cultured for 18 hours, coll...
Embodiment 3
[0052] Embodiment 3: the preparation of Lactobacillus reuteri composition preparation
[0053] With Lactobacillus reuteri CCFM14 and CCFM8631, respectively in the MRS medium after continuous activation for three generations, the strains after the activation were transferred to the MRS medium with 5% inoculum (V / V) (adding 0.5% cysteine Hydrochloride) to expand the culture. Centrifuge the expanded cultures of Lactobacillus reuteri CCFM14 and CCFM8631 respectively (5000g, 15min), discard the supernatant, resuspend the sludge with sterile physiological saline, and resuspend the sludge in 30% sterile sucrose solution after cleaning , making the concentration of each strain 5×10 10 CFU / mL, aliquoted, stored in a -80°C refrigerator, thawed before use, and diluted tenfold with sterile normal saline. The diluted cultures of the two strains were uniformly mixed at a ratio of 1:1 of viable bacteria, and placed in a 37°C incubator for 15 minutes to form a Lactobacillus reuteri composit...
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