A kind of Lactobacillus paracasei et-22 with immune regulation function
A technology of ET-22 and side cheese, applied in the field of microorganisms
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Embodiment 1
[0042] Example 1. Artificial gastric juice, intestinal juice tolerance, artificial bile tolerance
[0043] L.paracasei ET-22 was activated three times and divided into 8 tubes, and one tube was taken for colony counting to calculate the initial number of viable bacteria. For the remaining seven, centrifuge at 4000rpm for 10min, remove the supernatant, and culture in pH 2.5 medium at 37°C for 1 to 3 hours, take a tube of mixed solution every hour, centrifuge at 4000rpm for 10min, remove the supernatant , count the number of viable bacteria after washing twice with 5mL RO water. The other 4 tubes were centrifuged at 4000rpm for 10 min after 3 hours, and the supernatant (pH 2.5 MRS broth) was removed, and the 4 tubes were fully mixed with MRS broth containing 1.5% ox bile, and evenly distributed to 4 tubes, and incubated at 37°C for 1~ 4 hours. Take 1 tube of mixed solution per hour, centrifuge at 4000rpm for 10min, remove the supernatant, wash twice with 5mL RO Water, and calc...
Embodiment 2
[0045] Example 2. Adsorption effect of intestinal cells
[0046] Pick up the cover glass on the slide with tweezers, and put it into a 6-well dish for later use after being sterilized by an alcohol lamp. Take out the Caco-2 cells from the cryovial, adjust the cells, move them to a 6-well plate for culture, and perform the experiment after the plate is full. During the test, the liquid in the plate was removed, washed twice with PBS buffer, and 1.5 mL of bacterial solution (1×10 9 CFU / mL) and 1.5mL cell culture medium (containing 10% PBS and 1% penicilin-streptomycin) were mixed, cultured in a constant temperature incubator (37 ° C, 5% CO 2 ) after 4 hours, washed twice with sterile PBS, fixed the cells with methanol, stained with Gram, and counted the number of live bacteria under a microscope.
[0047] The adhesion of the strain to Caco-2 cells was detected by the adhesion test, and the results were as follows: figure 2 , L.paracaseiET-22 has strong adhesion to Caco-2 cel...
Embodiment 3
[0048] Example 3: Analysis of Immunomodulatory Activity
[0049] 3.1 Humoral immunity experiment
[0050] 3.1.1 Antibody producing cell detection experiment
[0051] The spleen cell suspension of mice immunized with sheep red blood cells (SRBC) is mixed with a certain amount of SRBC. With the participation of complement, the SRBC around the spleen cells that secrete antibodies are dissolved to form plaques visible to the naked eye. The number of hemolytic plaques can reflect Antibody producing cell number.
[0052] Use PBS to suspend and adjust the concentration of viable bacteria samples according to different gavage volumes before daily animal gavage (dosage is about 10 7 -10 9 CFU / day), after the animals were given samples continuously for 28 days, each mouse was injected with 0.2 mL of SRBC through the abdominal cavity for immunization. The mice 4 days after SRBC immunization were sacrificed, and the spleen was taken to make 5×10 6 cells / mL of cell suspension. After ...
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