Lactobacillus rhamnosus and application thereof
A technology of Lactobacillus rhamnosus, which is used in the field of microorganisms, can solve the problems of tissue destruction, harmfulness, and promotion of tumor occurrence, and achieve the effects of preventing or alleviating inflammation, high nutritional value, and good intestinal adhesion ability
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Embodiment 1
[0053] Example 1 Source and identification of Lactobacillus rhamnosus strain
[0054] Take the Inner Mongolia farmhouse yogurt fermented food as the sample, take 1g yogurt sample, make 10-fold gradient dilution with sterile physiological saline, dilute the sample 1000 times, spread it on the surface of the prepared MRS solid medium, and place it in the 37℃ incubator Incubate for 24 to 48 hours and observe the morphological characteristics of the colony. A single colony of suspected lactic acid bacteria was picked and streaked and separated on a new MRS medium. After overnight culture at 37°C, streaked and purified again. Use a sterile toothpick to pick several single colonies from the culture medium separated by the second streak, and number them respectively. Twenty single colonies were selected for 16s DNA sequencing.
[0055] Among them, 1L of MRS solid medium includes: casein peptone 10.0g / L, beef extract 10.0g / L, yeast extract 5.0g / L, glucose 20.0g / L, dipotassium hydrogen ph...
example 16
[0058] The primer sequences for 16s DNA amplification in this example are as shown in SEQ ID NO:1 and SEQ ID NO:2.
[0059] 5'-AGAGTTTGATCATGGCTCAG-3' (SEQ ID NO: 1).
[0060] 5'-TAGGGTTACCTTGTTACGACTT-3' (SEQ ID NO: 2).
[0061] The primers were synthesized by Shenzhen BGI Institute of Life Sciences.
[0062] The PCR reaction system is: 50μL reaction solution includes: 10mM dNTPs 1μL, 10×buffer 5μL, 10mM upstream and downstream primers 1μL, bacterial liquid PCR template 1μL, 5U / μL Taq enzyme 1μL, ddH 2 O was supplemented to 50 μL.
[0063] The PCR reaction conditions were: 94°C pre-denaturation for 5 minutes, and then 35 cycles: 94°C for 30s, 60°C for 30s, 72°C for 1 minute, after the cycle, extension at 72°C for 5 minutes, and 4°C for standby.
[0064] The PCR amplification product is cut and recovered, and the recovered PCR product is sequenced. In this example, the kit TaKaRa MiniBEST agarose GeL DNA Extraction kit was used for gel cutting and recovery. For detailed steps, see the k...
Embodiment 2
[0069] Example 2 Evaluation of anti-inflammatory and probiotic properties of Lactobacillus rhamnosus ASD-9
[0070] 1. HT-29 colon cancer cell line model to evaluate the intestinal adhesion ability of ASD-9
[0071] Colonization and adhesion are the prerequisites for strains to exert their probiotic functions. The inventor used HT-29 human colon cancer cells to approximate the body's colon cells and studied the adhesion ability of ASD-9 to the body's intestinal tract. The adhesion test process is as follows: first, digest HT-29 cells, dilute with 1640 medium without double antibody to adjust the cell concentration to 1×10 6 cell / mL; then, add 0.5 mL of the above-mentioned concentration of cells dropwise to a 12-well plate containing cell slides, and statically culture them to grow into a dense monolayer of cells. At the same time, culture and activate the tested strain for 2 generations, and resuspend the bacterial solution to 2×10 with 1640 culture broth. 8 The concentration of C...
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