Weight-losing composition containing bacillus subtilis and preparing method and application of weight-losing composition
A technology of Bacillus subtilis and composition, which is applied in the directions of medical preparations containing active ingredients, pharmaceutical combinations, active ingredients of phosphorus compounds, etc. Effects of body weight and weight reduction in rats
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Embodiment 1
[0042] The acquisition and morphological identification of embodiment 1 Bacillus subtilis
[0043] (1) Strain acquisition
[0044] The original seeds of Bacillus subtilis (Bacillus subtilis) Subtilisin-QK original seed batch were isolated from fermented soybeans by the laboratory of Professor Wang Yefu of Wuhan University, and went through sample collection → microbial isolation → cultivation → screening identification → positive results → strain identification And preservation → production of starting bacteria and other steps.
[0045] (2) Morphological identification of strains
[0046] Inoculate the isolated strains on NA solid medium and culture them at 30°C for 48 hours. The single colony is light white and opaque, the surface is not smooth, the edges are irregular and dull, and the texture is uniform; the colonies are inoculated on LB liquid medium and kept at a constant temperature of 30°C. Shake culture for 18-24 hours, the medium becomes turbid, and no bacterial fil...
Embodiment 2
[0048] The molecular identification of embodiment 2 Bacillus subtilis
[0049] The strain was streaked on a plate to obtain a single colony. Pick a single colony and place it in a shaking tube containing 2mL TSB, and place it at 28°C for shaking culture until turbid; take 1mL of the bacterial solution in a centrifuge tube, centrifuge at 10000rpm for 3min, discard the supernatant, and add 50 μL of sterile water, shake well, incubate at 95°C for 2 minutes, and place in a refrigerator at 0°C for later use;
[0050] According to literature records, universal primers 27F and 1492R were selected for PCR amplification of the strain genome. The reaction conditions were pre-denaturation at 94°C for 3 min, 35 cycles (denaturation at 94°C for 30 s, annealing at 50°C for 45 s, extension at 72°C for 100 s), and extension at 72°C for 7 min. PCR amplification primers are as follows:
[0051] 27F (SEQ ID NO: 1): 5'-TACGGYTACCTTGTTACGACTT-3';
[0052] 1492R (SEQ ID NO:2): 5'-TACGGYTACCTTGTTA...
Embodiment 3
[0055] Example 3 strain preservation
[0056] The present embodiment adopts the glycerol cryopreservation method to carry out bacterial strain preservation, and specific steps are as follows:
[0057] (1) Use a flame-sterilized inoculation loop to take the slant strains and streak on the LB medium plate to separate single colonies;
[0058] (2) Place the plate upside down in a constant temperature incubator at 37°C and incubate for 24-48 hours until the size of a single colony is about 3mm;
[0059] (3) Pick a single colony and inoculate it in a 250mL Erlenmeyer flask containing 50mL of LB medium, shake and culture at 37°C for 16 hours until the bacterial density is OD 600 for 3-5;
[0060] (4) Take a small amount of seed liquid with a flame-sterilized inoculation loop, and after smearing, do Gram staining, observe the shape of the thalline under a microscope, and whether there are any miscellaneous bacteria;
[0061] (5) Add 900 μL 50% sterile glycerol and 900 μL cultured ...
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