Artificial bear bile powder and quality evaluation method thereof
A kind of bear bile powder, artificial technology, applied in the field of biology
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Embodiment 1
[0096] 1 The effect of the secondary seed medium on the growth curve
[0097] 1) Apply the engineering bacteria stored in the -80°C refrigerator to the LB solid plate medium containing 100 mg / L ampicillin by streaking method, and activate and culture at 37°C for 12 hours;
[0098] The composition of the LB solid medium is as follows: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, agarose 15.0g / L.
[0099] 2) Pick a single colony from the LB solid plate medium, inoculate it in the LB liquid medium supplemented with 100mg / L ampicillin, and culture it with shaking at 225rpm and 37°C for 9 hours to obtain the first-grade seeds;
[0100] The composition of described LB liquid medium is as follows: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L;
[0101] 3) Inoculate the above culture into 2-YT liquid medium and the same LB liquid medium as described in 2) at a ratio of 1:20, culture with shaking at 225rpm / min and 37°C, and measure both Growth curve;
[010...
Embodiment 2
[0107] Optimization experiment of M9-GY optimized medium
[0108] 1) Apply the design method of Box-Behnken Design in the Design-Expert.V8.0.6.1 software, based on the composition of the M9 basic medium, design a test with glucose, glycerol, yeast extract and tryptone as four factors, Glucose, yeast extract, and tryptone all choose the three levels of 0, 10, and 20g / L, and glycerol chooses the three levels of 0, 15, and 30ml / L. The OD600 and conversion rate are used as the investigation indicators at the same time. The 29 groups of programs designed by the software and The results are as follows:
[0109]
[0110] 2) The specific fermentation process is as follows: Obtain first-grade seeds according to the operation in Example 1, inoculate the first-grade seeds into liquid 2-YT medium at a ratio of 1:20, and shake and culture at 225rpm and 37°C for 4 hours to obtain 2-YT. Grade seeds; the second grade seeds are inoculated into the above-mentioned various liquid mediums in ...
Embodiment 3
[0114] Optimization of induction culture phase
[0115] 1 Effect of IPTG concentration on conversion rate
[0116] 1) According to the operation in Example 1, the first-grade seeds were obtained, and the first-grade seeds were inoculated into the liquid 2-YT medium at a ratio of 1:20, and cultured with shaking at 225 rpm and 37° C. for 4 hours to obtain the second-grade seeds;
[0117] 2) Inoculate the secondary seeds into the M9-GY optimized medium liquid medium at a ratio of 1:20, in triplicate, and shake and culture at 225rpm and 37°C for 5 hours;
[0118] 3) Add IPTG to the final concentration of 0.1, 0.3, 0.6, 0.8, 1.0mM respectively, shake and culture at 225rpm, 30°C for 3 hours; Shake culture at 25°C for 6 hours for fermentation; the results are as follows figure 1 as shown in a:
[0119] 2. Combination optimization of induction conditions
[0120]Select the pre-log (4 hours), mid-log (5.5 hours), and late log (7.5 hours) of the growth curve of the recombinant engi...
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