Method for producing colanic acid by sucrose-induced bacteria solid-state fermentation and purifying colanic acid
A technology for solid-state fermentation and purification methods, applied in biochemical equipment and methods, microorganism-based methods, fermentation and other directions, can solve the problems of unsatisfactory bacterial growth state, increase the difficulty of product purification, and multiple impurities, and achieve high-efficiency production and purification methods. , the effect of high yield and short fermentation time
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[0034] The preparation of embodiment 1 sucrose solid medium
[0035] Prepare 200 mL of LB medium containing 1.5% (w / v) agar powder and 20% (w / v) sucrose, and sterilize it by autoclaving at 115°C for 15 minutes. After the medium in the plate is solidified, put it into a 4°C refrigerator for use.
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[0036] Activation and inoculation of embodiment 2 bacteria
[0037] Enterobacter sakazakii ATCC BAA-894 was purchased from American Type Culture Collection ATCC and preserved in glycerol in Tianjin Key Laboratory of Food Science and Health. Enterobacter sakazakii ATCC BAA-894 was cultured overnight in liquid LB medium at 37°C with shaking at 200rpm, then centrifuged and pelleted, washed with PBS repeatedly for 3 times, and finally re-suspended with PBS. Each LB solid plate with a diameter of 12 mm was inoculated with 50 μL of the bacterial suspension and spread evenly with a spreader, and a total of 10 plates were inoculated.
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[0038] The cultivation of embodiment 3 bacteria
[0039] Place the LB plates inoculated with bacteria in a constant temperature incubator at 37°C for 24-48 hours, and place the plates upright for culture, otherwise mucus-like bacteria will flow out of the plates. rt-qPCR was used to detect the effect of sucrose on the transcription level of colanic acid synthesis gene wcaB (ESA_01160), RNA was extracted, and after reverse transcription, primers Fwd: 5'-TATCGCATCGCGCACTTCTG-3' and Rev: 5'-GGCGGCCTGAATTTCATACC-3' were used to amplify Increased wcaB, extracted using primers Fwd: 5'-GAGT GGCGGACGGGTGAGT-3' and Rev: 5'-GTCCGTAGACGTTATGCGGTATTAG-3' to amplify 16s rDNA as a reference, it can be seen that the transcription level of sucrose-induced kolanic acid synthesis gene wcaB increased by 50% ( figure 1 ). After 24-48 hours, a large amount of mucus-like bacterial secretions can be seen on the plate ( figure 2 ), figure 2 Middle left is LB medium, right is LB medium with 20% s...
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