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

Inactive Publication Date: 2020-11-06
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In order to reduce the cost of subsequent purification, liquid fermentation often uses basal medium for fermentation, which leads to unsatisfactory growth of bacteria. According to existing literature reports, the OD600 of the strain is only about 5.0 when the co

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  • Method for producing colanic acid by sucrose-induced bacteria solid-state fermentation and purifying colanic acid
  • Method for producing colanic acid by sucrose-induced bacteria solid-state fermentation and purifying colanic acid
  • Method for producing colanic acid by sucrose-induced bacteria solid-state fermentation and purifying colanic acid

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Experimental program
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Example Embodiment

[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.

Example Embodiment

[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.

Example Embodiment

[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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Abstract

The invention provides a method for producing colanic acid by sucrose-induced bacteria solid-state fermentation and purifying the colanic acid; the method comprises the following steps: 1, putting proteobacteria with colanic acid synthetic gene as a production strain into a culture medium, carrying out solid-state culture, adding sucrose into the culture medium, and collecting thalli and secretions; 2, scraping thalli and secretions cultured on the solid, and dissolving the thalli and secretions in water; 3, performing boiling on water containing thalli and secretions, centrifugally precipitating the thalli, discarding precipitates, and reserving supernatant; step 4, adding glacial acetic acid into the supernatant obtained in the step 3 to centrifugally precipitate lipopolysaccharide, discarding the precipitate, and retaining the supernatant; and 5, centrifugally purifying the supernatant obtained in the step 4, and carrying out vacuum drying to obtain the colanic acid solid. Accordingto the method provided by the invention, the colanic acid is produced through solid-state culture for the first time and sucrose is used for inducing overexpression fermentation of the colanic acid gene, so that the yield is high, the fermentation time is short, and the product is easy to purify.

Description

technical field [0001] The invention belongs to the field of kolanic acid production, and in particular relates to a sucrose-induced bacterial solid-state fermentation production of kolamic acid and a purification method. Background technique [0002] Microbial exopolysaccharides are biopolymers produced by bacteria, fungi, cyanobacteria and other microorganisms during the metabolic process that have a protective effect on microorganisms. Under natural conditions, most bacteria are coated with polysaccharides. Polysaccharides coated on the surface of bacteria play an important role in the survival and growth of bacteria in a competitive environment. On the one hand, the presence of polysaccharides can improve the tolerance of bacteria to antibacterial substances, such as surfactants, antibiotics, and phagocytes. On the other hand, exopolysaccharides can improve the adaptability of bacteria to adverse environments, such as preventing cell dehydration, Prevent cell frostbite...

Claims

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Application Information

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IPC IPC(8): C12P19/04C08B37/00C12R1/01
CPCC12P19/04C08B37/0003C08B37/006
Inventor 季学猛王硕
Owner NANKAI UNIV
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