Bacillus licheniformis strain, application thereof and method for producing poly-gamma-glutamic acid thereby
A technology of Bacillus licheniformis and glutamic acid, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problem of high production cost, and achieves low production cost, short fermentation period and good genetic stability. Effect
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Embodiment 1
[0040] Embodiment 1: Isolation and screening of bacterial strains of the present invention
[0041]Weigh 1g of high-salt environment soil sample from Yingcheng Salt Mine in Hubei into a sterilized test tube, add 9mL of sterile water, seal it with a cotton plug, shake for 2min, then put it in a water bath, heat at 80°C for 10min, and then let it stand for 30min. After cooling, draw 0.1mL supernatant, dilute 10-2, 10-3, take 0.2mL respectively, spread on LB plate, incubate at 37°C for 48h to observe the results, and select a single colony that is emulsion-like and silk-drawing on LB medium , into fresh liquid seed culture medium, and after 12 hours of shaking culture at 37°C, they were inoculated to 50
[0042] In a 250mL shake flask containing sterilized basic fermentation medium, shake at 37°C and 190r / min for 36h to complete the fermentation. The fermentation products were sampled and centrifuged to remove the bacteria, and the supernatant was precipitated with 3 times of et...
Embodiment 2
[0043] Embodiment 2: the identification of bacterial strain of the present invention
[0044] Physiological and biochemical identification
[0045] The bacterium is straight rod-shaped, Gram staining is positive, and the spores are approximately round and partial. On LB solid medium, the colonies are round, with irregular edges, hairy, and the lawn is dry, opaque, spreading, off-white. The optimum growth temperature is 30-37℃, and the suitable pH is 6.8-7.2. The contact enzyme test was positive, the nitrate utilization was positive, the citrate utilization was positive, and the gelatin liquefaction test was positive.
[0046] Identification of Bacterial Species Using 16S rRNA Gene Sequence Analysis
[0047] Genomic DNA Extraction of Bacillus licheniformis WX-02
[0048] Streak Bacillus licheniformis WX-02 on an LB plate (containing peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 15 g / L, pH 7.0) and cultivate overnight at 37°C Finally, pick a small amount of bacteri...
Embodiment 3
[0081] Embodiment 3 adopts the yield determination of the poly-γ-glutamic acid produced by bacterial strain of the present invention
[0082] Its quantitative analysis of poly-γ-glutamic acid produced by bacterial strain of the present invention is completed by HPLC, chromatographic column Agilent1100, mobile phase is 100mmol.L -1 K H 2 PO 4 Add 5.0% methanol (adjust the pH value to 2.5 with phosphoric acid) to filter, degas by ultrasonic, the ultraviolet detection wavelength is 210nm, the flow rate is 1mL / min, and glutamic acid is used as a quantitative standard.
[0083] The specific measurement steps are as follows:
[0084] Take 5 ml of fermentation centrifuge supernatant, add 3 times the volume of alcohol to precipitate PGA, and dissolve the product, add an equal volume of concentrated hydrochloric acid to hydrolyze at 110°C for 24 hours under vacuum conditions, add 10mol.L of the hydrolyzed solution after hydrolysis -1 NaOH was neutralized to constant volume, filtere...
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