Screening method for producing chondroitin sulfate bacterial strain and application of bacterial strain fermentation method in production of chondroitin sulfate
A technology of chondroitin sulfate and screening method, which is applied in the field of bioengineering and can solve problems such as useless chondroitin sulfate
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Embodiment 1
[0044] Weigh about 0.2g fermented soya bean into 20ml 0.85% sterile saline, shake and heat the bacterial suspension in boiling water for 10min, then cool it down to room temperature rapidly. This bacterial suspension was diluted to 10 as a stock solution -8 , take 10 -6 -10 -8 The diluted bacterial solution was spread on the LB plate, cultured upside down at 37°C for 24 hours, single colonies with different colony shapes were picked for crystal violet staining, the spores were observed under the microscope, and the bacillus was selected for further fermentation and re-screening. The nutrient broth medium was cultured at 37°C for 24 hours, and the fermentation liquid was collected by centrifugation. The fermentation liquid was analyzed by the internal standard method in the high performance liquid phase method. Shark chondroitin sulfate was used as the internal standard to increase the peak height of the chondroitin sulfate standard product. That is, the chondroitin sulfate-p...
Embodiment 2
[0046] The physiological and biochemical characteristics of the screened BN strains were identified according to Microbial Taxonomy (see Tables 1 and 2), and genomic DNA was extracted according to the extraction method of the bacterial genome extraction kit, with P1 and P2 as forward and reverse primers:
[0047] P1: 5'-CAGATGGGAGCTTGCTCCCTG-3',
[0048] P2: 5'-CGACTTCACCCCAATCATCTG-3'.
[0049] The 16S rDNA gene was amplified by PCR, and the BGI Research Center was commissioned to perform 16S rDNA sequencing. After obtaining the partial 16S rDNA sequence of this strain (GenBank accession number: JF922967), the homology comparison analysis was performed on the NCBI website using the BLAST search tool. . Based on 16S rDNA sequence analysis and identification of physiological and biochemical characteristics, combined with the study of the growth and fermentation characteristics of the bacteria, it was considered to be Bacillus subtilis and named Bacillus.subtilis BN.
[0050] ...
Embodiment 3
[0055] The strains were stored in a glycerol tube with a final concentration of 15%. Take 100μl of the preserved bacteria liquid and insert it into 30ml of nutrient broth medium for seed culture. After culturing for 14 hours at 37°C and 200rpm, add 50ml of nutrient meat with an inoculum of 10%. Soup culture medium for fermentation. The nutrient broth consists of beef extract 3g / L, peptone 5g / L, and the initial pH of the medium is 7.2-7.4. Ferment for 24 hours at 37° C. and 200 rpm, and measure the content of chondroitin sulfate in the fermentation broth by HPLC. The yield was 177 mg / L.
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