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

Active Publication Date: 2011-10-19
HUNAN WUXING BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The research on the production of chondroitin by microbial fermentation is in the preliminary stage, and there is still no method that can directly produce chondroitin sulfate by microbial fermentation

Method used

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  • Screening method for producing chondroitin sulfate bacterial strain and application of bacterial strain fermentation method in production of chondroitin sulfate
  • Screening method for producing chondroitin sulfate bacterial strain and application of bacterial strain fermentation method in production of chondroitin sulfate
  • Screening method for producing chondroitin sulfate bacterial strain and application of bacterial strain fermentation method in production of 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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Abstract

The invention relates to a screening method for producing chondroitin sulfate bacillus subtilis and application of a bacterial strain fermentation method in the production of chondroitin sulfate, belonging to the technical field of biological engineering. The bacterial strain disclosed by the invention is bacillus subtilis which is obtained by the following steps of: diluting and coating fermented soya beans subjected to a boiling water bath, dyeing and carrying out microscopic examination on an obtained soporiferous strain, then carrying out primary fermentation and shaking culture one by one on screened bacterial strains, adding chondroitin sulfate standard substance of sharks in a formation liquor to be used as an internal standard, carrying out qualitative analysis and screening by adopting a high performance liquid chromatography, and carrying out morphological, physiological and biochemistric and molecular biological identification on the screened bacterial strains to obtain thebacillus subtilis for producing the chondroitin sulfate. Meanwhile, the invention discloses a method for producing the chondroitin sulfate by using the bacterial strain; and the method is used for fermenting the chondroitin sulfate for 24h, wherein the yield of the chondroitin sulfate is 177mg / L.

Description

technical field [0001] The invention discloses a method for screening Bacillus subtilis producing chondroitin sulfate and using the strain to produce chondroitin sulfate, belonging to the field of bioengineering, in particular to a Bacillus subtilis producing chondroitin sulfate. Background technique [0002] Chondroitin sulfate (CS), also known as Kangdeling, is a glycosaminoglycan polysaccharide whose disaccharide monomer consists of glucuronic acid (D-GlcUA) and N-acetylgalactosamine (GalNAc) Linked by β-1,3 bonds, after the sugar chain is generated, it is sulfated at carbon 4 or carbon 6 of GalNAc by sulfotransferase, such as figure 1 . According to its chemical composition and structural differences, it can be divided into A, B, C, D, E, F, H, etc. Chondroitin sulfate, which is usually extracted from mammalian tissues, is mainly CS-A and CS-C. [0003] CS is a natural acidic mucopolysaccharide drug extracted from animal cartilage, which is mainly used for neuropathic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P19/04C12R1/125
Inventor 刘立明吴秋林刘佳陈坚
Owner HUNAN WUXING BIOLOGICAL TECH CO LTD
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