Technique for preparing antimicrobial peptide by using Bacillus subtilis

A technology of Bacillus subtilis and process methods, applied in the direction of peptide preparation methods, microorganism-based methods, biochemical equipment and methods, etc., to achieve the effects of high safety, no drug resistance, and improved preparation efficiency

Inactive Publication Date: 2013-10-02
XUZHOU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem solved by the present invention is to overcome the deficiencies of the prior art, use Bacillus subtilis producing broad-spectrum high-efficiency antimicrobial peptides as the production strain, prepare liquid seeds, prepare fermentation broth, pretreat fermentation broth, extract and refine antimicrobial peptides, Preparation of antibacterial peptide dry powder and other processes to prepare high-purity antibacterial peptide products, providing a high-yield, simple extraction process, and less environmental pollution antibacterial peptide preparation process

Method used

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  • Technique for preparing antimicrobial peptide by using Bacillus subtilis
  • Technique for preparing antimicrobial peptide by using Bacillus subtilis
  • Technique for preparing antimicrobial peptide by using Bacillus subtilis

Examples

Experimental program
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Embodiment 1

[0043] A process for preparing antimicrobial peptides by using Bacillus subtilis according to the present invention The production strain is specifically Bacillus subtilis (Bacillus subtilis) BRT39. , the deposit number is CGMCC NO.5702.

[0044] The seed medium consists of: glucose 2g, peptone 1g, NaH 2 PO 4 2H 2 O0.2g, Na 2 HPO 4 2H 2 O0.4g, MgSO 4 ·7H 2 O0.05g, dH 2 O100mL, pH 7.5, sterilized at 121°C for 20min.

[0045] The shake flask fermentation medium consists of: soybean meal powder 1-2g, maltose 0.5g, glucose 0.7-1g, ammonium sulfate 0.3g, ammonium nitrate 0.2g, peptone 0.4-1g, beef extract 0.5g, NaCl 0.5g, KH 2 PO 4 0.1g, MgSO 4 ·7H 2 O0.04g, dH 2 O100mL, pH8.0, 121°C, sterilize for 20min.

[0046] The fermentation medium of the fermenter is composed of: soybean meal powder 1-2g, corn flour 0.5-1g, maltose 0.5g, soluble starch 0.5-0.7g, glucose 0.7-1g, ammonium sulfate 0.3g, ammonium nitrate 0.2g, peptone 0.4 -1g, beef extract 0.5g, NaCl 0.5g, KH 2 P...

Embodiment 2

[0048] A kind of antimicrobial peptide preparation process method, comprises the steps:

[0049] 1) Strain activation

[0050] The well-preserved slant strain CGMCC NO.5702 was inoculated on the slant medium and cultured at 35°C for 16 hours to activate the strain.

[0051] 2) Liquid seed culture

[0052] Insert a single colony of activated Bacillus subtilis into the sterilized seed medium, and culture it on a rotary shaker at 180r / min and 35°C for 16 hours, and the bacterial content of the seed fermentation liquid is 10 7 cfu / mL;

[0053] 3) Shake flask fermentation culture

[0054] The seed solution obtained in step 2) was inserted into the Erlenmeyer flask with a 5% inoculum amount, the 1000mL Erlenmeyer flask contained a fermentation liquid medium of 200mL, the Erlenmeyer flask stopper was a latex stopper, the fermentation temperature was 32°C, and the rotation speed was 160r / min, and the shaking table was cultivated for 60h;

[0055] 4) Fermentation culture in 15-lite...

Embodiment 3

[0066] A kind of antimicrobial peptide preparation process method, comprises the steps:

[0067] 1) Strain activation

[0068] The well-preserved slant strain CGMCC NO.5702 was inoculated on the slant medium and cultured at 36°C for 18 hours to activate the strain.

[0069] 2) Liquid seed culture

[0070] Insert a single colony of activated Bacillus subtilis into the sterilized seed culture medium, and cultivate it in a rotary shaker at 190r / min and 36°C for 18 hours, and the bacterial content of the seed fermentation liquid is 10 7 cfu / mL;

[0071] 3) Shake flask fermentation culture

[0072] The seed liquid obtained in step 2) was inserted into the Erlenmeyer flask with an inoculum amount of 8%, and the 1000mL Erlenmeyer flask contained a fermentation liquid medium of 200mL, and the Erlenmeyer flask stopper was made of latex, the fermentation temperature was 35°C, and the rotating speed was 180r / min, and the shaking table was cultivated for 61h;

[0073] 4) Fermentation cu...

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Abstract

The invention discloses a technique for preparing antimicrobial peptide by using Bacillus subtilis, belonging to the technical field of bioengineering. By using Bacillus subtilis BRT39 capable of generating broad-spectrum high-efficiency antimicrobial peptide as a production strain, liquid seed preparation, fermentation liquid preparation, fermentation liquid pretreatment, antimicrobial peptide extraction and refinement, antimicrobial peptide dry powder preparation and other techniques are carried out to prepare the high-purity antimicrobial peptide finished product, wherein the activity recovery rate is up to higher than 43.5%. The invention has the advantages of simple technique, high yield of antimicrobial peptide, and high purity; the prepared antimicrobial peptide dry powder belongs to a natural antimicrobial agent and has no toxic or side effect, and thus, can be used as a food preservative, feed additive, agricultural biological control agent or animal disease preventing agent; and the antimicrobial peptide has the advantages of no toxicity, no residue, no drug resistance and high safety.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a process method for preparing antibacterial peptides by using Bacillus subtilis. Background technique: [0002] Antimicrobial peptides (AMP) are small molecular polypeptides with biological activity induced in vivo, with a molecular weight of about 2000-7000 and composed of 20-60 amino acid residues. Most of these active peptides have the characteristics of strong alkalinity, thermal stability and broad-spectrum antibacterial properties. Compared with antibiotics, antimicrobial peptides are highly safe and harmless to normal cells of humans and animals. They are a type of biological preservatives that have no toxic side effects, no residue, and no bacterial resistance. They are widely used in the pharmaceutical industry, food industry, and feed industry. has broad application prospects. [0003] In the field of medicine, antimicrobial peptides can neutralize bacterial s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/00C07K1/36C07K1/34C07K1/30C07K1/18C07K1/16A23L3/3526C12R1/125
Inventor 高兆建樊陈鞠民友孙会刚侯进慧李同祥杜永凯纪伟
Owner XUZHOU UNIV OF TECH
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