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Method for producing antibiotic peptide by using Bacillus amyloliquefaciens and product thereof

A technology of bacillus and starch liquefaction, applied in microorganism-based methods, biochemical equipment and methods, applications, etc., to achieve the effects of stable antibacterial activity, broad antibacterial spectrum, and high safety

Inactive Publication Date: 2006-10-04
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] According to reports, many microorganisms can produce antimicrobial peptides, but there is no report on the use of Bacillus amyloliquefaciens to produce antimicrobial peptides for food preservation and feed additives

Method used

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  • Method for producing antibiotic peptide by using Bacillus amyloliquefaciens and product thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ES-2 strain (common public strain, 16SrRNA gene sequence GenBank accession number is DQ177319) through the expansion culture of seed, seed medium: tryptone 10.0g, yeast extract 5.0g, NaCl 5.0g , distilled water 1000ml, pH7.2;

[0027] Then in the fermenter at 28-35°C, carry out liquid submerged fermentation for 30-48h (liquid fermentation medium: glucose 20.0g, tryptone 5.0g, MgSO 4 0.5g, KCl 0.5g, KH 2 PO 4 1g, FeSO 4 0.15mg, MnSO 45.0mg, CuSO 4 0.16mg, distilled water 1000ml, pH7.0. ) to accumulate antibacterial metabolites, the fermentation broth is centrifuged to remove bacteria, and then the supernatant is concentrated and dried to obtain the finished antibacterial peptide. As determined by high performance liquid chromatography, the antimicrobial peptide content is not less than 100g / kg. As determined by LC-MS, the antimicrobial peptide product is a mixture of two homologues of lipopeptide surfactin...

Embodiment 2

[0029] Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ES-2 is expanded and cultivated through seeds, and then submerged in a fermenter at 28-35°C for 30-48 hours (liquid fermentation medium: glucose 20.0g, tryptone 5.0g, MgSO 4 0.5g, KCl 0.5g, KH 2 PO 4 1g, FeSO 4 0.15mg, MnSO 4 5.0mg, CuSO 4 0.16mg, distilled water 1000ml, pH7.0. ), accumulate antibacterial metabolites, the fermentation liquid is centrifuged to remove the bacteria, then add HCl to the supernatant to adjust the pH to 2, centrifuge after standing, and the precipitate is further extracted with methanol, and the extract is adsorbed and dried by the excipient to obtain The finished product of antimicrobial peptide, as determined by high performance liquid chromatography, has an antimicrobial peptide content of not less than 100g / kg.

Embodiment 3

[0031] Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ES-2 is expanded and cultivated through seeds, and then submerged in a fermenter at 28-35°C for 30-48 hours (liquid fermentation medium: glucose 20.0g, tryptone 5.0g, MgSO 4 0.5g, KCL 0.5g, KH 2 PO 4 1g, FeSO 4 0.15mg, MnSO 4 5.0mg, CuSO 4 0.16mg, distilled water 1000ML, pH7.0. ), accumulating antibacterial metabolites, the fermentation liquid is centrifuged to remove the thalline, then adding 25% ethyl acetate by volume to the supernatant for extraction, drying after extraction, and the extract is adsorbed by excipients to obtain the finished product of antibacterial peptides, As determined by high performance liquid chromatography, the antimicrobial peptide content is not less than 100g / kg.

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Abstract

The related preparation method for antibiotic peptide comprises: with ES-2 strain of Bacillus amyloliquefaciens, amplifying culture with medium, taking 2% inoculation, and deep fermenting for 30-48h at 28-35h, or taking inoculation with solid fermentation medium for culturing 2-4day at 28-37Deg; extracting, concentrating, adsorbing, and drying to obtain the product with molecular weight as 994, 1008, 1022, 1036 and 1449, 1463, 1477, 1491 Da as the mixture of surfactin and fangycin. This product can bear heating at 100Deg for 30min, keeps activity in pH 2-12, and can be used as food preservative and additive.

Description

technical field [0001] The invention relates to a method for producing antimicrobial peptides by using Bacillus amyloliquefaciens to produce antimicrobial peptides. The antimicrobial peptides have potential as food preservatives and feed additives. technical background [0002] In recent years, due to the increasing problems of drug abuse, drug residues and bacterial drug resistance, which has aroused people's attention to food safety, more and more countries have begun to call for the banning of antibiotics, and antimicrobial peptides are And the special mechanism of action different from traditional antibiotics has aroused people's great research interest and has become one of the hot spots in the research field. Antibacterial peptides (Antibacterial Peptides) are cationic polypeptides with strong antibacterial effects produced by organisms. Generally, their molecular weight is less than 5kD and the number of amino acids is less than 100. They are important components of b...

Claims

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Application Information

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IPC IPC(8): C12P21/02A23K1/16A23L3/3463C12R1/07A23K10/12A23K10/37A23K20/147
CPCY02P60/87
Inventor 陆兆新孙力军别小妹吕凤霞方传记刘姝黄现青
Owner NANJING AGRICULTURAL UNIVERSITY
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