Composite antibacterial peptide produced by bacillus natto NT-6 and production method thereof

Abstract

The invention relates to a composite antibacterial peptide produced by bacillus natto NT-6 and a production method thereof. The composite antibacterial peptide product is a mixture of three kinds of homologs of antibacterial lipopeptide substances iturins, fengycins and surfactins in the proportions of 10-20 percent, 25-35 percent and 50-60 percent respectively; the antibacterial peptide pure product is a yellow viscous substance which can resist heating of 30 minutes at the temperature of 100 DEG C, can keep activity at the pH of 2-12, has broad-spectrum antibacterial activity for bacteria and fungi and has strong killing effect on protozoa and cancer cells; and experiments prove that the peptide has high safety. The invention also discloses the production method of the composite antibacterial peptide. The peptide can be easily produced by using the process, is relatively low in cost, and can be used as a food preservative, a feed additive, an agricultural biological control agent, a medicament, an animal disease preventing agent and the like.

Description

technical field

[0001] The invention relates to a compound antibacterial peptide produced by Bacillus natto NT-6 strain and a method thereof, belonging to the field of biotechnology. The antibacterial lipopeptide produced by the method has broad-spectrum antibacterial activity and biological surface activity, and has broad application prospects in the fields of medicine, agriculture, food and environmental governance. Background technique

[0002] Bacterial tempeh (called natto in Japan) is a very common flavor-rich fermented soybean food in China with a history of more than 2,000 years. Its fermentation strain is Bacillus natto ( Bacillus natto). Bacillus natto can produce many functional factors, so that fermented products have many beneficial functions for the human body. At present, domestic and foreign research on Bacillus natto mainly focuses on the metabolite nattokinase with thrombolytic effect. And its antibacterial effect has only been paid attention to by peo...

Examples

Embodiment 1

[0027] After expanded cultivation of seeds, submerged fermentation was carried out in a fermenter at 28-35°C for 30-48 hours to accumulate antibacterial metabolites. The fermentation liquid was centrifuged to remove bacteria, and then HCl was added to the supernatant to adjust the pH to 2. Centrifuge after placing, the precipitate is further extracted with 3-5 times of methanol or ethanol, the extract is adsorbed by excipients and dried to obtain the finished product of antibacterial peptide, which is determined by high performance liquid chromatography, and the content of antibacterial peptide is not less than 100g / kg.

Embodiment 2

[0029] Bacillus natto ( Bacillus natto ) After the NT-6 bacterial strain is expanded through the above-mentioned seed medium, carry out liquid submerged fermentation in the above-mentioned liquid fermentation medium in a fermenter at 28-35° C., accumulate antibacterial metabolites for 30-48 hours, and remove the bacteria through centrifugation of the fermentation liquid. Then the supernatant is concentrated and dried to obtain the finished product of the antimicrobial peptide, and the content of the antimicrobial peptide is not less than 100 g / kg as determined by high performance liquid chromatography.

Embodiment 3

[0031] Bacillus natto ( Bacillus natto ) After the NT-6 bacterial strain is expanded through the above-mentioned seed medium, carry out liquid submerged fermentation in the above-mentioned liquid fermentation medium in a fermenter at 28-35° C., accumulate antibacterial metabolites for 30-48 hours, and remove the bacteria through centrifugation of the fermentation liquid. Then the supernatant is concentrated, extracted with 3-5 times of methanol, and dried to obtain the finished antimicrobial peptide. The content of the antimicrobial peptide is not less than 500g / kg as determined by high performance liquid chromatography.

technical field

[0001] The invention relates to a compound antibacterial peptide produced by Bacillus natto NT-6 strain and a method thereof, belonging to the field of biotechnology. The antibacterial lipopeptide produced by the method has broad-spectrum antibacterial activity and biological surface activity, and has broad application prospects in the fields of medicine, agriculture, food and environmental governance. Background technique

[0002] Bacterial tempeh (called natto in Japan) is a very common flavor-rich fermented soybean food in China with a history of more than 2,000 years. Its fermentation strain is Bacillus natto ( Bacillus natto). Bacillus natto can produce many functional factors, so that fermented products have many beneficial functions for the human body. At present, domestic and foreign research on Bacillus natto mainly focuses on the metabolite nattokinase with thrombolytic effect. And its antibacterial effect has only been paid attention to by peo...

Examples

Embodiment 1

[0027] After expanded cultivation of seeds, submerged fermentation was carried out in a fermenter at 28-35°C for 30-48 hours to accumulate antibacterial metabolites. The fermentation liquid was centrifuged to remove bacteria, and then HCl was added to the supernatant to adjust the pH to 2. Centrifuge after placing, the precipitate is further extracted with 3-5 times of methanol or ethanol, the extract is adsorbed by excipients and dried to obtain the finished product of antibacterial peptide, which is determined by high performance liquid chromatography, and the content of antibacterial peptide is not less than 100g / kg.

Embodiment 2

[0029] Bacillus natto ( Bacillus natto ) After the NT-6 bacterial strain is expanded through the above-mentioned seed medium, carry out liquid submerged fermentation in the above-mentioned liquid fermentation medium in a fermenter at 28-35° C., accumulate antibacterial metabolites for 30-48 hours, and remove the bacteria through centrifugation of the fermentation liquid. Then the supernatant is concentrated and dried to obtain the finished product of the antimicrobial peptide, and the content of the antimicrobial peptide is not less than 100 g / kg as determined by high performance liquid chromatography.

Embodiment 3

[0031] Bacillus natto ( Bacillus natto ) After the NT-6 bacterial strain is expanded through the above-mentioned seed medium, carry out liquid submerged fermentation in the above-mentioned liquid fermentation medium in a fermenter at 28-35° C., accumulate antibacterial metabolites for 30-48 hours, and remove the bacteria through centrifugation of the fermentation liquid. Then the supernatant is concentrated, extracted with 3-5 times of methanol, and dried to obtain the finished antimicrobial peptide. The content of the antimicrobial peptide is not less than 500g / kg as determined by high performance liquid chromatography.

Images