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A kind of fermentation method that improves the production of bacillus antibacterial lipopeptide

A technology for antibacterial lipopeptide and bacillus, which is applied in the field of fermentation, can solve the problems of less consideration of the influence of the composition change of lipopeptide homologues on the bacteriostatic activity, and achieves the effect of simple method and improved yield

Active Publication Date: 2020-11-20
THIRD INST OF OCEANOGRAPHY MINIST OF NATURAL RESOURCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the above studies have improved the yield of lipopeptide compounds, they are generally only one type of lipopeptide compound and less consideration has been given to the compositional changes of lipopeptide homologues and their effects on antibacterial activity

Method used

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  • A kind of fermentation method that improves the production of bacillus antibacterial lipopeptide
  • A kind of fermentation method that improves the production of bacillus antibacterial lipopeptide
  • A kind of fermentation method that improves the production of bacillus antibacterial lipopeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1. Separation, purification and identification of Bacillus Pc3 lipopeptide compound

[0021] Bacillus Pc3 was inoculated into a 5mL LB liquid test tube, cultured overnight at 28°C, 180r / min, and then inoculated into LB medium at 1% inoculum for fermentation, cultured at 28°C, 180r / min for 96 minutes. The formula of LB medium is as follows: yeast powder 1%, peptone 0.5%, sodium chloride 1%, pH 7.0-7.2. After fermentation, the fermentation broth was centrifuged at 8500r / min at room temperature for 10min to remove the bacteria. The fermentation supernatant was collected and the pH of the supernatant was adjusted to 2.0 with 6M hydrochloric acid. The supernatant was precipitated overnight at 4℃ and centrifuged at 9500r / min for 15min at 4℃. Collect the acid precipitation, freeze-dry the acid precipitation, extract the acid precipitation with methanol three times, combine the extracts, and use a rotary evaporator to concentrate the methanol extract to obtain a crude extr...

Embodiment 2

[0024] Example 2: The addition of heptadecanoic acid can increase the yield of lipopeptides produced by Bacillus Pc3 fermentation

[0025] Bacillus Pc3 strain was inoculated into LB liquid medium, cultured at 28°C and 180r / min for 36 minutes, and heptadecanoic acid was added to the medium at a final concentration of 0.01 g / L, and the culture was continued for 60 minutes. After fermentation, the bacteria were removed by centrifugation, the supernatant was acidified with hydrochloric acid, the acid precipitate was collected by centrifugation, freeze-dried, methanol extraction and rotary evaporation were used to obtain the crude lipopeptide extract, and the change of lipopeptide content was analyzed by RP-HPLC. RP-HPLC analysis results showed that the addition of heptadecanoic acid increased the yield of iturin A by 1.1 times, of which iturin A (C14) was increased by 3.3 times, and iturin A (C15) was increased by 0.85 times. Bacteriocin A (C17) increased by about 1.33 times ( figur...

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Abstract

The invention discloses a fermenting method for improving bacillus sp. antimicrobial lipopeptide yield, and relates to the technical field of fermentation. Bacillus sp. Pc3 is fermented in an LB liquid medium, fatty acid is added in the fermenting process, and antimicrobial lipopeptide is separated. The separating method includes the steps: centrifuging fermented liquid, removing bacteria and collecting fermented supernatant; adjusting the pH (potential of hydrogen) value of the fermented supernatant to be 2.0 by hydrochloric acid, performing acid precipitation, staying overnight at the temperature of 4 DEG C, centrifuging the fermented supernatant and collecting precipitates; freeze-drying the precipitates, performing extraction by the aid of methanol and performing vacuum concentration on methanol extracts to obtain lipopeptide crude extracts; separating the lipopeptide crude extracts, collecting chromatographic peaks, separating activity peaks and collecting chromatographic peaks for mass spectrum identification; performing mass spectrum identification. Compared with existing other fermenting technologies for improving lipopeptide yield, the fermenting method has the advantagesthat a small quantity of fatty acid is added in the fermenting method, the lipopeptide yield can be remarkably improved, and the method is simple and applicable to scale production.

Description

Technical field [0001] The present invention relates to the field of fermentation technology, in particular to a fermentation method for increasing the production of Bacillus antibacterial lipopeptides. Background technique [0002] Lipopeptides are a class of linear or cyclic compounds consisting of a hydrophobic fatty acid chain and a hydrophilic amino acid chain. Bacterial lipopeptides are mainly synthesized by non-ribosomal peptide synthase (NRPSs) catalyzed by strains of Bacillus and Paenibacillus. In the context of biological control, lipopeptides derived from Bacillus are mainly divided into three families based on the differences in synthetic gene clusters and chemical structures: iturin, fentocin and surfactin. Iturin is composed of cycloheptapeptide (amino acid sequence LDDLLDL) and β-amino fatty acid. It has antifungal activity and strong hemolytic activity, but its inhibitory activity against bacteria and viruses is weak. Fencin is composed of a 10-amino acid polype...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/00C12N1/20C12R1/07
CPCC07K14/32C12N1/20
Inventor 陈新华丁连帅郭文斌
Owner THIRD INST OF OCEANOGRAPHY MINIST OF NATURAL RESOURCES