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Antifungal peptide high-yield strain and method for preparing antibacterial peptide

A technology of high-yield strains and antifungal peptides, applied in the direction of microorganism-based methods, biochemical equipment and methods, chemicals for biological control, etc., can solve the problem of low production of antibacterial peptide iturin A, and achieve improved The effect of utilization

Inactive Publication Date: 2014-05-07
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Another object of the present invention is to provide a method for preparing the antibacterial peptide iturin A (iturin A) by culturing the Bacillus bacillus BH072 and its purification and extraction method, so as to solve the problem of low production of the antibacterial peptide iturin A in the prior art question

Method used

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  • Antifungal peptide high-yield strain and method for preparing antibacterial peptide
  • Antifungal peptide high-yield strain and method for preparing antibacterial peptide
  • Antifungal peptide high-yield strain and method for preparing antibacterial peptide

Examples

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Effect test

Embodiment 1

[0024] Identification of Bacillus BH072

[0025] Among the microorganisms isolated from honey, a microorganism with antifungal effect was accidentally discovered. After morphological and physiological and biochemical tests, it was found that the bacterium was Bacillus sp. BH072 (Bacillus sp.BH072 ). Further identified by molecular biology, the bacteria with antifungal activity was identified as Bacillus BH072.

[0026] The specific operation method of the identification process is as follows.

[0027] (1) Isolation of Bacillus BH072

[0028] The laboratory has isolated the target strain from a certain honey and named it Bacillus BH072.

[0029] ①Separate honey from honey into solid beef extract peptone plate, place at 37°C, and incubate for 24 hours.

[0030] ②Pure culture: Pick a single colony from the above plate and inoculate it on a solid slant medium, place it at 37°C, and incubate for 24 hours. (repeated twice).

[0031] ③ Glycerol storage: Pick bacteria from the a...

Embodiment 2

[0095] Cloning of Antibacterial Substance Genes

[0096] Primers were designed according to the published genes of several antibacterial substances of Bacillus, DNA of Bacillus was used as a template, PCR method was used to amplify, and the genes of antibacterial substances were cloned and determined. By consulting relevant literature, 6 antifungal gene primers were designed, as shown in Table 3. Using the DNA of Bacillus bacillus BH072 as a template, the antibacterial gene was amplified by PCR reaction. The conditions of the PCR reaction were as follows: pre-denaturation at 95°C for 3 min, then denaturation at 95°C for 30 s, renaturation at 51°C for 60 s, extension at 72°C for 90 s, and extension at 72°C for 5 min after 30 cycles. PCR amplification products were observed after 1% agarose gel electrophoresis. The PCR product was cloned into the pUCm-T Vector vector, the recombinant plasmid was transformed into Escherichia coli DH5α, the transformant containing the recombinan...

Embodiment 3

[0109] Determination of Inhibition Spectrum of Bacillus BH072

[0110] Bacteriostasis tests were carried out using the Oxford cup method.

[0111] In order to test the antifungal activity, the BH072 strain was inoculated into 10 mL of LB liquid medium, cultured at 37 °C, 150 rpm for 12 h, and then transferred to a 500 mL shake flask for fermentation for 24 h. Centrifuge the fermented liquid for 30 minutes at 4200 rpm and 0° C., and the sterile supernatant is the crude extract of antibacterial substances. Next, the activity of antibacterial substances was detected by the Oxford cup method. First, 1 mL of fungal spore suspension was evenly added to 100 mL of 40-50 ° C PDA medium, and spread on a solid beef extract peptone plate with an Oxford cup. After solidification, pull out the Oxford cup, add 5 μL of crude extract into the hole left by the Oxford cup, incubate at 30°C for 48 hours, and measure the diameter of the inhibition zone. The indicator bacteria are Aspergillus ni...

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Abstract

The invention discloses a bacillus BH072 (Bacillus sp.BH072) and a method for preparing antibacterial peptide iturin A through culturing the strain bacillus BH072 (Bacillus sp.BH072). According to the invention, iturin A is generated by adopting a fermentation method, 6%-7% inoculum size of bacillus BH072 (Bacillus sp.BH072) is taken from a seed culture medium to be inoculated to a fermentation culture medium to culture for 60 hours at the culture temperature of 29-30 DEG C at the pH of 6.4-6.5 at the rotation speed of 150rpm, and purification and extraction are carried out. The bacillus BH072 (Bacillus sp.BH072) disclosed by the invention has an obvious bactericidal effect on phytopathogens such as Aspergillus niger, Fusarium oxysporum, Botrytis cinerea and pythium, has broad-spectrum antibacterial property; through the optimization of fermentation conditions, the utilization rate of the strain is improved, a reference is provided for other similar tests; the iturin A separated from fermentation liquor by adopting an effective separation method has strong antifungal activity; after identification and yield optimization, the yield of the iturin A is 52.21mg / ml and is higher than that of reported strain iturin A; and the iturin A is suitable for large-scale industrial production after development.

Description

technical field [0001] The invention relates to a new strain of Bacillus sp. BH072, a high-yield antifungal peptide strain of the genus Bacillus, and a method for preparing the antibacterial peptide iturin A (iturin A) by cultivating the strain. Background technique [0002] Fungi widely exist in nature. It is reported that there are about 1.5 million species of fungi in nature, but only 74,000 species are currently known. Some fungi can cause plant diseases, reduce crop yields, and cause huge economic losses. Therefore, it is a very difficult task for us to control fungal pollution in food crops and food, control its growth and reproduction and the production of toxins. Among the biological control methods of fungi, the research on antifungal peptides has become a hot spot in current research. According to the APD (the Antimicrobial Peptide Database) database, 756 different antifungal peptides have been isolated from different organisms such as humans, animals, reptiles, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A01P3/00C12P21/04C12R1/07
Inventor 周志江赵鑫韩烨
Owner TIANJIN UNIV
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