Chili antibacterial peptide extraction and purification method

A purification method and antimicrobial peptide technology, which is applied in the field of extraction and purification of capsicum antimicrobial peptides, and can solve problems such as unsatisfactory extraction methods

Active Publication Date: 2018-09-28
SOUTHWEST UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, current extraction

Method used

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  • Chili antibacterial peptide extraction and purification method
  • Chili antibacterial peptide extraction and purification method
  • Chili antibacterial peptide extraction and purification method

Examples

Experimental program
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Effect test

Embodiment 1

[0135] 1. Test material

[0136] Pepper seeds: the varieties are Tianying Xiaojiao, Dianthus, Xinhua, Bullet, Indian Pepper and Guizhou Bell Pepper, purchased from Chongqing Murong Agricultural Products Sales Co., Ltd.

[0137] Tested strain: Aspergillus flavus isolated and screened from corn in the early stage, from the Grass Industry Teaching and Research Office of the School of Animal Science, Southwest University.

[0138] 2. Main reagents

[0139] Sample buffer: 10% SDS (W / V), 0.25mol / L Tris, 5% mercaptoethanol (V / V), 50% glycerol (W / V), 0.5% bromophenol blue (W / V), pH Adjusted to 6.8.

[0140] Tris-HCL buffer: 0.02mol / L Tris-HCL, pH adjusted to 7.6.

[0141] PBS buffer: 0.02mol / L, add distilled water to 1L, adjust pH to 7.6

[0142] EDTA buffer: 10mM Na 2 HPO 4 , 15mM NaH 2 PO 4 , 100mM KCl, 1.5% EDTA.

[0143] Acrylamide stock solution: Liquid A (49.5%T, 3%C): 48g of acrylamide, 1.5g of methylene bisacryloyl, distilled water to volume 100mL. Solution B (49.5%T...

Embodiment 2

[0149] Example 2 Antibacterial activity of different varieties of capsicum seed antimicrobial peptides to Aspergillus flavus

[0150] Pepper seeds: the varieties are Tianying Xiaojiao, Dianthus, Xinhua, Bullet, Indian Pepper and Guizhou Bell Pepper, purchased from Chongqing Murong Agricultural Products Sales Co., Ltd.

[0151] Tested strain: Aspergillus flavus isolated and screened from corn in the early stage, from the Grass Industry Teaching and Research Office of the School of Animal Science, Southwest University.

[0152] Pepper seeds were crushed and sieved, extracted with anhydrous ether overnight to remove crude fat. Add it to the EDTA buffer solution at a material-to-liquid ratio of 1:5, extract overnight at 4°C, filter the extract and centrifuge at 8,000 rpm for 15 minutes to take the supernatant, and measure the amount of solid ammonium sulfate required for 40% saturation. After grinding and pulverizing, add slowly, stir evenly to make it fully dissolved, stand stil...

Embodiment 3

[0157] Example 3 Optimization of Extraction Conditions for Capsicum Seed Antimicrobial Peptides——Selection of Buffer

[0158] Take 10 g of degreased chili seed powder, and use 50 mL of A. sterile water; B. PBS; C. EDTA; D. Tris-HCl; E. 75% ethanol. Soak overnight at 4°C, stir and homogenate, filter through 4 layers of gauze, and then centrifuge at 12000rpm for 20min at 4°C. Ammonium sulfate was added to the supernatant to make the concentration reach 70%. After magnetic stirring for 30 minutes, it was allowed to stand at 4° C. for 4 hours, and then centrifuged at 4° C. at 12,000 rpm for 20 minutes. After the precipitate was redissolved with 10 mL of distilled water, 200 μL of the filtrate was taken to measure the zone of inhibition by the Oxford cup double-layer agar method with Aspergillus flavus as the indicator bacterium.

[0159] The effects of different buffer leaching on the extraction of antimicrobial peptides are as follows: figure 1 and figure 2 shown. Antimicrob...

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Abstract

The invention discloses an optimized chili antibacterial peptide extraction and purification method. Particularly, an inventor acquires the best conditions of chili antibacterial peptide extraction byresearching the influence of different extracting liquid, solid-liquid ratios, extracting time and ammonium sulfate saturability on chili seed antibacterial peptide extraction, and further, components with good antibacterial effects are selected after ultrafiltration and crude separation and subjected to Sephadex gel filtration chromatography to obtain an electrophoresis pure antibacterial peptide sample. The extraction and purification method is simple, the antibacterial effect of an extracted antibacterial peptide is improved by 50.32% as compared with the antibacterial effect before optimization, the antibacterial peptide has a wide antibacterial spectrum, particularly has a strong inhibiting effect on Aspergillus flavus, can effectively inhibit Aspergillus flavus spore germination andmycelial growth and can further inhibit absorption and utilization of nutrient substances in a culture medium by the Aspergillus flavus. The antibacterial peptide extracted by the method is used as acorn mildew preventive, corn quality can be effectively kept, the service life is prolonged, and the antibacterial peptide is better in effect when being used with other mildew preventives.

Description

technical field [0001] The invention relates to the field of separation and purification of antimicrobial peptides, in particular to a method for extracting and purifying capsicum antimicrobial peptides. Background technique [0002] Antimicrobial peptides (AMPs), a naturally evolved innate immune barrier, are produced by all organisms. The first antimicrobial peptide discovered in the world was discovered by Swedish scientist G.Boman and others in the silkworm chrysalis of Xigubi, and then people successively discovered it from bacteria, fungi, amphibians, insects, higher plants, mammals and even humans. And isolate the polypeptide with antibacterial activity. As an important part of the innate immune system, antimicrobial peptides have natural antimicrobial effects and are not prone to drug resistance. Due to the protein characteristics, non-pollution, safety and antibacterial properties of antimicrobial peptides, it has a good advantage in research on the substitution o...

Claims

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Application Information

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IPC IPC(8): C07K1/36C07K1/34C07K1/30C07K1/16
CPCC07K1/16C07K1/303C07K1/34C07K1/36
Inventor 韩玉竹杨叶梅曾兵赵建军赵展展陈强廖欢科吴长凤
Owner SOUTHWEST UNIVERSITY
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