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Biological magnetic separation method for screening antimicrobial peptide with antifungal activity

A biomagnetic separation and anti-fungal technology, applied in the direction of fermentation, etc., to achieve the effects of reducing production costs, improving efficiency, and low price

Inactive Publication Date: 2015-12-09
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no relevant literature reports on the preparation of antifungal active antimicrobial peptides by magnetic liposomes at home and abroad

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: The preparation method of tartary buckwheat antifungal active antimicrobial peptide is as follows:

[0023] (1) Take 100g of tartary buckwheat powder as raw material, add 600mL of petroleum ether, reflux defatting with petroleum ether (boiling range: 30-60°C) in a water bath at 40°C for 6 hours to obtain defatted tartary buckwheat powder, add 100g of defatted tartary buckwheat powder Add 1000mL of distilled water, adjust the pH to 8.5, extract the protein at 25°C and 300rpm for 3h, then centrifuge at 8000g at 25°C for 30min to get the supernatant, adjust the pH of the supernatant to 4.0 to precipitate protein, and wash the protein precipitate with water for 2 The second time, after adding water to adjust the pH to neutral, place it in a pre-cooling temperature of -80°C in an ultra-low temperature refrigerator for 3 hours, freeze-dry it at -55°C and a pressure of 0.2mBar for 48 hours to obtain tartary buckwheat crude protein, and store it at 4°C for future u...

Embodiment 2

[0028] Embodiment 2: The preparation method of the antifungal active antimicrobial peptide of green thorn fruit meal is as follows:

[0029] (1) Take 100g of green thorn fruit meal as raw material, add 500mL of petroleum ether, and degrease with petroleum ether (boiling range: 30~60°C) in a water bath at 40°C for 5 hours to obtain defatted green thorn fruit meal powder. Add 700mL of distilled water to the thorn fruit meal, adjust the pH value to 9.0, extract the protein at 20°C and 400rpm for 4 hours, centrifuge at 9000g at 20°C for 25min to take the supernatant, adjust the pH of the supernatant to 4.5 to precipitate the protein, The protein precipitate was washed 3 times with water, mixed with water to adjust the pH to neutral, then placed in a pre-cooled temperature of -80°C in an ultra-low temperature refrigerator for 2 hours, and then freeze-dried at -50°C and a pressure of 0.1mBar for 45 hours to obtain the green thorn fruit meal Crude protein, stored at 4°C for later use...

Embodiment 3

[0034] Embodiment 3: The preparation method of soybean meal antifungal active antimicrobial peptide is as follows:

[0035] (1) Take 100g of soybean meal as raw material, add 800mL of petroleum ether, use petroleum ether (boiling range: 30-60°C) to reflux and degrease in a water bath at 40°C for 3 hours to obtain defatted soybean meal powder, add 2000mL of distilled water, adjust the pH to 9.0, extract the protein at 4°C and 500rpm for 6h, then centrifuge at 10,000g at 4°C for 15min to get the supernatant, adjust the pH of the supernatant to 4.5 to precipitate protein, wash the protein precipitate 3 times with water , add water and mix to adjust the pH to neutral, put it in a pre-cooling temperature of -80°C ultra-low temperature refrigerator for 6 hours, freeze-dry at -45°C and a pressure of 0.03mBar for 60 hours to obtain soybean meal crude protein, and store it at 4°C for later use;

[0036] (2) Prepare an aqueous solution of soybean meal crude protein with a mass percentag...

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PUM

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Abstract

The invention discloses a biological magnetic separation method for screening an antimicrobial peptide with antifungal activity and belongs to the field of preparing and purifying of a biological active peptide. In the method, food-borne protein is taken as a raw material, and is subjected to enzymolysis by protease to prepare a component containing antifungal peptide, and then biological magnetic separation of a magnetic fungal cytomembrane lipidosome is combined with a high performance liquid phase fingerprint technique to fast enrich, separate and purify tartary buckwheat crude protein hydrolysis antimicrobial peptide with antifungal activity. The magnetic fungal cytomembrane lipidosome is used for enriching the antifungal activity antimicrobial peptide, the whole separation process mainly involves two steps of biological magnetic separation and RP-HPLC, and compared with traditional tedious steps of protease release, ion exchange column chromatography, gel filtration and reversed phase chromatography for screening the antimicrobial peptide, the method has the advantages of being capable of furthest simulating physiological conditions of the action between the antimicrobial peptide and the fungal cytomembrane, fast and efficient, mild and accurate and simple in operation.

Description

technical field [0001] The invention relates to a process for rapid, accurate and large-scale screening and preparation of tartary buckwheat crude proteolytic antifungal and antibacterial peptides combined with biomagnetic separation combined with high-performance liquid phase fingerprinting technology, which belongs to the field of preparation of bioactive peptides. Background technique [0002] The incidence of human infection by pathogenic fungi has been increasing, especially some immunocompromised patients are facing a situation where there is no cure, and the spread of incurable fungal infection diseases and fungal drug-resistant strains has become a major problem in human health. big threat. [0003] Antimicrobial peptides, also known as host defense peptides, are the first line of defense against the threat of foreign microorganisms. They belong to the family of antibiotics. They are toxic to bacteria, fungi, protozoa, tumor cells, viruses, etc., and can be used as c...

Claims

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Application Information

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IPC IPC(8): C12P21/06C07K1/16C07K1/14
Inventor 李莉蓉陈璇韩鹏宋风霞
Owner KUNMING UNIV OF SCI & TECH
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