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Extraction method of porcine blood antibacterial peptide

An extraction method and technology of antimicrobial peptides, applied in the field of extraction of porcine blood antimicrobial peptides, can solve the problems of low enzymatic hydrolysis efficiency, achieve high yield and purity, easy industrial scale production, and good effect

Inactive Publication Date: 2018-06-15
金华市铁骑士生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the pig blood antibacterial agent has not been anticoagulated before enzymolysis, and the enzymolysis efficiency is low

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] A method for extracting pig blood antimicrobial peptides, comprising pig blood anticoagulant treatment, enzymatic hydrolysis, separation, and purification, the specific steps of which are:

[0016] 1) Pig blood anticoagulant treatment: Add 2% sodium hexametaphosphate aqueous solution to fresh pig blood at a volume ratio of 1:6, stir evenly, and then place it in a centrifuge at a temperature of 5°C and a speed of 4000r / min. Discard the precipitate after centrifuging in the machine for 40 minutes, adjust the pH value of the supernatant to 8.0, and obtain the anticoagulated pig blood. The presence of triethylamine can promote the PO in the molecular structure of sodium hexametaphosphate 3 - Ions combine with calcium ions in pig blood to prevent the catalytic action of calcium ions and prevent blood coagulation. At the same time, it can inhibit the activity of thrombin in pig blood and prevent soluble fibrinogen from becoming insoluble under the activation of thrombin. It...

Embodiment 2

[0021] A method for extracting pig blood antimicrobial peptides, comprising pig blood anticoagulant treatment, enzymatic hydrolysis, separation, and purification, the specific steps of which are:

[0022] 1) Pig blood anticoagulant treatment: Add 4% sodium hexametaphosphate aqueous solution to fresh pig blood at a volume ratio of 1:4, stir evenly, and then place it in a centrifuge at a temperature of 2°C and a speed of 6000r / min. After centrifuging in the machine for 20 minutes, the precipitate was discarded, and the pH of the supernatant was adjusted to 9.0 to obtain anticoagulated pig blood for later use. The above-mentioned sodium hexametaphosphate aqueous solution contained 0.02mM fatty acid methyl ester and 0.018mM triethylamine;

[0023] 2) Enzymolysis: Add Alcalase and trypsin at a weight ratio of 1:0.9 to anticoagulated pig blood at an enzyme dosage of 6%, and then microwave enzymolysis at a temperature of 35°C and a microwave power of 180W for 3 hours , after the enzy...

Embodiment 3

[0027] A method for extracting pig blood antimicrobial peptides, comprising pig blood anticoagulant treatment, enzymatic hydrolysis, separation, and purification, the specific steps of which are:

[0028] 1) Pig blood anticoagulant treatment: add 3% sodium hexametaphosphate aqueous solution to fresh pig blood at a volume ratio of 1:5, stir evenly, and then place it in a centrifuge at a temperature of 4°C and a speed of 5000r / min. After centrifuging in the machine for 30 minutes, the precipitate was discarded, and the pH value of the supernatant was adjusted to 8.5 to obtain anticoagulated pig blood for later use. The above aqueous solution of sodium hexametaphosphate contained 0.025 mM fatty acid methyl ester and 0.016 mM triethylamine;

[0029] 2) Enzymolysis: Add Alcalase and trypsin at a weight ratio of 1:0.8 to anticoagulated pig blood at an enzyme dosage of 6.5%, and then microwave enzymolysis at a temperature of 37°C and a microwave power of 160W for 4 hours , after the ...

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Abstract

The invention discloses an extraction method of porcine blood antibacterial peptide. The method comprises the following specific steps: adding a sodium hexametaphosphate aqueous solution into fresh porcine blood, uniformly stirring, removing precipitate after centrifugalization, and adjusting the pH value of supernate, so as to obtain anti-freezing porcine blood; adding Alcalase enzyme and trypsininto the anti-freezing porcine blood, performing microwave enzymolysis, adjusting the pH value after enzymolysis is completed, heating the anti-freezing porcine blood to be boiled, and filtering, soas to obtain filtrate namely enzymatic hydrolysate; performing ultrafiltration and nanofiltration on the enzymatic hydrolysate, so as to obtain antibacterial peptide crude extract; performing gel chromatography on the antibacterial peptide crude extract, washing through double distilled water after loading, collecting components with high antibacterial activity, and performing freeze drying, so asto obtain the porcine blood antibacterial peptide. The method has the benefits that the method is simple and practical, the extraction efficiency, yield and purity are high, and the formed antibacterial peptide is high in activity, can rapidly kill bacteria and has broad-spectrum antibacterial property.

Description

technical field [0001] The invention relates to the technical field of biological extraction, in particular to a method for extracting porcine antimicrobial peptides. Background technique [0002] Since the discovery of antibiotics, it has been widely used in medical treatment, traditional veterinary medicine and feed additives due to its good antibacterial effect. The more serious it is, it has gradually attracted people's attention. It is extremely urgent to find effective alternatives to antibiotics and solve the problems of drug resistance and drug residues. Antimicrobial peptides (AMPs) are widely distributed and diverse polypeptide substances with broad-spectrum antibacterial activity that can be produced by both prokaryotes and eukaryotes. Negative bacteria, fungi, protozoa, cancer cells, viruses, etc. all have obvious inhibitory effects, and are regarded as a substitute for antibiotics with great application prospects, and have become a research hotspot at home and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/06C07K1/34
CPCC12P21/06
Inventor 全盈园
Owner 金华市铁骑士生物科技有限公司
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