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Separation and purification method of antibacterial peptides in bodily wall of Apostichopus japonicus

A technology for separation, purification and antimicrobial peptides, applied in the field of separation and purification of antimicrobial peptides in the body wall of Apostichopus japonicus, can solve problems such as toxicity, and achieve the effects of small molecular weight, stable effect, and reduced drug residues

Inactive Publication Date: 2018-09-25
MARINE FISHERIES RES INST OF ZHEJIANG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in recent years, with the continuous expansion of the scale of sea cucumber farming, the quality of sea water has become worse and worse, and exogenous poisons have gradually increased, and the drugs used, such as chlorine dioxide, potassium permanganate, and formosan Malin and antibiotics have certain toxic effects on sea cucumbers to varying degrees

Method used

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  • Separation and purification method of antibacterial peptides in bodily wall of Apostichopus japonicus
  • Separation and purification method of antibacterial peptides in bodily wall of Apostichopus japonicus

Examples

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Embodiment 1

[0032] The fresh and live imitation japonicus was temporarily raised in seawater for 10 days in a holding tank, and Bacillus subtilis was added to the seawater to stimulate it to produce more antimicrobial peptides. After 10 days, it was rinsed with filtered seawater, removed from the intestinal tract, weighed, and cut. Grinding with a colloid mill, and then using a tissue masher for three consecutive times, each time for 3 minutes, and then using a cell wall crusher for three consecutive times, and set aside.

[0033] Add 5% acetic acid to the crushed imitation sea cucumber body wall tissue at a volume ratio of 1:1, mix with a high-speed disperser, place it in a 4°C refrigerator for extraction for 24 hours, then put the mixture into a high-speed refrigerated centrifuge, and centrifuge Rotate at 8000r / min, centrifuge at 4°C for 20min, take the supernatant, and store at 4°C. Add the same amount of 5% acetic acid to the precipitate again, leaching again at 4°C for 24 hours, cent...

Embodiment 2

[0042] The fresh and live imitation japonicus was temporarily raised in seawater for 8 days in a holding tank, and Bacillus subtilis was added to the seawater to stimulate it to produce more antimicrobial peptides. After 8 days, it was rinsed with filtered seawater, removed from the intestinal tract, weighed, and cut. Grinding with a colloid mill, and then using a tissue masher for 2 consecutive times, each time for 5 minutes, and then using a cell wall crusher for 5 consecutive crushing times, for later use.

[0043] Add 5% acetic acid to the crushed imitation sea cucumber body wall tissue at a volume ratio of 1:0.8, mix evenly with a high-speed disperser, place it in a refrigerator at 3°C ​​for extraction for 28 hours, then put the mixture into a high-speed refrigerated centrifuge, and centrifuge Rotate at 8000r / min, centrifuge at 3°C ​​for 25min, take the supernatant, and store at 3°C. Add the same amount of 5% acetic acid to the precipitate again, leaching again at 3°C ​​f...

Embodiment 3

[0049] Fresh and live imitation sea cucumbers were temporarily raised in seawater for 12 days in a holding tank, and Bacillus subtilis was added to the seawater to stimulate them to produce more antimicrobial peptides. After 12 days, they were rinsed with filtered seawater, removed from the intestinal tract, weighed, and cut. Grinding with a colloid mill, and then using a tissue masher for 4 consecutive crushes, each time for 2 minutes, and then using a cell wall crusher for 1 consecutive crush, for later use.

[0050] Add 5% acetic acid to the crushed imitation sea cucumber body wall tissue at a volume ratio of 1:1.2, mix evenly with a high-speed disperser, place it in a refrigerator at 5°C for extraction for 20 hours, then put the mixture into a high-speed refrigerated centrifuge, and centrifuge Rotate at 8000r / min, centrifuge at 5°C for 15min, take the supernatant, and store at 5°C. Add the same amount of 5% acetic acid to the precipitate again, extract again at 5°C for 20 ...

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Abstract

The invention relates to separation and purification of polypeptides and discloses a separation and purification method of antibacterial peptides in bodily wall of Apostichopus japonicus, comprising the steps of 1) pretreating a material; 2) performing acetic acid extraction; 3) performing ultrafiltration; 4) performing macroporous adsorbing resin chromatography; 5) performing high-speed counter-current chromatography purification; 6) performing sephadex chromatography. Live Apostichopus japonicus is stimulated to produce massive antibacterial peptides; a most suitable separation and purification method is selected according to the properties of the most effective ingredient T1 in the antibacterial peptides in the bodily wall of Apostichopus japonicus to acquire high-purity antibacterial peptides; the antibacterial peptides have best antibacterial effect on Staphylococcus aureus, Salmonella enteritidis and Bacillus subtilis and can replace drugs fed during farming of Apostichopus japonicus, thereby reducing medicine residue in Apostichopus japonicus, and promoting the ensuring of quality safety of Apostichopus japonicus products and healthy development of the food industry.

Description

technical field [0001] The invention relates to the separation and purification of polypeptides, in particular to a method for separation and purification of antibacterial peptides in the body wall of sea cucumbers. Background technique [0002] Antimicrobial peptides, also known as antimicrobial peptides, can resist external microbial invasion and remove mutated cells in the body, usually small molecule peptides. It widely exists in organisms, such as: insects, fish, birds, crustaceans, amphibians and mammals. Although there are still many problems to be solved in the popularization and application of marine animal active peptides, my country has abundant marine animal resources, and the huge application potential of active peptides derived from marine animals in functional food, health food, beauty and skin care and pharmaceutical industries will continue to grow. fully excavated. [0003] Sea cucumber is a very valuable tonic with high nutrition, also known as "sea ginse...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K1/36C07K1/34C07K1/16
CPCC07K14/43504
Inventor 孟春英
Owner MARINE FISHERIES RES INST OF ZHEJIANG
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