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Preparation method of porphyra protein antibacterial peptide

A technology of bacteriostatic peptide and seaweed, which is applied in the field of natural active substance extraction, can solve the problem of low added value and achieve the effect of small molecular weight, good application prospect and good antibacterial activity

Active Publication Date: 2014-06-18
NANTONG CHITSURU FOODS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] my country is a big country in the production, processing and utilization of seaweed resources. Zhejiang, Guangdong, Jiangsu and other coastal provinces have large-scale plantings. The production of seaweed ranks first in the world. According to statistics, the annual production of seaweed in China is about 60,000 tons, and the products are exported to Japan and Southeast Asia. And Europe and the United States and other countries and regions, but the current domestic products are mainly processed into rough processed foods or seasonings with relatively low added value after simple drying

Method used

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  • Preparation method of porphyra protein antibacterial peptide
  • Preparation method of porphyra protein antibacterial peptide
  • Preparation method of porphyra protein antibacterial peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Preparation and enzymatic hydrolysis of laver protein:

[0038] Pepsin, trypsin, acid protease, neutral protease, alkaline protease and papain were used to hydrolyze laver protein respectively, and compared with unhydrolyzed samples, the results were as follows figure 2 shown. Among several enzymes, papain has the best antibacterial activity of enzymatic hydrolysis of laver, so papain was selected as the enzyme for research.

[0039] a. Use Porphyra zebra as raw material, crush it after drying, sieve, take 60-80 mesh part and dissolve it in Na with a mass volume ratio of 1:25 2 HPO 4 -KH 2 PO 4 (0.02mol / L, pH 7.0) buffer solution, under the condition of ice bath, use 350 W power ultrasonic wave for 15 min (working mode: working time 3 s, time interval 3 s), and the material after ultrasonic treatment After centrifugation (4000 r / min, 15 min), discard the precipitate, and add ammonium sulfate to the supernatant to 60% saturation to extract the target lave...

Embodiment 2

[0041] Example 2 Purification and preparation of laver protein bacteriostatic peptide:

[0042] a. DEAE-52 anion exchange chromatography: using stage elution method, mobile phase: 0~74 min with 10 mmol / mL equilibrium solution (Na at pH 6.8 2 HPO 4 - citric acid) for 74-120 min with 0.2 mol / mL NaCl, 120-200 min with 0.4 mol / mL NaCl, 200-250 min with 0.6 mol / mL NaCl Buffer elution, flow rate: 1 mL / min, detection wavelength: 220 nm, collect the peak components, and compare the antibacterial activity of the collected peak components (6 mg / mL), and take the antibacterial activity The preferred peak fractions were lyophilized for later use.

[0043] b. Sephadex G-25 gel chromatography: take the above freeze-dried sample and dissolve it in ultrapure water at a concentration of 0.1 mg / mL. After the Sephadex G-25 gel chromatography column was equilibrated with ultrapure water, the injection volume was 2 mL, and eluted with ultrapure water. The detection wavelength is 220 nm, the fl...

Embodiment 3

[0044] Example 3 The molecular weight distribution of laver protein bacteriostatic peptide

[0045] Cytochrome (MW1250), ethanine-ethanine-tyrosine-arginine (MW451), bacillus enzyme (MW1450) After the μm microporous membrane treatment, the sample was loaded under the following chromatographic conditions: chromatographic column TSKgel 2000 SWxl (300 mm×7.8 mm, 5 μm); column temperature 30 ℃, mobile phase: water: trichloroacetic acid: acetonitrile=550:450: 1 (V:V:V); the flow rate is 1 mL / min, the injection volume is 5 μL, the detection wavelength is 220 nm, and the relative molecular mass calibration curve is drawn according to the peak pattern; 5 mL of chloroacetic acid (TCA) was mixed evenly, placed on the tabletop for 10 min, centrifuged at 8000 r / min for 10 min, the supernatant was treated with a 0.22 μm microporous membrane, and the molecular weight distribution of bacteriostatic peptide was measured under the same chromatographic conditions. Peptide molecular weight dist...

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Abstract

The invention discloses a preparation method of porphyra protein antibacterial peptide, and belongs to the field of natural active material extraction. The method comprises the following steps: with porphyra yezoensis as a raw material, drying and crushing, performing ultrasonic crushing and ammonium sulfate protein precipitation on the raw material, enzymatically hydrolyze the porphyra yezoensis raw material by using papain under a specific condition to obtain a crude product antibacterial peptide, separating and purifying the crude product through chromatographic processes such as ion-exchange column chromatography and G-25 gel chromatography to obtain the porphyra protein antibacterial peptide. The porphyra protein antibacterial peptide molecular weight is mainly distributed at 1000Da and lower, and the optimum effect is that the pH is 6.5, and the heat stability is good; the porphyra protein antibacterial peptide has the effect on gram-negative bacteria and gram-positive bacteria, and has a stronger inhibition effect on the gram-positive bacteria, the antibacterial effect is achieved through the destructive effect on the cell membrane. The porphyra protein antibacterial peptide is small in molecular weight, good in antibacterial activity, and good in application prospect in the fields of preparing antibacterial medicines and food preservatives.

Description

technical field [0001] The invention relates to the extraction of active components of marine plants, in particular to a preparation method of laver protein bacteriostatic peptide, which belongs to the field of extraction of natural active substances. Background technique [0002] With the abuse of antibiotics, there are more and more drug-resistant microorganisms in nature. In 2013, superbugs that appeared in the UK were discovered in many countries. Since the 1990s, due to the spread of global bird flu, many scientists worried that drug-resistant diseases in poultry and various animals would be transmitted to humans through the food chain, causing infections of patients that were difficult to cure. People began to pay attention to efficient and safe inhibitors. Bacterial drugs. Because the development of new chemically synthesized antibacterial drugs requires a long cycle, high costs, and demanding technical requirements. Therefore, at present, many pharmaceutical compan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/06C07K1/30C07K1/18C07K1/16
Inventor 田亚平刘冬冬周锋
Owner NANTONG CHITSURU FOODS
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