Antifreeze polypeptide prepared by utilizing alkaline protease to carry out enzymolysis on collagens from fish skins

A technology of fish skin collagen and antifreeze peptides, which is applied in the preparation method of peptides, peptides, organic chemistry, etc., and can solve problems such as loss of quality, damage to cell and tissue structures, etc.

Active Publication Date: 2013-12-25
FUZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Repeated rise and fall of temperature causes ice crystals to grow continuously, freeze-thaw and recrystallize, which seriously damages the cell and tissue structure and loses the quality of the product.

Method used

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  • Antifreeze polypeptide prepared by utilizing alkaline protease to carry out enzymolysis on collagens from fish skins
  • Antifreeze polypeptide prepared by utilizing alkaline protease to carry out enzymolysis on collagens from fish skins
  • Antifreeze polypeptide prepared by utilizing alkaline protease to carry out enzymolysis on collagens from fish skins

Examples

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preparation example Construction

[0028] The antifreeze activity detection system for preparing antifreeze polypeptides of the present invention uses low-temperature freeze-thawed bacteria to detect the growth protection effect of adding antifreeze polypeptides on bacteria after low-temperature freeze-thaw. Inoculate the activated Lactobacillus bulgaricus into the liquid medium at 37°C, and cultivate overnight on a shaker at 130r / min as the seed liquid, inoculate the seed liquid into a new liquid medium at a ratio of 1:100, at 37°C, 130r / min Shaker culture to OD 600 =1.0 or so. Take a number of 1.5mL sterilized centrifuge tubes, add 900μL of antifreeze peptide concentration of 250μg / mL tested sample that has been filtered and sterilized, and dilute the bacterial solution by 10 4times, pipette 100 μL and add it to the sample to be tested, mix evenly, spread, incubate upside down in an incubator at 37°C for 18 hours, and count the number of colonies. Put the remaining sample-bacteria solution mixture at -20°C ...

Embodiment 1

[0034] Weigh 1.65 g of fish skin collagen and dissolve it in 6 ml of Mili-Q water, then adjust its pH to 9.0 with 2 mol / L NaOH. First heat the solution to 45°C in a water bath, then add a corresponding amount of alkaline protease according to the enzyme-substrate ratio of 1:20, and the enzymatic hydrolysis time is 30 minutes. Then inactivate the enzyme in a boiling water bath for 10 minutes, then centrifuge at 14000 rpm for 10 minutes after cooling, and collect the supernatant for later use.

[0035] The supernatant was separated by Sephadex G-50 gel chromatography (length 100cm, diameter 2.6cm), the eluent was deionized water, the flow rate was 2mL / min, and the elution peak was measured at 225nm. Elution peak of antifreeze activity.

[0036] The elution peak with the best antifreeze activity separated by Sephadex G-50 gel chromatography is separated in the next step, separated by Sulfopropyl-Sepadex C-25 cation exchange chromatography (length 55cm, diameter 2.0cm), and elute...

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Abstract

The invention provides an antifreeze polypeptide prepared by utilizing alkaline protease to carry out enzymolysis on collagens from fish skins. A preparation method is characterized by using the collagens from fish skins as raw materials, adopting alkaline protease to carry out enzymolysis on the collagens from fish skins and then carrying out separation and purification, thus obtaining a purified specific antifreeze polypeptide. The amino acid complete sequence of the antifreeze polypeptide is la-Asp-Gly-Gln-Thr-Gly-Gln-Arg-Gly-Glu-Lys-Gly-Pro-Ala-Gly-Val-Lys-Gly-Asp-Ala. By adopting the antifreeze polypeptide, the research ideas and methods of antifreeze proteins (polypeptides) existing at home and abroad are broken through, limitation of the quantity of the antifreeze proteins purified form natural organisms and considerations of international FDA (food and drug administration) for security of transgenic antifreeze proteins in food application are overcome, an efficient antifreeze polypeptide based on food sources is obtained, and a theoretical foundation is laid for developing the antifreeze polypeptide based on food sources and exploring extensive application of the antifreeze polypeptide to food and medicines.

Description

technical field [0001] The invention relates to an antifreeze polypeptide, more specifically to an antifreeze polypeptide prepared by hydrolyzing fish skin collagen with alkaline protease, and belongs to the field of biotechnology. Background technique [0002] The problem that food and pharmaceutical products repeatedly suffer from ice crystal growth and recrystallization due to fluctuations in ambient temperature during cryogenic storage and transportation has attracted increasing attention. The repeated rise and fall of temperature causes ice crystals to grow continuously, freeze-thaw and recrystallize, which seriously damages the cell and tissue structure and loses the quality of the product. Scientists around the world are facing serious challenges: how to control the growth and recrystallization of ice crystals, and realize the control of ice crystal growth on the low-temperature cold chain is the key to restricting the quality of many food and pharmaceutical products....

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K1/20C07K1/18C07K1/16C12P21/06
Inventor 汪少芸赵立娜邵彪饶平凡
Owner FUZHOU UNIVERSITY
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