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Anti-freeze polypeptide prepared by enzymolysis of cow leather collagen through papain

An antifreeze polypeptide and amino acid technology, applied in the biological field, can solve problems such as small quantities and limited application prospects of large laws

Inactive Publication Date: 2011-10-12
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the discovery and research of various antifreeze proteins, two key issues restricting the research and application of biological antifreeze proteins have become increasingly prominent: 1. The amount obtained by natural isolation and purification is very small and very limited 2. When scientists are working on transgenic technology to expand the production of antifreeze protein from organisms, the safety concerns of transgenic antifreeze protein in food applications have become a major concern for consumers. , the focus of common concern of EU organizations and international FDA organizations

Method used

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  • Anti-freeze polypeptide prepared by enzymolysis of cow leather collagen through papain
  • Anti-freeze polypeptide prepared by enzymolysis of cow leather collagen through papain
  • Anti-freeze polypeptide prepared by enzymolysis of cow leather collagen through papain

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preparation example Construction

[0034] The antifreeze activity detection system for preparing antifreeze polypeptide of the present invention adopts a combined cold-heat-stage system, multi-fold microscope and automatic camera system to construct an antifreeze activity inhibition ice crystal growth detection system platform. The cryogenic environment during operation was maintained with liquid nitrogen. Simulate temperature fluctuations of products during cryogenic storage. Through the system program setting, set the temperature fluctuation of the sample and repeat it continuously for 7 to 25 cycles, connect a multi-fold microscope and a real-time photographic recording system, and record the real-time ice crystal growth of the sample during the cold-hot cycle.

[0035] Sephadex G-50 molecular sieves, Sepharose SP C-25 ion exchange chromatography, RP-HPLC reversed-phase high-performance liquid chromatography, dialysis and other separation and purification methods are used to achieve efficient separation and ...

Embodiment 1

[0040] Weigh 1.65 g bovine collagen and dissolve it in 6 ml Mili-Q water, then adjust its pH to 7.0 with 2 mol / L NaOH. First heat the solution to 37°C in a water bath, then add the corresponding amount of enzyme according to the enzyme-substrate ratio of 1:10, 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. Collect the supernatant for later use.

[0041] 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, see figure 1 . Peaks were collected and assayed for antifreeze activity (eg figure 2 shown). It can be seen that Fraction2 has the best antifreeze activity. Statistical analysis shows that the average diameter of ice crystals is 4.62um, which is significantly smaller than Fraction1 (19.68um) and Frac...

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Abstract

The invention provides anti-freeze polypeptide and a preparation method thereof. In the preparation method, the specific anti-freeze polypeptide is prepared by optimizing the enzymatic condition of the papain and separating and purifying the cow leather collagen serving as a raw material, wherein the molecular weight of the anti-freeze polypeptide is 877Da, and an amino acid complete sequence of the anti-freeze polypeptide is HyP-Gly-Met-Hyl-Gly-His-Arg-Gly. The anti-freeze polypeptide and the preparation method break through the study concept and method of the conventional anti-freeze protein (polypeptide) at home and abroad, eliminate the limitation on the number of purifying the anti-freeze protein from natural organisms and the safety worry of International FDA Organization about the transgenic anti-freeze protein in food application, obtain food source-based high-efficiency anti-freeze polypeptide, and establish theoretical basis for developing the food source-based anti-freeze polypeptide and exploring the wide application of the food source-based anti-freeze polypeptide in food and medical science.

Description

technical field [0001] The invention provides an antifreeze polypeptide and a preparation method thereof, and more specifically relates to an antifreeze polypeptide prepared by enzymolyzing bovine hide collagen with papain, belonging 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 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06C07K1/20C07K1/18C07K1/16C12P21/06
Inventor 汪少芸陈琳郑邦晓饶平凡
Owner FUZHOU UNIV
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