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Method for extracting main egg yolk protein of sea cucumber body walls by three-step precipitation

A technology of egg yolk protein and sea cucumber, applied in the field of protein extraction, can solve the problems of unsuitability for industrialized mass production, low purity of egg yolk protein, low extraction efficiency, etc., and achieves the effects of low cost, increased protein precipitation rate, and improved extraction rate.

Active Publication Date: 2021-03-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the extraction of main vitellin is concentrated on the method of acid precipitation or organic reagent precipitation, the main vitellin obtained is low in purity and incomplete in structure; while the main vitellin purified by ion column etc. is of high purity, but the extraction efficiency is low and not Suitable for industrial mass production

Method used

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  • Method for extracting main egg yolk protein of sea cucumber body walls by three-step precipitation
  • Method for extracting main egg yolk protein of sea cucumber body walls by three-step precipitation
  • Method for extracting main egg yolk protein of sea cucumber body walls by three-step precipitation

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Step 1) sea cucumber body wall pretreatment: fresh sea cucumbers are dissected to remove body cavity contents, washed and drained, and crushed by a homogenizer to obtain sea cucumber mud;

[0036] Step 2) Crude protein extraction: mix the sea cucumber mud obtained in step (1) with PBS buffer solution (pH=7.4) at a mass volume ratio of 1:5g / mL, extract at 4°C for 18 hours, and then centrifuge. The protein supernatant is the protein bold liquid, this process is repeated 3 times, and the supernatants are combined;

[0037] Step 3) Concentrate the mixed precipitation of trichloroacetic acid and acetone: Concentrate the mixed supernatant obtained in step 2) at 60°C to 1 / 4 of the original volume, and add the obtained concentrated solution while stirring to a final concentration of 10 % TCA + 1 times the volume of acetone as a precipitation reagent, let stand at 4°C for 3 hours, collect the precipitate after centrifugation, repeat this process twice, and combine the precipitat...

Embodiment 2

[0049] Change the precipitation reagent in step 3) to verify its precipitation effect. Precipitation reagents were adding trichloroacetic acid (TCA) to a final concentration of 10%, 10% TCA+1 volume of acetone, adding 1M hydrochloric acid to adjust the pH to 3.7, adding 1M hydrochloric acid to adjust the pH to 3.7+1 volume of acetone, and 2 times volume of acetone.

[0050] Comparing the effects of choosing different precipitants on crude protein, observing the precipitated protein after centrifugation, after adding 2 times the volume of acetone alone, the protein precipitated the largest amount, but when adding the final concentration of 10% TCA + 1 times the volume of acetone as When the precipitating agent is used, the precipitated protein is whiter, which shows that 10% TCA + 1 volume of acetone can cooperate to precipitate, so that the precipitated protein content is high.

[0051] In addition, 20% TCA was added during the experiment, and the amount of precipitation was ...

Embodiment 3

[0053] Change the final concentration of ammonium sulfate in step 5) to be 0, 20%, 40%, 60%, 80%, 100% respectively. By comparing the effect of different concentration gradients of ammonium sulfate on the protein precipitation results ( Figure 5 and Figure 6 ), found that with the increase of ammonium sulfate amount, the amount of protein precipitation increased gradually, when the mass volume ratio of ammonium sulfate and water was 0.4, the protein content of precipitation was more, and the bands were cleaner without impurity pollution. At the same time, considering the difficulty of the next step of dialysis desalination, the final concentration of ammonium sulfate and ammonium sulfate is selected to be 30-40% in this method.

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Abstract

The invention discloses a method for extracting main egg yolk protein of sea cucumber body walls by three-step precipitation, which belongs to the field of protein extraction, and comprises the following steps: preparing sea cucumber body wall protein crude extract by using sea cucumber body walls as raw materials, carrying out acid precipitation on mixed acetone precipitated protein, removing impure protein by Sevage, precipitating target protein by ammonium sulfate, performing desalting at low temperature by a dialysis method, carrying out freeze drying, obtaining the main egg yolk protein with high purity, wherein the main egg yolk protein of the protein is identified as a glyphosate protein through the cooperation of mass spectrometry and electrophoresis technologies. According to theinvention, high-valued utilization of sea cucumbers is improved, protein resources are fully developed and utilized, and a prerequisite basis is provided for research of protein targeting functional activity; the whole process is simple in experimental operation, the protein extraction rate and purity are high, special advanced instrument assistance is not needed, target protein is precipitated and separated through three steps, a large amount of main egg yolk protein can be obtained at a time, and the method is suitable for large-scale and high-precision purity research. The protein identification specificity is high, results are clear and visual, and operation is easy and convenient.

Description

technical field [0001] The invention relates to a three-step precipitation method for extracting the main yolk protein of sea cucumber body wall, belonging to the field of protein extraction. Background technique [0002] The main vitellin in the body wall of sea cucumber is a kind of glycophospholipid protein, which mainly exists in two forms, namely the main vitellin with low molecular weight and the main vitellogenin with high molecular weight. The main vitellogenin is the precursor of the main vitellin. substance. The main vitellin in the body wall of sea cucumber exists in the form of high polymers, which generally contain relatively high molecular weight. Studies have shown that major vitellin is closely related to the transport of amino acids, fats, carbohydrates, vitamins, minerals and other nutrients and functional substances in organisms, and major vitellin can be used as an immune factor to resist the invasion of external pathogenic microorganisms. Therefore, ho...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K1/14C07K1/30
CPCC07K14/43504
Inventor 周鹏冯建慧张丽娜
Owner JIANGNAN UNIV
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