阅读说明：本技术 鱼鳞角蛋白的制备方法 (Preparation method of fish scale keratin ) 是由 刘焱 李秋雨 陈韬 于 2020-06-03 设计创作，主要内容包括：本发明提供了一种鱼鳞角蛋白的制备方法,包括以下步骤：a)将鱼鳞进行预处理；b)提取预处理后的鱼鳞中的胶原蛋白；c)采用碱性蛋白酶法提取步骤b)获得的残渣,得到鱼鳞角蛋白；d)采用碱法提取步骤c)获得的残渣,得到鱼鳞角蛋白。本发明首先将鱼鳞预处理后提取其中的胶原蛋白,然后通过两步法提取剩余残渣中的角蛋白,第一步采用碱性蛋白酶法提取残渣中的角蛋白,第二步继续用碱法提取残渣中得角蛋白,本发明将碱性蛋白酶法和碱法联用提取角蛋白,不仅提高了角蛋白的收率,而且提高了其收率。(The invention provides a preparation method of fish scale keratin, which comprises the following steps: a) preprocessing fish scales; b) extracting collagen from the pretreated fish scales; c) extracting the residue obtained in the step b) by adopting an alkaline protease method to obtain fish scale keratin; d) extracting the residue obtained in the step c) by an alkaline method to obtain the fish scale keratin. The method comprises the steps of firstly pretreating fish scales, extracting collagen from the fish scales, then extracting keratin from the residual residues through a two-step method, extracting the keratin from the residues through an alkaline protease method in the first step, and continuously extracting the keratin from the residues through the alkaline method in the second step.)

1. A preparation method of fish scale keratin is characterized by comprising the following steps:

a) preprocessing fish scales;

b) extracting collagen from the pretreated fish scales;

c) extracting the residue obtained in the step b) by adopting an alkaline protease method to obtain fish scale keratin;

d) extracting the residue obtained in the step c) by an alkaline method to obtain the fish scale keratin.

2. The method for preparing according to claim 1, wherein the step c) comprises:

adding 0.005-0.2 mol/L sodium hydroxide solution and 1-5 wt% of alkaline protease in the residue obtained in the step b), reacting at 50-80 ℃, filtering, centrifuging the filtrate, adjusting the pH value of the supernatant to the isoelectric point of keratin, centrifuging, dissolving, dialyzing and freeze-drying the obtained precipitate to obtain the fish scale keratin.

3. The method of claim 2, wherein the step d) comprises:

adding a sodium hydroxide solution with the mass concentration of 7-9 wt% into the residue obtained in the step c), reacting at 55-65 ℃, filtering, adjusting the pH value of the filtrate to an isoelectric point, and centrifuging, dissolving, dialyzing and freeze-drying the obtained precipitate to obtain the fish scale keratin.

4. The method according to any one of claims 1 to 3, wherein the amount of the sodium hydroxide solution added in step c) is 15 to 25 times the weight of the residue obtained in step b).

5. The method according to claim 3, wherein the amount of the sodium hydroxide solution added in step d) is 15 to 25 times the weight of the residue obtained in step c).

6. The method according to claim 1, wherein step a) comprises in particular:

a1) soaking fish scales in a sodium chloride solution to remove foreign proteins;

a2) soaking the fish scales obtained in the step a1) in a sodium hydroxide solution for degreasing;

a3) soaking the fish scales obtained in the step a2) in a hydrogen peroxide solution for decoloring;

a4) soaking the fish scales obtained in the step a4) in a citric acid solution for deliming.

7. The preparation method according to claim 6, wherein in the step a1), the concentration of sodium chloride is 80-120 g/L;

in the step a2), the concentration of the sodium hydroxide solution is 0.5-1.5 mol/L;

in the step a3), the concentration of the hydrogen peroxide solution is 0.5-1 wt%;

in the step a4), the concentration of the citric acid solution is 3-4 wt%.

8. The method according to claim 1, wherein step b) comprises in particular:

extracting collagen in the pretreated fish scales by adopting an acid method and/or an enzyme method.

9. The method according to claim 8, wherein step b) comprises in particular:

soaking and extracting the fish scales obtained in the step a) in a citric acid solution.

10. The method according to claim 8, wherein step b) comprises in particular:

soaking the fish scales obtained in the step a) in a citric acid solution of pepsin for extraction.

Technical Field

The invention relates to the field of protein, in particular to a preparation method of fish scale keratin.

Background

Keratin is a fibrous scleroprotein with connective and protective functions, has stable structure and high denaturation temperature, and is often used in medicines and biological materials. At present, researches on keratin mainly focus on the extraction of animal hair keratin, wherein the researches on the extraction of wool keratin by a reduction method are the most, and the selection and optimization of a reducing agent and the like are hot spots.

In recent years, researches show that fish scales contain various active ingredients, and researches on fish scale collagen, fish scale hydroxyapatite and the like are more and more, so that the added value of the fish scales is improved, and waste is changed into valuable. However, after the collagen is extracted from the fish scales, a large amount of residues still remain, and the residues also contain rich keratin which is an effective substance. The keratin in the residues is extracted, so that the added value of the fish scales can be greatly improved, and the wastes are changed into valuables.

Disclosure of Invention

In view of the above, the invention provides a preparation method of fish scale keratin, and the fish scale keratin obtained by the method has high purity, high yield and high molecular weight.

The invention provides a preparation method of fish scale keratin, which comprises the following steps:

a) preprocessing fish scales;

b) extracting collagen from the pretreated fish scales;

c) extracting the residue obtained in the step b) by adopting an alkaline protease method to obtain fish scale keratin;

d) extracting the residue obtained in the step c) by an alkaline method to obtain the fish scale keratin.

The method comprises the steps of firstly pretreating fish scales, extracting collagen from the fish scales, then extracting keratin from the residual residues through a two-step method, extracting the keratin from the residues through an alkaline protease method in the first step, and continuously extracting the keratin from the residues through the alkaline method in the second step.

Specifically, the invention firstly preprocesses the fish scales, and the steps are as follows:

the step a) specifically comprises the following steps:

a1) soaking fish scales in a sodium chloride solution to remove foreign proteins;

a2) soaking the fish scales obtained in the step a1) in a sodium hydroxide solution for degreasing;

a3) soaking the fish scales obtained in the step a2) in a hydrogen peroxide solution for decoloring;

a4) soaking the fish scales obtained in the step a4) in a citric acid solution for deliming.

In one embodiment, in the step a1), the concentration of sodium chloride is 80-120 g/L.

In one embodiment, in the step a2), the concentration of the sodium hydroxide solution is 0.5-1.5 mol/L;

in one embodiment, in the step a3), the concentration of the hydrogen peroxide solution is 0.5-1 wt%;

in one embodiment, in the step a4), the citric acid solution has a concentration of 3 to 4 wt%.

Specifically, the step a) may include:

a1) removing hybrid protein: cleaning fresh fish scales, and soaking the fresh fish scales in 100g/L sodium chloride solution at 4 ℃ for 12 hours;

a2) degreasing: after removing the foreign proteins, the fish scales are washed by tap water and rinsed by distilled water for three times, and then are soaked in 0.1mol/L sodium hydroxide solution for 12 hours at 4 ℃.

a3) And (3) decoloring: after degreasing, the fish scales are washed by tap water and rinsed for three times by distilled water, and then are decolorized for 6 hours at 4 ℃ by 0.5-1% hydrogen peroxide solution.

a4) Deliming: washing the decolored fish scales with tap water, rinsing the fish scales with distilled water for three times, and soaking the fish scales in 3-4% citric acid solution with the volume being 12-14 times of that of the fish scales at 4 ℃ for 6 hours.

After fish scales are pretreated, collagen in the fish scales is extracted, and the collagen in the fish scales can be extracted by an enzyme method or an acid method.

Specifically, the acid method for extracting collagen comprises the following steps: soaking and extracting the fish scales obtained in the step a) in a citric acid solution.

The method for extracting collagen by the enzyme method comprises the following steps: soaking the fish scales obtained in the step a) in a citric acid solution of pepsin for extraction.

The residues obtained after the collagen extraction can be used for continuously extracting the keratin by adopting an alkaline protease method and an alkaline method in sequence, and the yield and the purity of the keratin extracted by the subsequent alkaline method can be improved by firstly extracting the keratin by adopting the alkaline protease method.

Specifically, the process for extracting keratin by using the alkaline protease method comprises the following steps:

adding 0.005-0.2 mol/L sodium hydroxide solution and 1-5 wt% of alkaline protease in the residue obtained in the step b), reacting at 50-80 ℃, filtering, centrifuging the filtrate, adjusting the pH value of the supernatant to the isoelectric point of keratin, centrifuging, dissolving, dialyzing and freeze-drying the obtained precipitate to obtain the fish scale keratin.

Specifically, the process for extracting keratin by the alkaline method comprises the following steps:

adding a sodium hydroxide solution with the mass concentration of 7-9 wt% into the residue obtained in the step c), reacting at 55-65 ℃, filtering, adjusting the pH value of the filtrate to an isoelectric point, and centrifuging, dissolving, dialyzing and freeze-drying the obtained precipitate to obtain the fish scale keratin.

In one embodiment, in the step c), the amount of the sodium hydroxide solution added is 15 to 25 times of the weight of the residue obtained in the step b).

In one embodiment, the amount of sodium hydroxide solution added is 15 to 25 times the weight of the residue obtained in step c).

The invention provides a preparation method of fish scale keratin, which comprises the following steps: a) preprocessing fish scales; b) extracting collagen from the pretreated fish scales; c) extracting the residue obtained in the step b) by adopting an alkaline protease method to obtain fish scale keratin; d) extracting the residue obtained in the step c) by an alkaline method to obtain the fish scale keratin. The method comprises the steps of firstly pretreating fish scales, extracting collagen from the fish scales, then extracting keratin from the residual residues through a two-step method, extracting the keratin from the residues through an alkaline protease method in the first step, and continuously extracting the keratin from the residues through the alkaline method in the second step.

Detailed Description