阅读说明：本技术 鱼皮胶原蛋白多肽的提取方法及其提取物和应用 (Extraction method of fish skin collagen polypeptide, extract and application thereof ) 是由 王代军 于 2019-10-29 设计创作，主要内容包括：本发明提供了一种鱼皮胶原蛋白多肽的提取方法及其提取物和应用,涉及蛋白多肽生产技术领域。该提取方法包括使用微生物水解经过预处理的鱼皮,然后使用酶膜耦合法纯化经微生物水解的鱼皮；所述预处理包括使用酶制剂酶解鱼皮。该提取方法无需酸性或碱性条件便可完成水解鱼皮,减少了酶的用量和整体的水解时间,能够使鱼皮水解彻底,提高胶原蛋白多肽的提取率,提取得到的提取物中小分子鱼皮胶原蛋白多肽含量高,分子量均一,经济价值高。(The invention provides an extraction method of fish skin collagen polypeptide, an extract and application thereof, and relates to the technical field of protein polypeptide production. The extraction method comprises hydrolyzing pretreated fish skin with microorganism, and purifying the fish skin hydrolyzed with microorganism by enzyme membrane coupling method; the pretreatment comprises enzymolysis of the fish skin by using an enzyme preparation. The extraction method can hydrolyze the fish skin without acid or alkaline conditions, reduces the dosage of enzyme and the whole hydrolysis time, can hydrolyze the fish skin thoroughly, improves the extraction rate of collagen polypeptide, and obtains the extract with high content of small-molecular fish skin collagen polypeptide, uniform molecular weight and high economic value.)

1. A method for extracting fish skin collagen polypeptide is characterized by comprising the steps of hydrolyzing pretreated fish skin by using microorganisms, and then purifying the fish skin hydrolyzed by the microorganisms by using an enzyme membrane coupling method; the pretreatment comprises enzymolysis of the fish skin by using an enzyme preparation.

2. The extraction process according to claim 1, wherein the microorganisms comprise Acetobacter pasteurianus, Bifidobacterium, Lactobacillus plantarum, Saccharomyces cerevisiae, Aspergillus niger, Pediococcus pentosaceus, Lactobacillus casei, Mucor, Lactobacillus paracasei, Bacillus sphaericus, Brevibacterium, Alcaligenes, Citrobacter, Pseudomonas sp, Arthrobacter sp, and stenotrophomonas maltophilia;

preferably, the microorganisms comprise 1-5 parts of acetobacter pasteurianus, 1-5 parts of bifidobacterium, 1-5 parts of lactobacillus plantarum, 1-5 parts of saccharomyces cerevisiae, 1-5 parts of aspergillus niger, 1-5 parts of pediococcus pentosaceus, 1-5 parts of lactobacillus casei, 1-5 parts of mucor, 1-2 parts of lactobacillus paracasei, 1-2 parts of bacillus sphaericus, 1-2 parts of brevibacterium, 1-2 parts of alcaligenes, 1-2 parts of bacillus citrate, 1-2 parts of pseudomonas, 1-2 parts of arthrobacter and 1-2 parts of stenotrophomonas maltophilia by weight;

preferably, the enzyme preparation comprises at least one of a neutral protease, an alkaline protease, trypsin and pepsin.

3. The extraction method according to claim 1, wherein the skin is derived from at least one of squid, grass carp, cod and tilapia.

4. The extraction method according to claim 1, wherein in the enzyme membrane coupling method, a filter membrane is used to pass polypeptides with molecular weight not more than 1000 Dal;

preferably, the enzyme membrane coupling method comprises a multi-stage enzyme membrane coupling method;

preferably, the pH condition of the enzyme membrane coupling method is 2.5-10;

preferably, the temperature condition of the enzyme membrane coupling method is 15-60 ℃;

preferably, the loading capacity of the protease in the enzyme membrane coupling method is 0.1-50 mg/g;

preferably, the pH of the elution buffer is 4-8;

preferably, a three-step gradient elution is used.

5. The extraction method according to any one of claims 1 to 4, wherein the pretreatment further comprises mixing the pulped fish skin with water to prepare a fish skin slurry, and then subjecting the fish skin slurry to enzymatic hydrolysis using an enzyme preparation; wherein the mass ratio of the beaten fish skin to the water is 1: (5-15);

preferably, the beaten fish skin is sieved by a sieve of 200-400 meshes and then mixed with water.

6. The extraction method according to claim 5, wherein in the enzymolysis of the fish skin by using the enzyme preparation, the dosage of the enzyme preparation is 0.1-3% of the mass of the fish skin pulp;

preferably, when the fish skin is derived from tuna, the enzyme preparation comprises pepsin; the dosage of the enzyme preparation is 1 to 2 percent of the mass of the fish skin pulp;

preferably, when the fish skin is derived from a squid, the enzyme preparation comprises an alkaline protease and a pepsin; the dosage of the enzyme preparation is 0.5 to 3 percent of the mass of the fish skin pulp;

preferably, the ratio of the amount of alkaline protease to the amount of pepsin is 1: 1-3: 1;

preferably, when the fish skin is derived from carp, the enzyme preparation comprises trypsin and neutral protease; the dosage of the enzyme preparation is 0.5 to 3 percent of the mass of the fish skin pulp;

preferably, the ratio of trypsin to neutral protease is 1: 1-1: 5;

preferably, the enzymolysis temperature is 45-60 ℃, and the enzymolysis time is 2-4 h;

preferably, the enzymatic hydrolysis is carried out with stirring.

7. The extraction method according to claim 5, wherein the pretreated fish skin is hydrolyzed by using microorganisms, and the amount of the microorganisms is 1-5% of the mass of the fish skin slurry;

preferably, the hydrolysis temperature is 35-45 ℃, and the hydrolysis time is 2-6 h;

preferably, the hydrolysis is carried out with stirring.

8. The extraction method according to claim 5, wherein the fish skin collagen polypeptide is obtained by solid-liquid separation of fish skin slurry hydrolyzed by microorganisms and purification of the separated liquid;

preferably, the solid-liquid separation comprises the steps of firstly carrying out first solid-liquid separation on the fish skin slurry subjected to microbial hydrolysis to obtain a first separated liquid, then using water to extract and precipitate, then carrying out solid-liquid separation to obtain a second separated liquid, and then combining the first separated liquid and the second separated liquid;

preferably, in the step of leaching the precipitate by using water, the using amount of the water is 5-15 times of the mass of the precipitate;

preferably, the solid-liquid separation comprises centrifugation;

preferably, the rotation speed of the centrifugation is 8000-10000 rpm;

preferably, the separation liquid to be purified is concentrated prior to purification; preferably concentrating until the content of solid matters in the separation liquid is 15-20%;

preferably, concentrating the separated liquid comprises concentrating the separated liquid to be concentrated using reduced pressure evaporation; preferably, the separation liquid is concentrated by using a reduced-pressure vacuum single-effect evaporator;

preferably, the temperature of the reduced pressure evaporation is 55-65 ℃;

preferably, the vacuum degree of the reduced pressure evaporation is 0.07-0.095 MPa;

preferably, the separation liquid is subjected to decolorization treatment before purification; activated carbon is preferably used for decolorization;

preferably, the particle size of the activated carbon is 50-100 meshes;

preferably, the using amount of the activated carbon is 1-3% of the mass of the separation liquid;

preferably, the protein is adsorbed by using a static adsorption method at the temperature of 40-60 ℃;

preferably, the extraction method further comprises preparing the fish skin collagen polypeptide into fish skin polypeptide powder after purification;

preferably, the fish skin protein polypeptide powder is prepared by adopting a spray drying method;

preferably, the temperature of a feed inlet of the spray drying equipment is 160-180 ℃;

preferably, the temperature of a discharge port of spray drying equipment is 75-80 ℃;

preferably, the proportion of the auxiliary materials in the dry powder obtained by spray drying is 0-20%.

9. An extract prepared by the extraction method of any one of claims 1 to 8.

10. Use of the extraction method according to any one of claims 1 to 8 or the extract according to claim 9 in (x1) to (x4) below;

(x1) preparing a cosmetic;

(x2) preparing a medical material;

(x3) preparing a food product;

(x4) preparing a medicament.

Technical Field

The invention relates to the technical field of protein polypeptide production, in particular to a method for extracting fish skin collagen polypeptide, an extract and application thereof.

Background

The collagen polypeptide as a natural degradable polymer has the advantages of high biological activity, good biocompatibility, proper degradation speed and the like, is easy to process and produce, is one of ideal biomedical materials at present, has excellent affinity with fish skin collagen polypeptide with the molecular weight of less than 1000, is easy to be absorbed by a human body, and can well play the functions of permeation, moisture retention, repair and the like. The fish resources in China are very rich, a large amount of leftovers such as fish skin, fish head, fish bones, fish scales and the like are generated in the aquaculture processing process, the wastes account for 45-70% of the total weight of the fish body and are basically directly discarded, how to change the leftovers generated in the aquatic product processing process into valuables is realized, the full utilization of biological resources is one of the problems to be solved urgently by scientific researchers, considerable economic benefits can be generated, and the method has great significance for protecting the ecological environment.

At present, the extraction method of fish skin collagen polypeptide can be divided into 5 types: acid, alkaline, enzymatic, salt and hot water extraction. In the actual extraction process, most of collagen extracted by a hot water extraction method is denatured into gelatin, and the temperature needs to be strictly controlled; the salt extraction method can affect the stability of collagen conformation and reduce the bioactivity; alkaline extraction destroys the amino acid sequence of collagen and produces toxic substances; the collagen extracted by the acid method can keep a triple helix structure, but the extraction time is long and the extraction rate is low; enzymatic extraction, although the hydrolysis conditions are relatively mild, the hydrolysis is not complete. In order to improve the collagen yield on the basis of maintaining the triple helical structure of collagen, different extraction methods are often combined with each other. At present, an extraction method combining acid and enzyme is often adopted, but the problems of large enzyme dosage, long hydrolysis time, strict requirement on reaction pH value and low yield still exist, the obtained collagen has a large molecular weight distribution range and a high structural damage degree, and meanwhile, industrial production brings more strong acid and strong alkali wastewater, and the wastewater treatment cost is high. Therefore, an improved method for extracting fish skin collagen polypeptide is needed.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first purpose of the invention is to provide a method for extracting fish skin collagen polypeptide, which has high production efficiency and extraction efficiency.

The second purpose of the invention is to provide an extract prepared by the method for extracting the fish skin collagen polypeptide.

The third purpose of the invention is to provide an extraction method of the fish skin collagen polypeptide or application of the extract.

In order to solve the technical problems, the invention adopts the following technical scheme:

according to one aspect of the present invention, there is provided a method for extracting fish skin collagen polypeptide, comprising hydrolyzing pretreated fish skin using a microorganism, and then purifying the microbial-hydrolyzed fish skin using an enzyme membrane coupling method; the pretreatment comprises enzymolysis of the fish skin by using an enzyme preparation.

Preferably, the microorganisms include acetobacter pasteurianus, bifidobacterium, lactobacillus plantarum, saccharomyces cerevisiae, aspergillus niger, pediococcus pentosaceus, lactobacillus casei, mucor, lactobacillus paracasei, bacillus sphaericus, brevibacterium, alcaligenes, citrobacter, pseudomonas, arthrobacter, and stenotrophomonas maltophilia;

preferably, the microorganisms comprise 1-5 parts of acetobacter pasteurianus, 1-5 parts of bifidobacterium, 1-5 parts of lactobacillus plantarum, 1-5 parts of saccharomyces cerevisiae, 1-5 parts of aspergillus niger, 1-5 parts of pediococcus pentosaceus, 1-5 parts of lactobacillus casei, 1-5 parts of mucor, 1-2 parts of lactobacillus paracasei, 1-2 parts of bacillus sphaericus, 1-2 parts of brevibacterium, 1-2 parts of alcaligenes, 1-2 parts of bacillus citrate, 1-2 parts of pseudomonas, 1-2 parts of arthrobacter and 1-2 parts of stenotrophomonas maltophilia by weight;

preferably, the enzyme preparation comprises at least one of a neutral protease, an alkaline protease, trypsin and pepsin.

Preferably, the fish skin is derived from at least one of squid, grass carp, cod, and tilapia.

Preferably, in the enzyme membrane coupling method, a filter membrane is used for passing polypeptides with the molecular weight not more than 1000 Dal;

preferably, the enzyme membrane coupling method comprises a multi-stage enzyme membrane coupling method;

preferably, the pH condition of the enzyme membrane coupling method is 2.5-10;

preferably, the temperature condition of the enzyme membrane coupling method is 15-60 ℃;

preferably, the loading capacity of the protease in the enzyme membrane coupling method is 0.1-50 mg/g;

preferably, the pH of the elution buffer is 4-8;

preferably, a three-step gradient elution is used.

Preferably, the pretreatment further comprises mixing the pulped fish skin with water to prepare fish skin pulp, and then carrying out enzymolysis on the fish skin pulp by using an enzyme preparation; wherein the mass ratio of the beaten fish skin to the water is 1: (5-15);

preferably, the beaten fish skin is sieved by a sieve of 200-400 meshes and then mixed with water.

Preferably, in the enzymolysis of the fish skin by using the enzyme preparation, the dosage of the enzyme preparation is 0.1-3% of the mass of the fish skin pulp;

preferably, when the fish skin is derived from tuna, the enzyme preparation comprises pepsin; the dosage of the enzyme preparation is 1 to 2 percent of the mass of the fish skin pulp;

preferably, when the fish skin is derived from a squid, the enzyme preparation comprises an alkaline protease and a pepsin; the dosage of the enzyme preparation is 0.5 to 3 percent of the mass of the fish skin pulp;

preferably, the ratio of the amount of alkaline protease to the amount of pepsin is 1: 1-3: 1;

preferably, when the fish skin is derived from carp, the enzyme preparation comprises trypsin and neutral protease; the dosage of the enzyme preparation is 0.5 to 3 percent of the mass of the fish skin pulp;

preferably, the ratio of trypsin to neutral protease is 1: 1-1: 5;

preferably, the enzymolysis temperature is 45-60 ℃, and the enzymolysis time is 2-4 h;

preferably, the enzymatic hydrolysis is carried out with stirring.

Preferably, in the method for hydrolyzing the pretreated fish skin by using the microorganisms, the using amount of the microorganisms is 1-5% of the mass of the fish skin slurry;

preferably, the hydrolysis temperature is 35-45 ℃, and the hydrolysis time is 2-6 h;

preferably, the hydrolysis is carried out with stirring.

Preferably, the fish skin slurry subjected to microbial hydrolysis is subjected to solid-liquid separation, and the separation liquid is purified to obtain the fish skin collagen polypeptide;

preferably, the solid-liquid separation comprises the steps of firstly carrying out first solid-liquid separation on the fish skin slurry subjected to microbial hydrolysis to obtain a first separated liquid, then using water to extract and precipitate, then carrying out solid-liquid separation to obtain a second separated liquid, and then combining the first separated liquid and the second separated liquid;

preferably, in the step of leaching the precipitate by using water, the using amount of the water is 5-15 times of the mass of the precipitate;

preferably, the solid-liquid separation comprises centrifugation;

preferably, the rotation speed of the centrifugation is 8000-10000 rpm;

preferably, the separation liquid to be purified is concentrated prior to purification; preferably concentrating until the content of solid matters in the separation liquid is 15-20%;

preferably, concentrating the separated liquid comprises concentrating the separated liquid to be concentrated using reduced pressure evaporation; preferably, the separation liquid is concentrated by using a reduced-pressure vacuum single-effect evaporator;

preferably, the temperature of the reduced pressure evaporation is 55-65 ℃;

preferably, the vacuum degree of the reduced pressure evaporation is 0.07-0.095 MPa;

preferably, the separation liquid is subjected to decolorization treatment before purification; activated carbon is preferably used for decolorization;

preferably, the particle size of the activated carbon is 50-100 meshes;

preferably, the using amount of the activated carbon is 1-3% of the mass of the separation liquid;

preferably, the protein is adsorbed by using a static adsorption method at the temperature of 40-60 ℃;

preferably, the extraction method further comprises preparing the fish skin collagen polypeptide into fish skin polypeptide powder after purification;

preferably, the fish skin protein polypeptide powder is prepared by adopting a spray drying method;

preferably, the temperature of a feed inlet of the spray drying equipment is 160-180 ℃;

preferably, the temperature of a discharge port of spray drying equipment is 75-80 ℃;

preferably, the proportion of the auxiliary materials in the dry powder obtained by spray drying is 0-20%.

According to another aspect of the invention, the invention also provides the extract prepared by the extraction method.

According to another aspect of the invention, the invention also provides an extraction method of the fish skin collagen polypeptide or an extract prepared by the extraction method, and applications of the fish skin collagen polypeptide or the extract in the following (x1) to (x 4);

(x1) preparing a cosmetic;

(x2) preparing a medical material;

(x3) preparing a food product;

(x4) preparing a medicament.

Compared with the prior art, the invention has the following beneficial effects:

the extraction method of the fish skin collagen polypeptide provided by the invention firstly uses an enzyme preparation to carry out enzymolysis on the fish skin, and then uses microorganisms to hydrolyze the fish skin. The fish skin is hydrolyzed by combining the enzyme preparation and the microorganism without acid or alkaline conditions; and because of the combined use of microorganisms, the use amount of enzyme and the whole hydrolysis time are reduced; the fish skin is hydrolyzed completely by combining the enzyme preparation and the microorganism, so that the extraction rate of the fish skin collagen polypeptide is improved. And then purifying the fish skin hydrolyzed by the microorganisms by an enzyme membrane coupling method, so that the extracted fish skin collagen polypeptide has uniform and controllable molecular weight, wherein the ratio of the polypeptide with the molecular weight of 700-1000Dal is high, and the economic value is high. The fish skin collagen polypeptide content in the extract prepared by the extraction method is high, and the molecular weight is uniform. The extraction method of the fish skin collagen polypeptide or the extract prepared by the extraction method provided by the invention can be applied to a plurality of fields of preparing cosmetics, medical materials, foods, medicines and the like.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

According to one aspect of the present invention, there is provided a method for extracting collagen polypeptide from fish skin, comprising hydrolyzing pretreated fish skin with a microorganism, and purifying the microbial-hydrolyzed fish skin using an enzyme membrane coupling method; the pretreatment comprises enzymolysis of the fish skin by using an enzyme preparation.

The invention jointly uses the enzyme preparation and the microorganism to hydrolyze the fish skin, and the fish skin can be hydrolyzed without acid or alkaline conditions; and because of the combined use of microorganisms, the use amount of enzyme and the whole hydrolysis time are reduced; the fish skin is hydrolyzed completely by combining the enzyme preparation and the microorganisms, so that the extraction rate of the collagen polypeptide is improved; the fish skin hydrolyzed by the microorganisms is purified by an enzyme membrane coupling method, so that the molecular weight of the extracted fish skin collagen polypeptide is controllable, wherein the ratio of the polypeptide with the molecular weight of 700-1000Dal is high, and the economic value is high.

It should be noted that the present invention is not limited to the kind of fish with fish skin, and the fish that can be used to extract collagen from fish skin, which is conventional in the art, may include, but is not limited to, at least one of squid, grass carp, cod, and tilapia, for example. It is understood that only one kind of skin may be used as the raw material in the extraction, for example, the skin may be derived from squid, grass carp, cod or tilapia, or a mixture of several kinds of skins may be used, for example, the skin of squid and the skin of grass carp may be used as the raw material for the extraction; for example, the skin of carp and the skin of cod are used as extraction raw materials; for example, the mixture of squid skin, grass carp skin, cod skin and tilapia skin is used as the extraction raw material, but the present invention is not limited thereto.

In some preferred embodiments, the hydrolysis of the enzymatically hydrolyzed fish skin is more effective when the microorganisms include the following microorganisms: acetobacter pasteurianus, Bifidobacterium, Lactobacillus plantarum, Saccharomyces cerevisiae, Aspergillus niger, Pediococcus pentosaceus, Lactobacillus casei, Mucor, Lactobacillus paracasei, Bacillus sphaericus, Brevibacterium, Alcaligenes, Citrobacter, Pseudomonas sp, Arthrobacter sp, and stenotrophomonas maltophilia.

In some preferred embodiments, the microorganisms include acetobacter pasteurianus 1-5 parts by weight, such as but not limited to 1 part, 2 parts, 3 parts, 4 parts or 5 parts; 1-5 parts of bifidobacterium, such as but not limited to 1 part, 2 parts, 3 parts, 4 parts or 5 parts; 1-5 parts of lactobacillus plantarum, such as but not limited to 1 part, 2 parts, 3 parts, 4 parts or 5 parts; 1-5 parts of saccharomyces cerevisiae, such as but not limited to 1 part, 2 parts, 3 parts, 4 parts or 5 parts; 1-5 parts of Aspergillus niger, such as but not limited to 1 part, 2 parts, 3 parts, 4 parts or 5 parts; 1-5 parts of pediococcus pentosaceus, for example but not limited to, 1 part, 2 parts, 3 parts, 4 parts or 5 parts; 1-5 parts of lactobacillus casei, such as but not limited to 1 part, 2 parts, 3 parts, 4 parts or 5 parts; 1-5 parts of mucor, such as but not limited to 1 part, 2 parts, 3 parts, 4 parts or 5 parts; 1-2 parts of lactobacillus paracasei, such as but not limited to 1 part, 1.2 parts, 1.5 parts, 1.8 parts or 2 parts; 1-2 parts of bacillus sphaericus, for example but not limited to, 1 part, 1.2 parts, 1.5 parts, 1.8 parts or 2 parts; 1-2 parts of brevibacterium, such as but not limited to 1 part, 1.2 parts, 1.5 parts, 1.8 parts or 2 parts; 1-2 parts of Alcaligenes, such as but not limited to 1 part, 1.2 parts, 1.5 parts, 1.8 parts or 2 parts; 1-2 parts of citric acid bacillus, for example but not limited to, 1 part, 1.2 parts, 1.5 parts, 1.8 parts or 2 parts; 1-2 parts of pseudomonas, such as but not limited to 1 part, 1.2 parts, 1.5 parts, 1.8 parts or 2 parts; 1-2 parts of arthrobacter, such as but not limited to 1 part, 1.2 parts, 1.5 parts, 1.8 parts or 2 parts; and 1-2 parts of stenotrophomonas maltophilia, such as but not limited to 1 part, 1.2 parts, 1.5 parts, 1.8 parts or 2 parts.

In some preferred embodiments, the enzyme preparation comprises at least one of a neutral protease, an alkaline protease, trypsin, and pepsin; in some alternative embodiments, the fish skin is hydrolyzed using one of neutral protease, alkaline protease, trypsin or pepsin as an enzyme preparation; in other alternative embodiments, the fish skin is hydrolyzed using a combination of several of neutral protease, alkaline protease, trypsin and pepsin as the enzyme preparation. The kind of enzyme in the enzyme preparation can be adjusted according to the source of the fish skin: in some preferred embodiments, the skin of tuna is preferably hydrolyzed with pepsin; preferably, the squid skin is enzymolyzed by alkaline protease and pepsin; preferably, the carp skin is enzymolyzed with trypsin and neutral protease.

The enzyme membrane coupling method depends on the specificity, the catalysis and the specific function of the enzyme, realizes the coupling of the reaction and the reaction in the biological reaction process, integrates the in-situ separation and the concentration of the product and the recycling of the enzyme, on the basis, the enzyme is fixed by a column, macromolecular components which do not pass through the membrane after the enzymolysis reaction and the membrane filtration enter an enzymolysis system again, and pass through the membrane filtration after the degradation until the macromolecular substances are almost completely degraded into target-size substances which can pass through the membrane, the multistage enzyme membrane coupling technology can greatly strengthen the reaction process, control the reaction process, regulate and control the composition of the product, reduce the generation of by-products, improve the conversion rate of raw materials and enrich and purify the polypeptide product. In some preferred embodiments, the enzyme membrane coupling method comprises a multistage enzyme membrane coupling method, preferably using a filter membrane through which polypeptides having a molecular weight of not more than 1000Dal pass. The molecular weight distribution range of the fish skin collagen polypeptide obtained by purifying the fish skin hydrolyzed by the microorganisms is 0-1000Dal, wherein the polypeptide with the molecular weight of 700-1000Dal accounts for 98 percent, the molecular weight of the polypeptide product can be uniform and controllable, and the economic value of the obtained extract is improved. In the enzyme membrane coupling method, the pH condition is preferably 2.5 to 10, for example, but not limited to, 2.5, 5, 7, 8 or 10; the temperature condition is preferably 15 to 60 ℃, for example, but not limited to, 15 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃ or 60 ℃; the protease load is preferably 0.1-50mg/g, and may be, for example, but not limited to, 0.1mg/g, 0.5mg/g, 1mg/g, 2mg/g, 5mg/g, 10mg/g, 15mg/g, 20mg/g, 30mg/g, 40mg/g, 50mg/g or 60 mg/g.

In some preferred embodiments, the pre-treatment further comprises mixing the pulped fish skin with water to make a fish skin slurry, and then enzymatically hydrolyzing the fish skin slurry using an enzyme preparation; the fish skin is mixed with water to prepare fish skin slurry, which facilitates the reaction of enzyme and microorganism with substrate. Wherein the fish skin and the water after pulping are preferably mixed according to the mass ratio of 1: (5-15), for example, but not limited to, 1:5, 1:8, 1:10, 1:12 or 1: 15. Preferably, the beaten fish skin is firstly sieved by a 200-400 mesh sieve, for example, but not limited to 200 mesh, 260 mesh, 300 mesh, 325 mesh or 400 mesh, so as to filter out impurities in the raw material.

In some preferred embodiments, when the amount of the enzyme preparation used in the enzymatic hydrolysis of fish skin is 0.1% to 3% of the mass of the fish skin slurry, the hydrolysis effect is better, and may be, for example, but not limited to, 0.1%, 0.2%, 0.5%, 0.8%, 1%, 1.5%, 2%, 2.5% or 3%. When the fish skin comes from different fish species, the extraction effect can be optimized by adjusting the proportion and the dosage of the enzyme in the enzyme preparation. Preferably, when the fish skin is derived from tuna, the enzyme preparation comprises pepsin; the dosage of the enzyme preparation is 1 to 2 percent of the mass of the fish skin pulp; preferably, when the fish skin is derived from a squid, the enzyme preparation comprises an alkaline protease and a pepsin; the dosage of the enzyme preparation is 0.5-3% of the mass of the fish skin pulp, wherein the dosage ratio of the alkaline protease to the pepsin is preferably 1: 1-3: 1; preferably, when the fish skin is derived from carp, the enzyme preparation comprises trypsin and neutral protease; the dosage of the enzyme preparation is 0.5 to 3 percent of the mass of the fish skin pulp; wherein, the dosage ratio of the trypsin to the neutral protease is preferably 1: 1-1: 5.

in some preferred embodiments, the following enzymatic conditions are preferred: the enzymolysis temperature is 45-60 ℃, for example, but not limited to, 45 ℃, 48 ℃, 50 ℃, 55 ℃, 58 ℃ or 60 ℃; the enzymolysis time is 2-4 h, for example, but not limited to, 2h, 2.5h, 3h, 3.5h or 4 h. Preferably, during enzymolysis, a mode of enzymolysis while stirring can be adopted to promote full contact between enzyme and substrate so as to improve the enzymolysis efficiency.

In some preferred embodiments, in the hydrolysis of the pretreated fish skin by using the microorganism, the hydrolysis efficiency is better when the amount of the microorganism is 1 to 5 percent of the mass of the fish skin slurry, and the amount of the microorganism, namely the amount of the strain, if the microorganism exists in the form of a microbial inoculum, refers to the amount of the microorganism in the microbial inoculum; the amount of the microorganism may be, for example, but not limited to, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%.

In some preferred embodiments, the following hydrolysis conditions are preferred: the hydrolysis temperature is 35-45 ℃, for example, but not limited to, 35 ℃, 38 ℃, 40 ℃, 42 ℃ or 45 ℃; the hydrolysis time is 2-6 h, for example but not limited to 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h or 6 h; preferably, the hydrolysis is carried out under stirring, so that the microorganisms can be promoted to be fully contacted with the fish skin, and the hydrolysis efficiency is improved.

In some preferred embodiments, the fish skin collagen polypeptide is obtained by solid-liquid separation of fish skin slurry hydrolyzed by microorganisms and purification of the separated liquid.

In some preferred embodiments, the solid-liquid separation comprises subjecting the fish skin slurry hydrolyzed by the microorganism to a first solid-liquid separation to obtain a first separated liquid, then using water to extract the precipitate so as to more fully extract collagen from the fish skin, then performing solid-liquid separation to obtain a second separated liquid, and then combining the first separated liquid and the second separated liquid. Preferably, when water is used for leaching precipitation, the amount of water is 5 to 15 times of the mass of the precipitation, for example, but not limited to, 5 times, 8 times, 10 times, 12 times or 15 times.

In some preferred embodiments, the solid-liquid separation comprises centrifugation, and it is understood that centrifugation may be used in both of the above-described two solid-liquid separation operations, or centrifugation may be used in any one of the solid-liquid separation operations. The rotation speed of the centrifugation is preferably 8000-10000rpm, for example but not limited to 8000rpm, 8500rpm, 9000rpm, 9500rpm or 10000 rpm.

In some preferred embodiments, the separation to be purified is concentrated prior to purification; preferably, the concentration is performed until the content of solids in the separation liquid is 15 to 20%, for example, but not limited to, 15%, 16%, 17%, 18%, 19%, or 20%, which is beneficial for the subsequent purification operation.

In some preferred embodiments, concentrating the separated liquid comprises concentrating the separated liquid to be concentrated using evaporation under reduced pressure. The reduced pressure evaporation can reduce the boiling point of the liquid, prevent collagen in the separated liquid from denaturation and decomposition, increase the heat transfer temperature difference, strengthen the evaporation operation and reduce the requirement of a heat source, and preferably a reduced pressure vacuum single-effect evaporator is used for concentrating the separated liquid. The temperature of the reduced pressure evaporation is preferably 55 to 65 ℃, and for example, but not limited to, 55 ℃, 58 ℃, 60 ℃, 62 ℃ or 65 ℃; the degree of vacuum for reduced pressure evaporation is preferably 0.07 to 0.095MPa, and may be, for example, but not limited to, 0.07MPa, 0.075MPa, 0.08MPa, 0.085MPa, 0.09MPa, or 0.095 MPa.

In some preferred embodiments, the separation solution is subjected to a decolorization treatment prior to purification; activated carbon which has no toxic action and good adsorption effect is preferably used for decoloring. The particle size of the activated carbon is preferably 50-100 meshes, and for example, but not limited to, the particle size can be 50 meshes, 60 meshes, 70 meshes, 80 meshes, 90 meshes or 100 meshes; the amount of activated carbon used is preferably 1% to 3% by mass of the separation liquid, and may be, for example, but not limited to, 1%, 1.5%, 2%, 2.5%, or 3%; preferably, the protein is adsorbed by static adsorption at 40-60 deg.C, such as but not limited to 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C or 60 deg.C.

In some preferred embodiments, the extraction method further comprises purifying the fish skin collagen polypeptide to obtain fish skin polypeptide powder, preferably by spray drying. The temperature of the feed inlet of the spray drying equipment is preferably 160-180 ℃, for example, but not limited to 160 ℃, 165 ℃, 170 ℃, 175 ℃ or 180 ℃; the temperature of the discharge port of the spray drying equipment is preferably 75-80 ℃, for example, but not limited to, 75 ℃, 76 ℃, 77 ℃, 78 ℃, 79 ℃ and 80 ℃; the proportion of the auxiliary materials in the dry powder obtained by spray drying is 0-20%, for example, but not limited to, 0%, 5%, 10%, 15%, 20%, wherein 0% means no auxiliary materials.

According to another aspect of the invention, the invention also provides the extract prepared by the extraction method. The extract prepared by the extraction method has high fish skin collagen polypeptide content and uniform molecular weight.

According to another aspect of the invention, the invention also provides an extraction method of the fish skin collagen polypeptide or application of the extract prepared by the extraction method. The collagen polypeptide has good effects of moisturizing, locking water and whitening, and can be used in the field of cosmetics; the collagen polypeptide can also be used for preparing biological materials such as artificial skin, artificial esophagus, artificial trachea and the like, and can also be used for cell culture, preparation of slow-release medicines and the like; the collagen polypeptide has the effects of repairing gastric mucosa and liver, promoting calcium absorption, lowering blood pressure and resisting oxidation. In summary, the extraction method of fish skin collagen polypeptide provided by the invention or the extract prepared by the extraction method can be used for preparing cosmetics, medical materials, foods, medicines and other fields.

The technical solution and the advantages of the present invention will be further explained with reference to the preferred embodiments.