阅读说明：本技术 一种鱼皮胶原蛋白肽的制备方法 (Preparation method of fish skin collagen peptide ) 是由 苏战华 苏仲民 王晓强 于 2021-04-13 设计创作，主要内容包括：本发明提供了一种鱼皮胶原蛋白肽的制备方法,属于海洋胶原肽制备技术领域。本发明将鱼皮依次经碱溶液和酸溶液浸泡预处理后,进行水提,将鱼皮中的有效成分转移至浸提液中,得到明胶浸提液；对所述明胶浸提液进行酶解,将大分子蛋白质有效酶解成肽类物质,得到酶解液；过滤去除酶解液中的原料渣与悬浮物；酶解液滤液经超滤分离其中分子量为7000～8000Da的胶原蛋白肽；超滤液脱色后进行纳滤脱盐,截留分子量为200～1000Da的胶原蛋白肽,得到鱼皮胶原蛋白肽。本发明的制备方法在多个环节对不同分子大小的杂质进行分步去除,能够有效减少胶原蛋白肽中的杂质。(The invention provides a preparation method of fish skin collagen peptide, belonging to the technical field of preparation of marine collagen peptide. The method comprises the steps of sequentially soaking and pretreating fish skin by an alkali solution and an acid solution, then carrying out water extraction, and transferring effective components in the fish skin into a leaching solution to obtain a gelatin leaching solution; carrying out enzymolysis on the gelatin leaching liquor, and effectively carrying out enzymolysis on macromolecular proteins to obtain an enzymolysis liquor; filtering to remove raw material slag and suspended matters in the enzymolysis liquid; performing ultrafiltration on the enzymolysis liquid filtrate to separate collagen peptide with the molecular weight of 7000-8000 Da; and (3) decolorizing the ultrafiltrate, performing nanofiltration and desalination, and intercepting the collagen peptide with the molecular weight of 200-1000 Da to obtain the fish skin collagen peptide. The preparation method provided by the invention can remove impurities with different molecular sizes step by step in multiple links, and can effectively reduce impurities in the collagen peptide.)

1. A preparation method of fish skin collagen peptide comprises the following steps:

1) sequentially soaking the fish skin in an alkali solution and an acid solution to obtain a pretreated fish skin; carrying out water extraction on the pretreated fish skin to obtain a gelatin leaching liquor;

2) mixing the gelatin leaching liquor and protease for enzymolysis, and inactivating the enzyme to obtain an enzymolysis liquid;

3) filtering the enzymolysis liquid to obtain filtrate; the aperture of the filtered sieve pore is 1 mu m;

4) carrying out ultrafiltration on the filtrate, and collecting ultrafiltrate; the cut-off molecular weight of the ultrafiltration is 7000-8000 Da;

5) decolorizing the ultrafiltrate to obtain a decolorized solution;

6) nano-filtering the decolored solution, and collecting a nano-filtrate; the nano filtrate contains fish skin collagen peptide; the nanofiltration interception molecular weight is 200-1000 Da.

2. The method of claim 1, wherein the alkali solution in step 1) comprises an aqueous NaOH solution; the concentration of the alkali solution is 0.03-0.08 mol/L;

the acid solution comprises H₂SO₄An aqueous solution; said H₂SO₄The volume concentration of the aqueous solution is 0.1-0.3%;

the time for soaking the fish skin in the alkali solution and the acid solution is independently 2-4 h.

3. The preparation method according to claim 1 or 2, wherein the leaching temperature is 48-52 ℃; the leaching time is 5-7 h; the mass ratio of the fish skin to the water is 1: (15-25).

4. The method according to claim 1, wherein the protease in step 2) comprises an alkaline protease.

5. The method according to claim 1 or 4, wherein the protease is added in an amount of 3600u/mL in step 2).

6. The preparation method according to claim 1 or 4, wherein the pH value of the enzymolysis in the step 2) is 7.8-8.2.

7. The preparation method according to claim 1, wherein the temperature of the enzymolysis in the step 2) is 48-52 ℃; the enzymolysis time is 1.5-2.5 h.

8. The method according to claim 1, wherein the ultrafiltration membrane used in the step 4) comprises a ceramic ultrafiltration membrane.

9. The preparation method according to claim 1, wherein the nanofiltration in step 6) has a molecular weight cut-off of 450 Da.

10. The method of claim 1, wherein after collecting the nanofiltration liquid in step 6), concentrating and drying the nanofiltration liquid to obtain a dried fish skin collagen peptide product.

Technical Field

The invention relates to the technical field of preparation of marine collagen peptides, in particular to a preparation method of fish skin collagen peptides.

Background

Collagen is a protein formed by winding 3 peptide chains around each other to form a helical structure, and is widely present in the body of animals. The collagen peptide is a product between amino acid and protein formed by aquatic collagen after peptide bond breakage under the action of enzyme, microorganism and chemistry.

The fish body leftovers contain a large amount of collagen, and the collagen content in the fish skin accounts for more than 80 percent of the total protein amount and is much higher than that of other parts of the fish body. Therefore, the collagen peptide is extracted by using the fish skin as a raw material, so that the resources can be fully utilized, and the additional value of fish processing can be improved.

Currently, the common method for extracting collagen peptide from fish skin is an alkaline protease hydrolysis extraction method, and most of the alkaline protease hydrolysis extraction methods mostly focus on optimization of enzymolysis conditions and improvement of extraction rate, for example, zhanghan et al have studied the optimal conditions for extracting collagen peptide from tilapia skin hydrolyzed by alkaline protease, and have obtained an enzymolysis system with the highest extraction rate (see [ zhanghanjun, populus delphinium, buddlein ] research on the optimal conditions for extracting collagen peptide from tilapia skin hydrolyzed by alkaline protease [ J ] food research and development, 2008(09):27-30 ]. However, the fish skin collagen peptide obtained in the prior art contains more impurities such as salt, raw material residue, suspended matters and the like, and the purity of the fish skin collagen peptide needs to be further improved.

Disclosure of Invention

The invention aims to provide a preparation method of fish skin collagen peptide, and the collagen peptide obtained by the preparation method has high purity.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a preparation method of fish skin collagen peptide, which comprises the following steps:

1) sequentially soaking the fish skin in an alkali solution and an acid solution to obtain a pretreated fish skin; carrying out water extraction on the pretreated fish skin to obtain a gelatin leaching liquor;

2) mixing the gelatin leaching liquor and protease for enzymolysis, and inactivating the enzyme to obtain an enzymolysis liquid;

3) filtering the enzymolysis liquid to obtain filtrate; the aperture of the filtered sieve pore is 1 mu m;

4) carrying out ultrafiltration on the filtrate, and collecting ultrafiltrate; the cut-off molecular weight of the ultrafiltration is 7000-8000 Da;

5) decolorizing the ultrafiltrate to obtain a decolorized solution;

6) nano-filtering the decolored solution, and collecting a nano-filtrate; the nano filtrate contains fish skin collagen peptide; the nanofiltration interception molecular weight is 200-1000 Da.

Preferably, the alkali solution in step 1) comprises an aqueous NaOH solution; the concentration of the alkali solution is 0.03-0.08 mol/L;

the acid solution comprises H₂SO₄An aqueous solution; said H₂SO₄The volume concentration of the aqueous solution is 0.1-0.3%;

the time for soaking the fish skin in the alkali solution and the acid solution is independently 2-4 h.

Preferably, the leaching temperature is 48-52 ℃; the leaching time is 5-7 h; the mass ratio of the fish skin to the water is 1: (15-25).

Preferably, the protease in step 2) comprises an alkaline protease.

Preferably, the amount of the protease added in step 2) is 3600 u/mL.

Preferably, the pH value of the enzymolysis in the step 2) is 7.8-8.2.

Preferably, the enzymolysis temperature in the step 2) is 48-52 ℃; the enzymolysis time is 1.5-2.5 h.

Preferably, the ultrafiltration membrane used in step 4) comprises a ceramic ultrafiltration membrane.

Preferably, the nanofiltration in step 6) has a molecular weight cut-off of 450 Da.

Preferably, after the nanofiltration liquid is collected in the step 6), concentrating and drying the nanofiltration liquid to obtain a dried product of the fish skin collagen peptide.

The invention provides a preparation method of fish skin collagen peptide. The method comprises the steps of sequentially soaking and pretreating fish skin by an alkali solution and an acid solution, then carrying out water extraction, and transferring effective components in the fish skin into a leaching solution to obtain a gelatin leaching solution; carrying out enzymolysis on the gelatin leaching liquor, and effectively carrying out enzymolysis on macromolecular proteins to obtain an enzymolysis liquor; filtering to remove raw material slag and suspended matters in the enzymolysis liquid; performing ultrafiltration on the enzymolysis liquid filtrate to separate collagen peptide with the molecular weight of 7000-8000 Da; and (3) decolorizing the ultrafiltrate, performing nanofiltration and desalination, and intercepting the collagen peptide with the molecular weight of 200-1000 Da to obtain the fish skin collagen peptide. The enzyme, polypeptide with large molecular weight, protein which is not completely enzymolyzed and aggregate formed in the enzymolysis process in the enzymolysis liquid filtrate can be removed through ultrafiltration treatment. The method removes the salt in the decolored liquid by nanofiltration. The preparation method provided by the invention can remove impurities with different molecular sizes step by step in multiple links, and can effectively reduce impurities in the collagen peptide. The preparation method can scientifically and reasonably utilize the collagen source in the fish skin, change the fish skin into things of value, solve the problems of waste of fish leftover resources and the like, and simultaneously improve the economic added value of fish product processing.

Detailed Description

The invention provides a preparation method of fish skin collagen peptide, which comprises the following steps:

1) sequentially soaking the fish skin in an alkali solution and an acid solution to obtain a pretreated fish skin; carrying out water extraction on the pretreated fish skin to obtain a gelatin leaching liquor;

2) mixing the gelatin leaching liquor and protease for enzymolysis, and inactivating the enzyme to obtain an enzymolysis liquid;

3) filtering the enzymolysis liquid to obtain filtrate; the aperture of the filtered sieve pore is 1 mu m;

4) carrying out ultrafiltration on the filtrate, and collecting ultrafiltrate; the cut-off molecular weight of the ultrafiltration is 7000-8000 Da;

5) decolorizing the ultrafiltrate to obtain a decolorized solution;

6) nano-filtering the decolored solution, and collecting a nano-filtrate; the nano filtrate contains fish skin collagen peptide; the nanofiltration interception molecular weight is 200-1000 Da.

Firstly, sequentially soaking fish skins in an alkali solution and an acid solution to obtain pretreated fish skins; and carrying out water extraction on the pretreated fish skin to obtain a gelatin leaching liquor. In the present invention, the fish skin preferably includes tilapia skin; the tilapia skin is preferably subjected to shearing treatment.

In the present invention, the alkali solution preferably includes Naqueous solution of aOH; the concentration of the NaOH aqueous solution is preferably 0.03-0.08 mol/L, and more preferably 0.05 mol/L; the acid solution preferably comprises H₂SO₄An aqueous solution; said H₂SO₄The volume concentration of the aqueous solution is preferably 0.1 to 0.3%, and more preferably 0.2%; the mass ratio of the volume of the fish skin to the alkali solution is preferably 1 g: 10-20 mL, more preferably 1 g: 15 mL; the volume of the fish skin and the mass ratio of the acid solution are preferably 1 g: 10-20 mL, more preferably 1 g: 15 mL; the time for soaking the fish skin in the alkali solution and the acid solution is preferably 2-4 hours, and more preferably 3 hours.

In the invention, the leaching temperature is preferably 48-52 ℃, and more preferably 50 ℃; the leaching time is preferably 5-7 h, and further preferably 6 h; the mass ratio of the volume of the fish skin to the water is preferably 1 g: 15-25 mL, more preferably 1 g: 20 mL.

After the gelatin leaching liquor is obtained, the gelatin leaching liquor and protease are mixed for enzymolysis and enzyme deactivation to obtain an enzymolysis liquid.

In the present invention, the protease preferably includes alkaline protease; the enzymolysis temperature is preferably 48-52 ℃, and is further preferably 50 ℃; the addition amount of the protease is preferably 3600 u/mL; the pH value of the enzymolysis is preferably 7.8-8.2, and further preferably 8.0; the enzymolysis time is preferably 1.5-2.5 h, and more preferably 2 h. Compared with other proteases, the alkaline protease has the advantages of good hydrolysis effect and high extraction rate.

In the invention, the enzyme deactivation mode is preferably high-temperature enzyme deactivation; the enzyme deactivation temperature is preferably 90-100 ℃; the enzyme deactivation time is preferably 10 min.

After the enzymolysis liquid is obtained, filtering the enzymolysis liquid to obtain a filtrate; the aperture of the filter mesh is 1 μm. In the present invention, the filtration mode preferably comprises plate and frame filtration, and the adopted equipment is preferably a plate and frame filter press.

After obtaining the filtrate, the invention carries out ultrafiltration on the filtrate and collects the ultrafiltrate; the cut-off molecular weight of the ultrafiltration is 7000-8000 Da. In the invention, the membrane element adopted by ultrafiltration is preferably an electrodeless ceramic membrane, and more preferably a delaunay Muller ceramic ultrafiltration membrane, and the delaunay Muller ceramic ultrafiltration membrane has the advantages of long service life and high mechanical strength; the mode of ultrafiltration is preferably a cross-flow filtration mode; the operating pressure of the ultrafiltration is preferably 8-10 bar; the enzyme, polypeptide with large molecular weight, protein which is not completely enzymolyzed and aggregate formed in the enzymolysis process in the enzymolysis liquid filtrate can be removed through ultrafiltration treatment.

After ultrafiltrate is obtained, the ultrafiltrate is decolorized by the method, and decolorized liquid is obtained. In the invention, activated carbon is preferably used for adsorbing and decoloring the ultrafiltrate.

In the present invention, the ratio of the mass of the activated carbon to the volume of the ultrafiltrate is preferably 3 g: 90-110 mL, more preferably 3 g: 100 mL.

The method decolors the ultrafiltrate by using the active carbon and removes the fishy smell.

In the present invention, the activated carbon is preferably powdered activated carbon, and the particle size of the powdered activated carbon is preferably 300 mesh undersize.

In the invention, the adsorption temperature is preferably 70-90 ℃, and more preferably 80 ℃; the adsorption time is preferably 50-70 min, and more preferably 60 min.

After a decolored solution is obtained, nano-filtering the decolored solution, and collecting a nano-filtrate; the nano filtrate contains fish skin collagen peptide; the nanofiltration precision was 5 μm. In the invention, the molecular weight cut-off of nanofiltration is preferably 200-1000 Da, and more preferably 450 Da; the operating pressure of the nanofiltration is preferably 15-20 bar; the nanofiltration membrane adopted by the nanofiltration preferably comprises a Dellamel ceramic nanofiltration membrane. The method removes the salt in the decolored liquid by nanofiltration.

After collecting the nanofiltration liquid, the invention preferably further comprises concentrating and drying the nanofiltration liquid to obtain a dried product of fish skin collagen peptide.

In the present invention, the concentration means preferably includes evaporation concentration; the evaporation concentration is preferably carried out by adopting a multi-stage evaporation system; the effect of the concentration is to further reduce the nanofiltration volume.

In the present invention, the drying means preferably includes spray drying.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the 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.

Example 1

1) Cutting 400kg tilapia skin into pieces, putting into an acid-base pool, adding 6t of 0.05mol/LNaOH aqueous solution into the acid-base pool, and continuously soaking for 3 h. 6t of H with a concentration of 0.2% by volume are added₂SO₄Soaking in water solution for 3 hr, and replacing acid solution once per hour. Adding 8t of clear water, adjusting the temperature to 50 ℃, and soaking for 6 h.

2) After pretreatment, adding gelatin leaching liquor into an enzymolysis tank, adding 2kg of alkaline protease, adjusting the pH to 8.0 and the temperature to 50 ℃, carrying out enzymolysis for 2h, stirring, raising the temperature to 100 ℃ after the enzymolysis is finished, and inactivating at high temperature for 10 min.

3) And filtering the enzymolysis solution by using a plate and frame filter press to remove substances with the size of more than 1 mu m to obtain filtrate.

4) The filtrate enters an inorganic ceramic membrane ultrafiltration system, the ultrafiltration interception molecular weight is 7000-8000 Da, and the system filter membrane area is 10m²And (3) operating at 8-10 bar, and concentrating by 10 times to obtain ultrafiltrate.

5) After ultrafiltration treatment, the ultrafiltrate enters an adsorption tank, the adding amount of the activated carbon powder is 24kg, the temperature is 80 ℃, and the adsorption time is 60 min.

6) After fishy smell is removed and decolored, the mixture enters a nanofiltration system, the intercepted molecular weight of nanofiltration is 200-1000 Da, and the concentrated solution is desalted collagen peptide feed liquid.

7) And then the mixture enters a multi-effect evaporator for further concentration, and finally enters a spray drying tower for powder spraying to obtain a collagen peptide product, wherein the content of the collagen peptide is 97 percent.

Example 2

1) Cutting 400kg tilapia skin into pieces, putting into an acid-base pool, adding 6t of 0.06mol/L NaOH aqueous solution into the acid-base pool, and continuously soaking for 3.5 h. 6t of H with a concentration of 0.3% by volume are added₂SO₄Soaking in water solution for 3.5 hr, and replacing acid solution once per hour. Adding 8t of clear water, adjusting the temperature to 50 ℃, and soaking for 6 h.

2) After pretreatment, adding gelatin leaching liquor into an enzymolysis tank, adding 2kg of alkaline protease, adjusting the pH to 8.0 and the temperature to 50 ℃, carrying out enzymolysis for 2h, stirring, raising the temperature to 100 ℃ after the enzymolysis is finished, and inactivating at high temperature for 10 min.

3) And filtering the enzymolysis solution by using a plate and frame filter press to remove substances with the size of more than 1 mu m to obtain filtrate.

4) The filtrate enters an inorganic ceramic membrane ultrafiltration system, the ultrafiltration interception molecular weight is 7000-8000 Da, and the system filter membrane area is 10m²Operating pressure is 10bar, and concentration times is 10 times to obtain ultrafiltrate.

5) After ultrafiltration treatment, the ultrafiltrate enters an adsorption tank, the adding amount of the activated carbon powder is 24kg, the temperature is 80 ℃, and the adsorption time is 60 min.

6) After fishy smell is removed and decolored, the mixture enters a nanofiltration system, the intercepted molecular weight of nanofiltration is 200-1000 Da, and the concentrated solution is desalted collagen peptide feed liquid.

7) And then the mixture enters a multi-effect evaporator for further concentration, and finally enters a spray drying tower for powder spraying to obtain a collagen peptide product, wherein the content of the collagen peptide is 97 percent.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.