CN107385005A - A kind of preparation method of sturgeon bone II Collagen Type VI peptides - Google Patents

Abstract

The invention belongs to the technical field of preparation of functional polypeptides, and specifically relates to a preparation method of sturgeon bone type II collagen peptide, which comprises preparing sturgeon bone type II collagen, preparation of sturgeon bone type II collagen peptide, desalination, concentration, Separation and purification, the finally prepared II collagen peptide is low molecular weight sturgeon cartilage II collagen peptide, the degree of hydrolysis is 9-17%, and the peptide chain length ranges from 3 to 24 amino acids. The collagen peptide prepared by the method is also rich in chondroitin sulfate and calcium, and has a good function of joint health care. In addition, the preparation process is suitable for industrial production.

Description

A preparation method of sturgeon bone type II collagen peptide

technical field

The invention belongs to the technical field of preparation of functional polypeptides, and in particular relates to a preparation method of sturgeon bone type II collagen peptide.

Background technique

Our country is extremely rich in sturgeon resources. There are a total of 27 species of sturgeon in the world, and 8 species can be found in my country, among which Chinese sturgeon, Sturgeon's sturgeon and Dabry's sturgeon are the three species with the largest proportion. Sturgeon is full of treasures. Collagen can be extracted from fish skin and swim bladder, and high-quality fish protein peptides can be extracted from fish meat. Sturgeon not only has rich nutritional value, but also unique medical and health value, known as "shark fin, sturgeon bone". Sturgeon has a large body, and cartilage accounts for a relatively large proportion (about 5.7% of the body). Except for the partially ossified fin and a piece of true bone in the head, the rest of the bones are cartilage, which is extracted from II It is a good raw material for type collagen, and artificial aquaculture of sturgeon has risen successively in many countries, and my country has become the largest country for aquaculture of sturgeon, which also provides a stable source of raw materials. Most of the collagen peptides extracted by the existing collagen peptide extraction and preparation methods have a wide molecular weight distribution and poor uniformity.

Contents of the invention

Aiming at the above problems, the present invention provides a preparation method of sturgeon bone type II collagen peptide.

The technical scheme adopted in the present invention is as follows: a preparation method of sturgeon bone type II collagen peptide, comprising the steps of:

1) The preparation of type II collagen of sturgeon bone, first select fresh or frozen sturgeon cartilage and/or sturgeon skull, rinse the surface of sturgeon cartilage and/or sturgeon skull fully with clean water, cut into small pieces, Weigh a certain mass of sturgeon cartilage and/or sturgeon skull, add 5-20 times the volume (w/v) of NaOH solution and stir for 1.5-2.5 hours, rinse with running water until neutral, homogenate, add purified water, 100- Treat at 130°C for 20-60min, cool down to 60-90°C for extraction for 3-6 hours, centrifuge and filter at 4000-8000 rpm, the filtrate is the sturgeon bone type II collagen extract, and the concentration of collagen is 0.5-2% ( w/v), pH 6-9;

2) Preparation of sturgeon bone type II collagen peptide, prepare the sturgeon bone type II collagen extract obtained in step 1 into a sturgeon bone type II collagen solution with a concentration of 1-5% (w/v), and use alkali One or two of neutral protease, trypsin or neutral protease for single-enzyme hydrolysis or compound enzyme hydrolysis, enzymatic hydrolysis pH 6-10, enzymatic hydrolysis temperature 40-60°C, enzymatic hydrolysis time 1-6h, enzymatic hydrolysis After the solution is completed, heat it to 90-100°C and keep it warm for 20-30 minutes to inactivate the enzyme, and then obtain the sturgeon bone II collagen peptide product;

3) Desalination and concentration of sturgeon bone type II collagen peptide, filter the sturgeon bone type II collagen peptide product obtained in step 2, add purified water to the filtrate, and use nanofiltration with a membrane molecular weight cut-off of 300 Daltons The membrane is desalted and concentrated, the conductivity of the concentrated solution is controlled, and the filtrate with large molecular weight is collected to obtain the concentrated solution of sturgeon bone type II collagen peptide;

4) Separation and purification of sturgeon bone type II collagen peptide, remove pigment, odor and macromolecular protein by coarse filtration, centrifugation and ultrafiltration, and concentrate the volume of collagen peptide solution to the original volume by membrane concentration or rotary evaporation 30-50% of the solvent is removed to obtain the sturgeon bone type II collagen peptide product.

Preferably, the volume of purified water added in step 1 is 1-8 times the volume of sturgeon bone type II collagen solution.

Preferably, the concentration of the NaOH solution in step 1 is 0.01-0.1 mol/L.

Preferably, in step 1, add 1-3 times the volume of water to the centrifuged filter residue and continue to extract at 60-90°C for 1-2 hours, centrifuge at 4000-8000 rpm to remove the residue, and the filtrate is the same as the first centrifuged filtrate The combination is the sturgeon bone type II collagen extract.

Preferably, the concentration of the enzyme added in step 2 in the collagen solution is 4000-12000U/gpro.

Preferably, in step three, the sturgeon bone type II collagen peptide product obtained in step two is filtered through a 0.45 μm nylon microporous membrane.

Preferably, the volume of purified water added in step 3 is 2-8 times the volume of sturgeon bone type II collagen peptide solution.

Preferably, the conductivity of the concentrated solution is controlled to be 100-300 μS/cm after desalting in step 3.

Preferably, in step 4, the concentrated collagen peptide solution is fractionated by selecting polysulfone ultrafiltration membranes with different pore sizes, and then the solvent is removed to obtain sturgeon bone type II collagen peptide products with different length peptides.

Preferably, in step 4, the method of removing the solvent from the collagen peptide solution is freeze-drying.

The purified water added above is the secondary water specified in GB/T6682

The beneficial effects of the present invention are as follows: the degree of hydrolysis of the low-molecular-weight sturgeon bone II collagen peptide prepared by the method is 9-17%, and the length of the peptide chain is 3-24 amino acids. The collagen peptide prepared by the method is also rich in chondroitin sulfate and calcium, and has a good function of joint health care. In addition, the preparation process is suitable for industrial production.

detailed description

Implementation example 1

Choose fresh sturgeon cartilage and sturgeon skull as raw materials for collagen extraction, wash them, chop them up, add 8 times the volume (w/v) of 0.05mol/L NaOH solution and stir for 2 hours, rinse with running water until neutral, and mix well. Add 4 times the volume of purified water to the slurry, treat at 110°C for 40 minutes, cool down to 75°C for 3 hours, centrifuge and filter at 4000 rpm, add 2 times the volume of water to the residue and continue to extract at 75°C for 1 hour, centrifuge at 4000rpm to remove the residue, and combine The two extracts are the sturgeon bone type II collagen extract; adjust the concentration of collagen in the concentrated solution to 4%, add trypsin to it, the addition amount is 6000U/gpro, adjust the pH to 7.5, and the reaction temperature 50°C, enzymatic hydrolysis time 4h, after enzymolysis, inactivate the enzyme at 90-100°C for 30min, cool to room temperature and centrifuge to obtain sturgeon bone type II collagen peptide solution, filter the supernatant with a nylon membrane with a pore size of 0.45μm , adding 5 times the volume of purified water to the filtrate, using a nanofiltration membrane with a membrane molecular weight cut-off of 300 Daltons for desalination and concentration, concentrating to 20% of the original volume, and controlling the conductivity of the concentrated solution to 100 μS/cm. Type II collagen peptides from sturgeon bone were obtained after freeze-drying. The molecular weight of the sturgeon bone type II collagen peptide is concentrated in 1000-3000 Daltons, and the number of amino acids is 8-24.

Implementation example 2

Choose fresh sturgeon cartilage as the raw material for collagen extraction, wash it, chop it up, add 10 times the volume (w/v) of 0.08mol/L NaOH solution and stir for 3 hours, rinse with running water until neutral, homogenate, add 5 Double the volume of purified water, treat at 120°C for 50 minutes, cool down to 65°C for extraction for 4 hours, centrifuge at 6000 rpm, add 2 times the volume of water to the residue and continue to extract at 65°C for 1 hour, remove the residue by centrifugation at 6000 rpm, and combine the two extractions solution, which is the sturgeon bone type II collagen extract; adjust the concentration of collagen in the concentrated solution to 2%, add alkaline protease to it, the addition amount is 8000U/gpro, adjust the pH to 9, and the reaction temperature is 50°C. The enzymolysis time is 4 hours. After the enzymolysis, inactivate the enzyme at 90-100°C for 30 minutes, cool to room temperature and centrifuge to obtain the sturgeon bone type II collagen peptide solution. Add 8 times the volume of purified water to the supernatant, use a nanofiltration membrane with a membrane molecular weight cut-off of 300 Daltons to desalt and concentrate, and concentrate to 30% of the original volume, and control the conductivity of the concentrated solution to 150 μS/cm. Type II collagen peptides from sturgeon bone were obtained after freeze-drying. The molecular weight of the sturgeon bone type II collagen peptide is concentrated at 2000-3000 Daltons, and the number of amino acids is 16-24.

Implementation example 3

Frozen sturgeon cartilage and sturgeon skull were selected as raw materials for collagen extraction, thawed and washed, chopped, added 10 times the volume (w/v) of 0.1mol/L NaOH solution, stirred for 2 hours, rinsed with running water until neutral , homogenate, add 4 times the volume of purified water, treat at 125°C for 60 minutes, cool down to 60°C for extraction for 4 hours, centrifuge and filter at 8000 rpm, add 2 times the volume of water to the residue and continue to extract at 60°C for 1 hour, centrifuge at 8000rpm to remove the residue , the combined extract twice is the sturgeon bone type II collagen extract; adjust the concentration of collagen in the concentrated solution to 5%, add trypsin and neutral protease to it, the addition amount is 8000U/gpro respectively, Adjust the pH to 7.5, the reaction temperature is 50°C, and the enzymatic hydrolysis time is 3 hours. After the enzymolysis is completed, the enzyme is inactivated at 90-100°C for 30 minutes, cooled to room temperature and then centrifuged to obtain the sturgeon bone type II collagen peptide solution. The supernatant is used Filter through a nylon membrane with a pore size of 0.45 μm, add 5 times the volume of purified water to the filtrate, use a nanofiltration membrane with a molecular weight cut-off of 300 Daltons for desalination and concentration, concentrate to 30% of the original volume, and control the conductivity of the concentrate It is 260μS/cm. Type II collagen peptides from sturgeon bone were obtained after freeze-drying. The molecular weight of the sturgeon bone type II collagen peptide is concentrated at 500-1000 Daltons, and the number of amino acids is 4-8.

The above description is only an embodiment of the present invention, and is not intended to limit the scope of protection of the present invention; the scope of protection of the present invention is defined by the claims in the claims, and all equivalent changes and modifications made according to the invention are included in Within the protection scope of the patent of the present invention.

Claims (10)

1. a kind of preparation method of sturgeon bone II Collagen Type VI peptides, it is characterised in that comprise the following steps：

1）The preparation of sturgeon bone II collagen types, fresh or frost sturgeon cartilage and/or sturgeon skull is selected first, with clear Water fully rinses sturgeon cartilage and/or sturgeon skull surface well, is cut into small pieces, weigh certain mass sturgeon cartilage and/ Or sturgeon skull, add 5-20 times of volume NaOH solution and stir 1.5-2.5h, flowing water is rinsed to neutrality, homogenate, adds purifying Water, 100-130 DEG C of processing 20-60min, is cooled to 60-90 DEG C of extraction 3-6 hour, 4000-8000 rpm centrifugal filtrations, filtrate As sturgeon bone II collagen type extract solutions；

2）The preparation of sturgeon bone II Collagen Type VI peptides, the sturgeon bone II collagen type extract solutions that step 1 obtains are modulated into 1-5% The sturgeon bone II collagen type solution of concentration, is entered using one or both of alkali protease, pancreatin or neutral proteinase The single enzyme enzymolysis of row or complex enzyme zymohydrolysis, digest pH 6-10,40-60 DEG C of hydrolysis temperature, enzymolysis time 1-6h, digest after terminating, Being heated to 90-100 DEG C of insulation 20-30min makes enzyme-deactivating, that is, obtains sturgeon bone II collagen peptides products；

3）Desalination, the concentration of sturgeon bone II Collagen Type VI peptides, the sturgeon bone II Collagen Type VIs peptides products that step 2 is obtained filter, and to Filtered solution adds purified water, carries out desalination, concentration for the NF membrane of 300 dalton using retaining molecular weight, controls concentrate Electrical conductivity, the filter liquor for collecting macromolecule obtains sturgeon bone II Collagen Type VI peptide concentrates；

4）Sturgeon bone II Collagen Type VI peptides isolate and purify, and remove depigmentation, peculiar smell by coarse filtration, centrifugation, ultrafiltration and divide greatly Sub- albumen, and the volume concentration of collagen peptide solution is made to the 30-50% of original volume by the method for film concentration or rotary evaporation, Remove solvent and obtain sturgeon bone II Collagen Type VI peptide products.

2. the preparation method of sturgeon bone II Collagen Type VI peptides according to claim 1, it is characterised in that：Purified water in step 1 Addition volume be sturgeon bone II collagen types liquid product 1-8 times.

3. the preparation method of sturgeon bone II Collagen Type VI peptides according to claim 1, it is characterised in that：NaOH is molten in step 1 The concentration of liquid is 0.01-0.1mol/L.

4. the preparation method of sturgeon bone II Collagen Type VI peptides according to claim 1, it is characterised in that：In step 1, centrifugation The water that filter residue after filtering adds 1-3 times of volume continues to extract at 60-90 DEG C 1-2 hours, and 4000-8000 rpm centrifugations remove Residue, filtrate merge with the filtrate of first time centrifugal filtration Ji Wei sturgeon bone II collagen type extract solutions.

5. the preparation method of sturgeon bone II Collagen Type VI peptides according to claim 1, it is characterised in that：Enzyme adds in step 2 Concentration of the dosage in collagen solution is 4000-12000U/gpro.

6. the preparation method of sturgeon bone II Collagen Type VI peptides according to claim 1, it is characterised in that：In step 3, it will walk The rapid two sturgeon bone II Collagen Type VIs peptides products filterings obtained are filtered by 0.45 μm of nylon micro porous filter membrane.

7. the preparation method of sturgeon bone II Collagen Type VI peptides according to claim 1, it is characterised in that：Purified water in step 3 Addition volume be sturgeon bone II Collagen Type VI peptides liquid product 2-8 times.

8. the preparation method of sturgeon bone II Collagen Type VI peptides according to claim 1, it is characterised in that：In step 3 after desalination The electrical conductivity for controlling concentrate is 100-300 μ S/cm.

9. the preparation method of sturgeon bone II Collagen Type VI peptides according to claim 1, it is characterised in that：In step 4, concentration Then collagen peptide solution afterwards removes solvent and obtains not having to length polypeptide by selecting the polysulphone super-filter membrane of different pore size to be classified The sturgeon bone II Collagen Type VI peptide products of section.

10. the preparation method of the sturgeon bone II Collagen Type VI peptides according to claim 1 or 9, it is characterised in that：In step 4, The mode that collagen peptide solution removes solvent is freeze-drying.