Preparation method and use of composition of triple-helix collagen and antarctic glycoprotein

A method for preparing a composition of triple-helix collagen and Antarctic glycoprotein, comprising:

• step S1, adding a lyophilized Antarctic krill powder to distilled water to obtain a first mixture, adjusting a pH value of the first mixture, then adding an enzyme mixture to the first mixture for enzymatic hydrolysis, and then performing inactivation and collecting a first supernatant;
• step S2, performing dialysis and concentration on the first supernatant, and then adding ethanol, standing still overnight, collecting a precipitate, and lyophilizing the precipitate to obtain a crude Antarctic krill glycoprotein product;
• step S3, dissolving the crude Antarctic krill glycoprotein product in distilled water to obtain a crude Antarctic krill glycoprotein solution, adding an MaAFP antifreeze protein solution into the crude Antarctic krill glycoprotein solution to obtain a second mixture, using a cation exchange medium to adsorb proteins in the second mixture, and then performing elution and separation, and collecting a first eluent which is an Antarctic glycoprotein solution;
• and
• step S4, mixing the Antarctic glycoprotein solution and a triple-helix collagen hydrogel solution at a volume ratio of (0.4-1.5):1 to obtain a third mixture, and lyophilizing the third mixture to obtain the composition of triple-helix collagen and Antarctic glycoprotein;
• wherein:
• the enzyme mixture in the step S1 consists of a cellulase, papain and a neutral protease, and a weight ratio of the cellulase to the papain to the neutral protease is 2:1.5:1;
• an amount of the enzyme mixture added in the step S1 is 0.5 wt % to 1.0 wt % of a mass of the lyophilized Antarctic krill powder;
• a volume ratio of the MaAFP antifreeze protein solution to the crude Antarctic krill glycoprotein solution in the step S3 is (0.3-1.0):1;
• an amino acid sequence of the MaAFP antifreeze protein in the step S3 is as shown in SEQ ID NO 1;
• and
• a method for preparing the triple-helix collagen hydrogel solution in the step S4 comprises:
• step (1) soaking pig skin in an ethylene diamine tetraacetic acid (EDTA)-containing trypsin solution, and then rinsing;
• then, soaking the pig skin in a detergent solution and shaking, and then rinsing;
• then, soaking the pig skin in a neutral protease solution, and then rinsing;
• and then soaking the pig skin in the detergent solution and shaking, and then rinsing;
• and repeating the above steps for 1-3 times to obtain non-immunogenic pig skin;
• step (2) soaking the non-immunogenic pig skin in an α-1,3-galactosidase solution, and then rinsing to obtain precipitate A;
• adding a filter aid solution to the precipitate A, then stirring, filtering, and collecting a second supernatant;
• concentrating the second supernatant, then using a cation exchange medium to adsorb collagen in the second supernatant, and then performing elution and separation to finally obtain a second eluent;
• step (3) adding a modified polysaccharide solution with a concentration of 10 mg/mL to 40 mg/mL to the second eluent from the step (2) for modification to obtain a hydrogel, and a mass ratio of the second eluent to the modified polysaccharide solution is 5:1-1:3;
• and
• step (4) performing filtration and sterilization on the hydrogel obtained in the step (3) with a 0.22 μm filter membrane to obtain the triple-helix collagen hydrogel solution;
• the detergent solution in the step (1) is a sodium phosphate buffer with a pH value of 6.0-8.0, containing 0.05%-0.15 wt % of an ionic detergent;
• and the ionic detergent is a mixture of sodium dodecyl sulfate and p-Octylphenoxyethoxyethyl sodium sulfonate at a mass ratio of 4:6;
• the modified polysaccharide in the step (3) is a mixture of methacrylic anhydride-modified hyaluronic acid, oat β-glucan and aldehyde-modified hyaluronic acid at a mass ratio of 2:5:3.