A phycocyanin-collagen composite hydrogel and its preparation method and application

Abstract

The present invention provides a phycocyanin-collagen composite hydrogel and its preparation method and application. The present invention utilizes transglutaminase to catalyze the combination of phycocyanin and collagen to form a new, more stable collagen; transglutaminase acts between protein molecules, utilizes lysine and glutamic acid in phycocyanin and collagen to transfer acyl groups, and cross-links to form new chemical bonds, thereby enhancing the binding strength of phycocyanin and collagen; the hydrogel prepared by the present invention can be used to prepare medical materials and combined with skin injury sites to solve the problem that traditional dressings are easy to adhere to wounds and easily penetrated by tissue fluid and become infected. In addition to ensuring basic air permeability, it reduces the adhesion of the dressing, increases moisture retention and antioxidant properties, and avoids secondary damage when replacing a new dressing. The hydrogel prepared by the present invention has a certain antioxidant capacity and can be used to prepare products with antioxidant effects.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogels, and in particular to a phycocyanin-collagen composite hydrogel and a preparation method and application thereof. Background Art

[0002] Extracting collagen from fish skin is a new approach to collagen production. Advances in genetic engineering have deepened our understanding of collagen's structure. Collagen exhibits a triple helical structure, consisting of three intertwined polypeptide chains. Each α-peptide chain is a left-handed helix, and the intertwining of the three α-peptide chains ultimately forms a right-handed helix approximately 300 nm long and 1.6 nm in diameter, also known as a superhelix. Hydrogen bonds between the carbon-oxygen double bonds and nitrogen-hydrogen bonds within the three α-peptide chains stabilize the triple helical structure. Collagen extracted from fish skin is more similar to human skin and may have fewer side effects. my country has abundant fish resources, and the scale of artificial aquaculture is continuously increasing, providing a rich source of raw materials. Moreover, compared to collagen from pigs, sheep, and cattle, fish skin is less susceptible to viruses that carry diseases such as mad cow disease, broadening its application. Phycocyanin (PC), a pigment protein found widely in cyanobacteria, possesses antioxidant, moisturizing, and anti-inflammatory properties. Naturally extracted PC is also highly safe.

[0003] While much research has been conducted on collagen, it primarily focuses on collagen itself, with less research on its combination with other proteins to form new collagens. Phycocyanin, a natural pigment protein found in seaweed, has been studied relatively little in its combination with collagen. Current research on the two primarily involves combining them through spontaneous assembly into hydrogels, but this integration is incomplete. For example, prior art discloses combining type I collagen extracted from tilapia skin with varying concentrations of phycocyanin, leveraging the self-assembly properties of collagen to create composite hydrogels. However, these hydrogels exhibit incomplete integration.

[0004] Based on the defects of the current hydrogel prepared by combining phycocyanin and collagen, it is necessary to improve it. Summary of the Invention

[0005] The present invention aims to solve a problem in the related art at least to a certain extent, and provides a phycocyanin-collagen composite hydrogel and a preparation method and application thereof.

[0006] In a first aspect, the present invention provides a method for preparing a phycocyanin-collagen composite hydrogel, comprising the following steps:

[0007] adding fish-derived collagen to acetic acid to obtain a collagen solution;

[0008] adding phycocyanin to a PBS solution to obtain a phycocyanin solution;

[0009] The collagen solution and the phycocyanin solution are mixed to obtain a mixed solution, the pH of the mixed solution is adjusted to 6.3-6.7, and then glutamine transaminase is added. After mixing, the pH is further adjusted to neutral, and the solution is allowed to stand to form a hydrogel, which is the phycocyanin-collagen composite hydrogel.

[0010] Preferably, the concentration of fish-derived type I collagen in the collagen solution is 5 to 10 mg / mL;

[0011] and / or, the phycocyanin concentration in the phycocyanin solution is 2 to 16 mg / mL;

[0012] And / or, the concentration of phycocyanin in the mixed solution is 1 to 8 mg / mL.

[0013] Preferably, the collagen solution and the phycocyanin solution are mixed to obtain a mixed solution, the pH of the mixed solution is adjusted to 6.3-6.7, and transglutaminase is added; wherein the amount of transglutaminase added is 0.2-0.5% of the mass of the mixed solution.

[0014] Preferably, the collagen solution and the phycocyanin solution are mixed to obtain a mixed solution, the pH of the mixed solution is adjusted to 6.3-6.7, and then transglutaminase is added. After mixing, the pH is further adjusted to neutral, and the solution is allowed to stand at 34-38°C for 22-26 hours to form a hydrogel, which is a phycocyanin-collagen composite hydrogel.

[0015] Preferably, the concentration of the acetic acid is 0.05 to 0.5 mol / L;

[0016] And / or, the concentration of the PBS solution is 0.01-0.05 mol / L.

[0017] Preferably, the preparation method of the fish-derived collagen is:

[0018] The fish skin is placed in an n-butanol aqueous solution for degreasing;

[0019] The defatted fish skin is immersed in a sodium hydroxide aqueous solution to remove non-collagenous components and washed to neutrality;

[0020] Add acetic acid solution and pepsin to the defatted fish skin, extract at 3-5°C for 36-72h, centrifuge, and collect the supernatant;

[0021] Add NaCl to the supernatant, centrifuge, collect the precipitate, dissolve the precipitate in acetic acid solution, dialyze with disodium hydrogen phosphate solution for 24-48 hours, and centrifuge to collect the precipitate again;

[0022] The collected precipitate is dissolved in acetic acid solution, dialyzed again with acetic acid solution for 24 to 48 hours, and then dialyzed with distilled water for 24 to 48 hours to obtain an enzyme-soluble collagen solution;

[0023] The enzyme-soluble collagen solution is freeze-dried to obtain a fish-derived collagen freeze-dried sponge, namely, fish-derived collagen.

[0024] Preferably, the fish skin is placed in an n-butanol aqueous solution and degreased for 24 to 48 hours, wherein the volume concentration of the n-butanol aqueous solution is 10 to 15%, and the mass volume ratio of the fish skin to the n-butanol aqueous solution is 1 g: (20 to 30) mL;

[0025] The defatted fish skin is placed in a sodium hydroxide aqueous solution with a concentration of 0.05 to 0.2 mol / L and soaked for 24 to 48 hours to remove non-collagenous components, and then washed to neutrality;

[0026] In the step of adding acetic acid solution and pepsin in the fish-skin that has been defatted, the mass volume ratio of the fish-skin that has been defatted and acetic acid solution is 1g:(30~50)mL, and the concentration of acetic acid solution is 0.2~0.8mol / L; Adding acetic acid solution and pepsin in the fish-skin that has been defatted makes the mass concentration of pepsin be 0.5~1%;

[0027] NaCl is added to the supernatant to a concentration of 0.5 to 1.5 mol / L, the mixture is centrifuged, and the precipitate is collected. The precipitate is dissolved in a 0.2 to 0.5 mol / L acetic acid solution, dialyzed against a 0.01 to 0.03 mol / L sodium hydrogen phosphate solution for 24 to 48 hours, and the precipitate is collected again by centrifugation.

[0028] The collected precipitate was dissolved in 0.2-0.8 mol / L acetic acid solution, dialyzed again with 0.05-0.1 mol / L acetic acid solution for 24-48 hours, and then dialyzed with distilled water for 24-48 hours to obtain an enzyme-soluble collagen solution.

[0029] Preferably, the collagen solution and the phycocyanin solution are mixed to obtain a mixed solution, the pH of the mixed solution is adjusted to 6.3-6.7, and then transglutaminase is added. After mixing, the pH is further adjusted to neutral; wherein, 1 mol / L-5 mol / L sodium hydroxide solution and 0.1 mol / L-0.5 mol / L acetic acid solution are used to adjust the pH.

[0030] In a second aspect, the present invention further provides a phycocyanin-collagen composite hydrogel prepared by the above-mentioned preparation method.

[0031] In a third aspect, the present invention also provides a phycocyanin-collagen composite hydrogel prepared by the preparation method or the use of the phycocyanin-collagen composite hydrogel in preparing medical dressings and products with antioxidant effects.

[0032] The phycocyanin-collagen composite hydrogel of the present invention and its preparation method and application have the following beneficial effects compared with the prior art:

[0033] The preparation method of the phycocyanin-collagen composite hydrogel of the present invention utilizes transglutaminase (TGase) to catalyze the combination of phycocyanin and collagen extracted from fish skin to form a novel, more stable collagen; transglutaminase (TGase) acts on protein molecules, utilizes lysine and glutamic acid in phycocyanin and collagen to transfer acyl groups, and cross-links to form new chemical bonds, thereby enhancing the strength of the combination of phycocyanin and collagen, and further improving the stability of the newly formed collagen; the binding strength and stability of phycocyanin and collagen are enhanced by transglutaminase (TGase), so that the newly formed collagen is more stable in the application of dressings and other aspects, and has stronger applicability. The novel collagen structure formed by the present invention is more stable, can be used for preparing medical materials, is combined at the skin injury site, can solve the problem that traditional dressings are easy to adhere to wounds, are easily soaked by tissue fluid and become infected, and outside the air permeability of the basis, reduce the adhesion of the dressing, increase moisture retention and antioxidant properties, and avoid the secondary damage caused when replacing a new dressing. The hydrogel prepared by the invention has certain antioxidant capacity and can be used to prepare products with antioxidant effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0035] Figure 1 The figures are the actual pictures of the hydrogels prepared in Examples 1 to 5 of the present invention and Comparative Example 1, and the actual pictures of the hydrogels immersed in 0.01 mol / L PBS solution;

[0036] Figure 2 Infrared spectra of the phycocyanin-collagen composite hydrogel, phycocyanin, and fish-derived collagen prepared in Example 1 of the present invention;

[0037] Figure 3Graph showing the air permeability results of the hydrogels prepared in Examples 1 to 5 of the present invention and Comparative Example 1;

[0038] Figure 4 This is a graph showing the swelling rate results of the hydrogels prepared in Examples 1 to 5 of the present invention and Comparative Example 1;

[0039] Figure 5 These are scanning electron micrographs of the hydrogels prepared in Examples 1 to 5 of the present invention and Comparative Example 1 at different magnifications;

[0040] Figure 6 This is a graph showing the results of reducing properties of the hydrogels prepared in Examples 1 to 5 of the present invention;

[0041] Figure 7 This is a graph showing the test results of the hydroxyl radical scavenging rate of the hydrogels prepared in Examples 1 to 5 of the present invention. DETAILED DESCRIPTION

[0042] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0043] It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of the embodiments. In addition, in the description of this application, the term "including" means "including but not limited to". Various embodiments of the present invention may be in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be understood as a rigid limitation on the scope of the invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the numbered ranges, such as 1, 2, 3, 4, 5 and 6, regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited numbers (fractions or integers) within the indicated range.

[0044] The present invention provides a method for preparing a phycocyanin-collagen composite hydrogel, comprising the following steps:

[0045] S1. Adding fish-derived collagen to acetic acid to obtain a collagen solution;

[0046] S2, adding phycocyanin to PBS solution to obtain a phycocyanin solution;

[0047] S3. Mix the collagen solution and the phycocyanin solution to obtain a mixed solution, adjust the pH of the mixed solution to 6.3-6.7, add transglutaminase, and continue to adjust the pH to neutral after mixing. Let it stand to form a hydrogel, which is the phycocyanin-collagen composite hydrogel.

[0048] The preparation method of the phycocyanin-collagen composite hydrogel of the present invention, transglutaminase (TGase) is a monomeric protein with a molecular weight of about 38,000 and an active center composed of 331 amino groups, which can catalyze covalent cross-linking within and between protein polypeptides, thereby improving the structure and function of the protein and increasing the stability of the protein; transglutaminase (TGase) is a transferase that can catalyze the transfer reaction of acyl groups, resulting in cross-linking within protein molecules, between protein molecules, and between amino acids and proteins, thereby improving the functional properties of the protein. Under the action of the hydrolysis reaction, the glutamic acid and lysine between the protein molecules undergo a transamination reaction to form an intermolecular network structure. The formed GL peptide bond not only constitutes a new connection point on the gel network, but also has a stronger connection force than the disulfide bond, thereby making the gel more stable. The fish-derived collagen freeze-dried sponge used in the present invention is made of collagen extracted from fish skin. Its structure and components are conducive to wound repair and cell proliferation, and it has high biosafety, abundant sources, low price, and is suitable for large-scale production. The phycocyanin (PC) used in the present invention is a natural pigment widely found in seaweed, which has excellent antioxidant, moisturizing and anti-inflammatory effects.

[0049] The present invention utilizes transglutaminase (TGase) to catalyze the combination of phycocyanin and collagen extracted from fish skin to form a novel, more stable collagen; utilizes transglutaminase (TGase) to act on protein molecules, utilizes lysine and glutamic acid in phycocyanin and collagen to transfer acyl groups, and cross-links to form new chemical bonds, thereby enhancing the strength of the combination of phycocyanin and collagen, and further improving the stability of the newly formed collagen; by transglutaminase (TGase), the binding strength and stability of phycocyanin and collagen are enhanced, making this newly formed collagen more stable in applications such as dressings and more suitable. The novel collagen structure formed by the present invention is more stable, can be used to prepare medical materials, and is combined at the site of skin injury to solve the problem that traditional dressings are easy to adhere to wounds and are easily penetrated by tissue fluid and infected. In addition to ensuring basic air permeability, the adhesiveness of the dressing is reduced, moisture retention and antioxidant properties are increased, and secondary damage caused when replacing a new dressing is avoided.

[0050] In some embodiments, the concentration of fish-derived collagen in the collagen solution is 5 to 10 mg / mL. Preferably, the concentration of fish-derived collagen in the collagen solution is 8 mg / mL.

[0051] In some embodiments, the concentration of phycocyanin in the phycocyanin solution is 2 to 16 mg / mL. Preferably, the concentration of phycocyanin in the phycocyanin solution is 2 mg / mL, 4 mg / mL, 8 mg / mL, 12 mg / mL, or 16 mg / mL.

[0052] In some embodiments, the concentration of phycocyanin in the mixed solution is 1 to 8 mg / mL. Preferably, the concentration of phycocyanin is 1 mg / mL, 2 mg / mL, 4 mg / mL, 6 mg / mL, or 8 mg / mL.

[0053] In some embodiments, a collagen solution and a phycocyanin solution are mixed to obtain a mixed solution, the pH of the mixed solution is adjusted to 6.3-6.7, and transglutaminase is added; wherein the amount of transglutaminase added is 0.2-0.5% of the mass of the mixed solution.

[0054] In some embodiments, a collagen solution and a phycocyanin solution are mixed to obtain a mixed solution, the pH of the mixed solution is adjusted to 6.3-6.7, and then transglutaminase is added. After mixing, the pH is further adjusted to neutral, and the solution is allowed to stand at 34-38°C for 22-26 hours to form a hydrogel, which is a phycocyanin-collagen composite hydrogel.

[0055] Preferably, the collagen solution and the phycocyanin solution are mixed to obtain a mixed solution, the pH of the mixed solution is adjusted to 6.5, and then transglutaminase is added. After sufficient mixing, the pH is further adjusted to 7.0, and the mixture is allowed to stand at 36°C for 24 hours to form a hydrogel, which is a phycocyanin-collagen composite hydrogel.

[0056] In some embodiments, a fish-derived collagen freeze-dried sponge is added to acetic acid to obtain a collagen solution; wherein the concentration of the acetic acid is 0.05 to 0.5 mol / L, preferably, the concentration of the acetic acid is 0.1 mol / L.

[0057] In some embodiments, phycocyanin is added to a PBS solution to obtain a phycocyanin solution; wherein the concentration of the PBS solution is 0.01 to 0.05 mol / L, preferably, the concentration of the PBS solution (i.e., phosphate buffer solution) is 0.01 mol / L, pH 7.2 to 7.4, and the brand is Perfemiker.

[0058] In some embodiments, the method for preparing fish-derived collagen is:

[0059] S11, placing fish skin in n-butanol aqueous solution and carrying out degreasing treatment;

[0060] S12, the fish skin of defatting process is placed in aqueous sodium hydroxide solution and soaked, to remove non-collagen components, washed to neutrality;

[0061] S13, in the fish-skin that defatting is complete in S12, add acetic acid solution and pepsin, extract 36~72h down at 3~5 ℃, after centrifugal, collect supernatant (being that supernatant is enzymatic soluble collagen);

[0062] S14, adding NaCl to the supernatant, centrifuging, collecting the precipitate, dissolving the precipitate in acetic acid solution, dialyzing with sodium hydrogen phosphate solution for 24 to 48 hours, and centrifuging to collect the precipitate again;

[0063] S15, dissolving the collected precipitate in acetic acid solution, dialyzing the solution again with acetic acid solution for 24 to 48 hours, and then dialyzing with distilled water for 24 to 48 hours to obtain an enzyme-soluble collagen solution;

[0064] S16. Freeze-drying the enzyme-soluble collagen solution to obtain a fish-derived collagen freeze-dried sponge, namely, fish-derived collagen.

[0065] The fish-derived collagen protein of the present invention is prepared by defatting fish skin, performing pepsin hydrolysis, salting out, multiple dialysis, and freeze-drying to obtain a fish-derived collagen protein freeze-dried sponge, i.e., the fish-derived collagen protein. In some embodiments, the fish skin used is tilapia skin.

[0066] In some embodiments, the fish skin is placed in an n-butanol aqueous solution at 3-5° C. for degreasing for 24-48 hours, the volume concentration of the n-butanol aqueous solution is 10-15%, and the mass volume ratio of the fish skin to the n-butanol aqueous solution is 1 g: (20-30) mL; specifically, the n-butanol aqueous solution is replaced every 8-12 hours.

[0067] In some embodiments, the defatted fish skin is immersed in a sodium hydroxide aqueous solution with a concentration of 0.05 to 0.2 mol / L for 24 to 48 hours to remove non-collagenous components and washed to neutrality; specifically, the NaOH aqueous solution is replaced every 8 to 12 hours.

[0068] In some embodiments, in the step of adding acetic acid solution and pepsin to the defatted fish skin, the mass volume ratio of the defatted fish skin and the acetic acid solution is 1g:(30-50)mL, and the concentration of the acetic acid solution is 0.2-0.8mol / L; the acetic acid solution and pepsin are added to the defatted fish skin so that the mass concentration of the pepsin is 0.5-1%.

[0069] In some embodiments, acetic acid solution and pepsin are added to defatted fish skin, extracted at 3-5° C. for 36-72 h, centrifuged at 8000-12000 r / min for 10-20 min, and the supernatant (the supernatant is the enzymatically soluble collagen) is collected.

[0070] In some embodiments, NaCl is added to the supernatant to a NaCl concentration of 0.5 to 1.5 mol / L, the mixture is centrifuged at 8000 to 12000 r / min for 10 to 20 min, the precipitate is collected, and the precipitate is dissolved in 0.2 to 0.5 mol / L acetic acid solution (the mass volume ratio of the precipitate to the acetic acid solution is 1 g: (20 to 30) mL). The precipitate is dialyzed for 24 to 48 hours using 2 to 3 L of 0.01 to 0.03 mol / L disodium hydrogen phosphate solution, the disodium hydrogen phosphate solution is replaced every 8 to 12 hours, the mixture is centrifuged at 8000 to 12000 r / min for 25 to 35 minutes, and the precipitate is collected again.

[0071] The collected precipitate was dissolved in 0.2-0.8 mol / L acetic acid solution (the mass volume ratio of precipitate to acetic acid solution was 1 g: (20-30) mL), dialyzed again with 2-3 L of 0.05-0.1 mol / L acetic acid solution for 24-48 h, and then dialyzed with 2-3 L of distilled water for 24-48 h to obtain an enzyme-soluble collagen solution; the acetic solution and distilled water were replaced every 8-12 h.

[0072] In some embodiments, the enzyme-soluble collagen solution is freeze-dried at -20°C to -30°C to obtain a fish-derived collagen freeze-dried sponge, namely, fish-derived collagen.

[0073] In some embodiments, a collagen solution and a phycocyanin solution are mixed to obtain a mixed solution, the pH of the mixed solution is adjusted to 6.3-6.7, and then glutamine transaminase is added. After mixing, the pH is further adjusted to neutral; wherein, 1 mol / L-5 mol / L sodium hydroxide solution and 0.1 mol / L-0.5 mol / L acetic acid solution are used to adjust the pH.

[0074] Based on the same inventive concept, the present invention also provides a phycocyanin-collagen composite hydrogel, which is prepared using the above-mentioned preparation method.

[0075] Based on the same inventive concept, the present invention also provides a phycocyanin-collagen composite hydrogel prepared by the above-mentioned preparation method or the use of the above-mentioned phycocyanin-collagen composite hydrogel in preparing medical dressings and in preparing products with antioxidant effects.

[0076] Specifically, the antioxidant effect includes scavenging hydroxyl free radicals.

[0077] The present invention treats the chemical bonds between phycocyanin and collagen, utilizing transglutaminase (TGase) to act on glutamic acid and lysine in the two protein molecules, thereby transferring acyl groups between different protein molecules, thereby forming a stable chemical bond and forming a new collagen protein combined with phycocyanin. The present invention enhances the strength and viscosity of the bond between phycocyanin and collagen by forming a new chemical bond. The formed collagen and phycocyanin are not easy to fall off and are more stable. This stable phycocyanin collagen protein has a wider range of applications, is subject to fewer limiting factors, and can better exert the effects of phycocyanin and collagen. The hydrogel prepared by the present invention is subjected to diffusivity, infrared spectroscopy, swelling rate, air permeability, antioxidant properties, and scanning electron microscopic measurements. After combining under certain conditions, the color of the formed hydrogel gradually deepens with the increase of phycocyanin concentration. After soaking in 0.01 mol / L PBS buffer for 4 hours, the hydrogel does not dissolve as a whole, and has a certain strength and stability. In the permeability test, as the concentration of phycocyanin increased, the permeability of the prepared hydrogel increased, but when the concentration of phycocyanin continued to increase, the permeability decreased to a certain extent. This was because the excess phycocyanin was cross-linked within the phycocyanin molecules under the action of transglutaminase (TGase), affecting the permeability of the formed network structure; when measuring the swelling rate of the hydrogel, the pH of the internal environment of human skin and wound healing was simulated, and it was found that the hydrogels prepared with different concentrations of phycocyanin had different swelling rates at different pH values, and all had a certain water absorption capacity as a whole. The appropriate phycocyanin concentration can be selected according to the pH value of the use environment; in terms of antioxidant properties, the reducing and hydroxyl radical scavenging abilities were tested. With ascorbic acid as a control, it was found that with the increase of phycocyanin concentration, the reducing property and hydroxyl radical scavenging ability of the prepared hydrogel showed an increasing trend. When the phycocyanin concentration was 8 mg / mL, the hydroxyl radical scavenging rate of the hydrogel prepared reached the maximum, which was 57.37%; the prepared hydrogel had a certain antioxidant capacity, but compared with ascorbic acid (Vc), its antioxidant capacity was relatively weak. Infrared spectroscopy showed that the hydrogel prepared by the present invention retained the basic structure of phycocyanin and collagen, and scanning electron microscopy microstructure analysis also showed the void structure of the hydrogel of the present invention, which corresponded to the measured air permeability, etc. The phycocyanin used in the present invention itself has a certain antioxidant property, and collagen has a certain biocompatibility and has a certain promoting effect on wound healing. Therefore, the hydrogel prepared by the present invention using transglutaminase (TGase) can be used as a medical dressing for wound healing. It can effectively protect the wound during the healing process of skin burns, injuries, etc., avoiding the secondary damage caused by traditional dressings.

[0078] The following further illustrates the preparation method of the phycocyanin-collagen composite hydrogel of the present invention using specific examples. This section further illustrates the present invention with reference to specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the techniques employed in the examples are conventional techniques well known to those skilled in the art. Unless otherwise specified, the reagents, methods, and equipment employed in the present invention are conventional in the art.

[0079] Example 1

[0080] The present invention provides a method for preparing a phycocyanin-collagen composite hydrogel, comprising the following steps:

[0081] S1. Dissolve fish-derived collagen in 0.1 mol / L acetic acid solution to obtain a collagen solution with a concentration of 8 mg / mL;

[0082] S2, adding phycocyanin to a 0.01 mol / L PBS solution to obtain a phycocyanin solution with a concentration of 2 mg / mL;

[0083] S3, mixing the collagen solution in S1 and the phycocyanin solution in S2 in equal volumes to obtain a mixed solution, wherein the concentration of phycocyanin in the mixed solution is 1 mg / mL;

[0084] The pH of the mixed solution was adjusted to 6.5 (using 1 mol / L sodium hydroxide solution and 0.5 mol / L acetic acid solution to adjust the pH), and then transglutaminase was added (the amount of transglutaminase added was 0.3% of the mass of the mixed solution). After thorough mixing, the pH was further adjusted to 7.0 and allowed to stand at 36°C for 24 hours to form a hydrogel, namely, phycocyanin-collagen composite hydrogel (denoted as P1);

[0085] The method for preparing fish-derived collagen comprises the following steps:

[0086] S11, 15g tilapia fish skin is placed in 4 ℃, volume concentration is 10% n-butanol aqueous solution and is carried out degreasing treatment 24h; Wherein, fish skin and n-butanol aqueous solution mass volume ratio is 1g:20mL; n-butanol aqueous solution is replaced once every 8h;

[0087] S12, the fish-skin of defatted process is placed in the sodium hydroxide aqueous solution that concentration is 0.1mol / L and soaks 24h, to remove non-collagenous components, is washed to neutrality; Concrete, the NaOH aqueous solution changes once every 8h, and the mass volume ratio of the fish-skin that defatted is complete and acetic acid solution is 1g:20mL;

[0088] S13, the acetic acid solution and the pepsin that are added to the neutral fish-skin that defatted in S12 are complete, extract 48h at 4 ℃, centrifugal 15min under 10000r / min rotating speed, collect supernatant; The mass volume ratio of the neutral fish-skin that defatted is complete and acetic acid solution is 1g:50mL; Pepsin is added to the neutral fish-skin that defatted is complete, so that the mass concentration of pepsin is 1% of a fish-skin quality;

[0089] S14, NaCl was added to the supernatant to make the NaCl concentration 0.9 mol / L, and the mixture was centrifuged at 10000 r / min for 15 min to collect the precipitate, which was dissolved in 0.5 mol / L acetic acid solution (the mass volume ratio of the precipitate to the acetic acid solution was 1 g:20 mL). After 24 h of dialysis with 2 L of 0.02 mol / L disodium hydrogen phosphate solution, the disodium hydrogen phosphate solution was replaced every 8 h, and the mixture was centrifuged at 10000 r / min for 30 min to collect the precipitate again.

[0090] S15. Dissolve the collected precipitate in 0.5 mol / L acetic acid solution (the mass volume ratio of precipitate to acetic acid solution is 1 g:20 mL), dialyze again with 2 L of 0.1 mol / L acetic acid solution for 24 h, and then dialyze with 2 L of distilled water for 48 h to obtain an enzyme-soluble collagen solution; replace the acetic solution and distilled water every 8 h;

[0091] S16. Freeze-dry the enzyme-soluble collagen solution at -20°C to obtain a fish-derived collagen freeze-dried sponge, namely, fish-derived collagen.

[0092] Example 2

[0093] The present invention provides a method for preparing a phycocyanin-collagen composite hydrogel, comprising the following steps:

[0094] S1. Dissolve fish-derived collagen in 0.1 mol / L acetic acid solution to obtain a collagen solution with a concentration of 8 mg / mL;

[0095] S2, adding phycocyanin to a 0.01 mol / L PBS solution to obtain a phycocyanin solution with a concentration of 4 mg / mL;

[0096] S3, mixing the collagen solution in S1 and the phycocyanin solution in S2 in equal volumes to obtain a mixed solution, wherein the concentration of phycocyanin in the mixed solution is 2 mg / mL;

[0097] The pH of the mixed solution was adjusted to 6.5 (using 1 mol / L sodium hydroxide solution and 0.5 mol / L acetic acid solution to adjust the pH), and then transglutaminase was added (the amount of transglutaminase added was 0.3% of the mass of the mixed solution). After thorough mixing, the pH was further adjusted to 7.0 and allowed to stand at 36°C for 24 hours to form a hydrogel, namely, phycocyanin-collagen composite hydrogel (denoted as P2);

[0098] The preparation method of fish-derived collagen is the same as that in Example 1.

[0099] Example 3

[0100] The present invention provides a method for preparing a phycocyanin-collagen composite hydrogel, comprising the following steps:

[0101] S1. Dissolve fish-derived collagen in 0.1 mol / L acetic acid solution to obtain a collagen solution with a concentration of 8 mg / mL;

[0102] S2, adding phycocyanin to a 0.01 mol / L PBS solution to obtain a phycocyanin solution with a concentration of 8 mg / mL;

[0103] S3, mixing the collagen solution in S1 and the phycocyanin solution in S2 in equal volumes to obtain a mixed solution, wherein the concentration of phycocyanin in the mixed solution is 4 mg / mL;

[0104] The pH of the mixed solution was adjusted to 6.5 (using 1 mol / L sodium hydroxide solution and 0.5 mol / L acetic acid solution to adjust the pH), and then transglutaminase was added (the amount of transglutaminase added was 0.3% of the mass of the mixed solution). After thorough mixing, the pH was further adjusted to 7.0 and allowed to stand at 36°C for 24 hours to form a hydrogel, namely, phycocyanin-collagen composite hydrogel (denoted as P3);

[0105] The preparation method of fish-derived collagen is the same as that in Example 1.

[0106] Example 4

[0107] The present invention provides a method for preparing a phycocyanin-collagen composite hydrogel, comprising the following steps:

[0108] S1. Dissolve fish-derived collagen in 0.1 mol / L acetic acid solution to obtain a collagen solution with a concentration of 8 mg / mL;

[0109] S2, adding phycocyanin to a 0.01 mol / L PBS solution to obtain a phycocyanin solution with a concentration of 12 mg / mL;

[0110] S3, mixing the collagen solution in S1 and the phycocyanin solution in S2 in equal volumes to obtain a mixed solution, wherein the concentration of phycocyanin in the mixed solution is 6 mg / mL;

[0111] The pH of the mixed solution was adjusted to 6.5 (using 1 mol / L sodium hydroxide solution and 0.5 mol / L acetic acid solution to adjust the pH), and then transglutaminase was added (the amount of transglutaminase added was 0.3% of the mass of the mixed solution). After thorough mixing, the pH was further adjusted to 7.0 and allowed to stand at 36°C for 24 hours to form a hydrogel, namely, phycocyanin-collagen composite hydrogel (denoted as P4);

[0112] The preparation method of fish-derived collagen is the same as that in Example 1.

[0113] Example 5

[0114] The present invention provides a method for preparing a phycocyanin-collagen composite hydrogel, comprising the following steps:

[0115] S1. Dissolve fish-derived collagen in 0.1 mol / L acetic acid solution to obtain a collagen solution with a concentration of 8 mg / mL;

[0116] S2, adding phycocyanin to a 0.01 mol / L PBS solution to obtain a phycocyanin solution with a concentration of 16 mg / mL;

[0117] S3, mixing the collagen solution in S1 and the phycocyanin solution in S2 in equal volumes to obtain a mixed solution, wherein the concentration of phycocyanin in the mixed solution is 8 mg / mL;

[0118] The pH of the mixed solution was adjusted to 6.5 (using 1 mol / L sodium hydroxide solution and 0.5 mol / L acetic acid solution to adjust the pH), and then transglutaminase was added (the amount of transglutaminase added was 0.3% of the mass of the mixed solution). After thorough mixing, the pH was further adjusted to 7.0 and allowed to stand at 36°C for 24 hours to form a hydrogel, namely, phycocyanin-collagen composite hydrogel (denoted as P5);

[0119] The preparation method of fish-derived collagen is the same as that in Example 1.

[0120] Comparative Example 1

[0121] This comparative example provides a method for preparing a hydrogel, comprising the following steps:

[0122] S1. Dissolve fish-derived collagen in 0.1 mol / L acetic acid solution to obtain a collagen solution with a concentration of 8 mg / mL;

[0123] S2, mixing equal volumes of the collagen solution in S1 and phycocyanin dissolved in 0.01 mol / L PBS solution to obtain a mixed solution;

[0124] The pH of the mixed solution was adjusted to 6.5 (using 1 mol / L sodium hydroxide solution and 0.5 mol / L acetic acid solution to adjust the pH), and then transglutaminase was added (the amount of transglutaminase added was 0.3% of the mass of the mixed solution). After thorough mixing, the pH was further adjusted to 7.0 and allowed to stand at 36°C for 24 hours to form a hydrogel (denoted as P0);

[0125] The preparation method of fish-derived collagen is the same as that in Example 1.

[0126] Performance Testing

[0127] Observation of hydrogel appearance and diffusion properties

[0128] The results of visual observation of the hydrogels prepared in Examples 1 to 5 and Comparative Example 1 are as follows: Figure 1 As shown in (a), at the same time, the hydrogels prepared in Examples 1 to 5 and Comparative Example 1 were immersed in 0.01 mol / L PBS solution and the diffusion properties of the hydrogels were observed after standing for 4 hours. The results are shown in FIG. Figure 1 (b) shown.

[0129] from Figure 1 As can be seen in (a), as the concentration of phycocyanin increases, the color of the hydrogel changes from light to dark; in addition, Figure (b) shows the state of hydrogels with different concentrations of phycocyanin after being immersed in 0.01 mol / L PBS buffer for a period of time. After immersion for 4 hours, the hydrogel still maintains its original shape and does not dissolve. Therefore, the hydrogel prepared by cross-linking with transglutaminase (TGase) has certain strength and stability.

[0130] FT-IR determination of hydrogel

[0131] The infrared spectra of the phycocyanin-collagen composite hydrogel, phycocyanin, and fish-derived collagen prepared in Example 1 are as follows: Figure 2 Specifically, the infrared spectrum was measured using a Fourier transform infrared spectrometer (FT-IR-4100) from JASCO, Japan, with a wavelength range of 4000 to 400 cm -1 ; Figure 2 Here, PC represents the phycocyanin in Example 1, Col represents the fish-derived collagen in Example 1, and PC / Col represents the phycocyanin-collagen composite hydrogel prepared in Example 1.

[0132] from Figure 2 It can be seen that phycocyanin is at 3299 cm-1 、3079cm -1 、1650cm -1 、1537cm -1 There are absorption peaks at the following locations; the absorption peak of fish collagen mainly appears at 3433 cm -1 、1558cm -1 、1413cm -1 The characteristic peaks of phycocyanin and collagen were mainly at 3462 cm-1 by chemical cross-linking of phycocyanin and collagen using transglutaminase (TGase). -1 、1643cm -1 、1554cm -1 、1414cm -1 The basic structures of the two proteins are retained to a certain extent.

[0133] Hydrogel gas permeability measurement

[0134] Prepare several identical centrifuge tubes and record the area of ​​the centrifuge tube opening as S (cm 2 ). Deionized water was added to the centrifuge tube, and the hydrogels prepared in Examples 1 to 5 and Comparative Example 1 were respectively adhered to the mouth of the centrifuge tube and accurately weighed, recorded as M1; the centrifuge tube with the hydrogel film was placed in a 36°C oven for 24 hours, and was continued to be accurately weighed, recorded as M2, and the experimental difference T (h) was recorded at the same time. The water vapor transmission rate (MVTR) was determined according to the formula of Wang Mingchao et al. (see reference: Preparation and research of an antioxidant phycocyanin / collagen composite hydrogel, Wang Mingchao et al., Functional Materials, Vol. 11, No. 49, 2018) for analysis. Calculation formula:

[0135]

[0136] The air permeability of the hydrogels prepared in Examples 1 to 5 and Comparative Example 1 was tested according to the above method. The results are as follows: Figure 3 shown. Figure 3 The vertical axis Permeability represents the water vapor transmission rate.

[0137] from Figure 3 It can be seen that the collagen hydrogel P0 prepared in Comparative Example 1 without adding phycocyanin has the lowest air permeability; the hydrogels P1 to P5 prepared in Examples 1 to 5, with the addition of phycocyanin, the gaps in the formed hydrogels increase and the air permeability increases. When the phycocyanin concentration reaches 4 mg / mL, the air permeability of the prepared hydrogel P3 reaches the maximum, which is 1964.98 g·m -2 12h- 1When the concentration of phycocyanin continues to increase, the permeability begins to decrease. This is because there is too much phycocyanin, and glutamine transaminase (TGase) can no longer catalyze the cross-linking of phycocyanin and collagen, resulting in cross-linking of internal molecules of phycocyanin. After cross-linking, phycocyanin fills the gaps in the hydrogel, causing the permeability of the hydrogel to decrease.

[0138] Determination of hydrogel swelling rate

[0139] The swelling rate (SR) of the prepared hydrogel was determined by measuring the water absorption of the hydrogel under different pH environments. Specifically, PBS buffer solutions were prepared to simulate the human body environment (pH = 5.0), human skin (pH = 7.4), and human skin healing (pH = 8.2). The prepared hydrogel was air-dried at 36°C for 24 hours, and a certain amount of hydrogel was weighed, recorded as W0. The hydrogel was then placed in PBS buffer solutions of different pH values. After 12 hours, the hydrogel was taken out, dried, and weighed, recorded as W0. t , calculate the swelling rate according to the following formula. Calculation formula:

[0140]

[0141] The swelling rates of the hydrogels prepared in Examples 1 to 5 and Comparative Example 1 were tested according to the above method. The results are as follows: Figure 4 shown. Figure 4 The vertical axis Swelling Rate represents the swelling rate.

[0142] from Figure 4 It can be seen that in three different pH environments, the overall swelling rates of the hydrogels P1 to P5 prepared by adding phycocyanin in Examples 1 to 5 are higher than the swelling rate of the hydrogel P0 prepared in Comparative Example 1 without adding phycocyanin. Therefore, the addition of phycocyanin can increase the swelling rate of collagen. In addition, in three different pH environments, with the increase of phycocyanin concentration, the swelling rate of the hydrogel generally increases. In general, the swelling rate of the hydrogel P3 prepared at a phycocyanin concentration of 4 mg / mL in Example 3 is the highest, which corresponds to the measurement results of the gas permeability of the gel.

[0143] Microscopic morphology of hydrogel

[0144] The scanning electron micrographs of the hydrogels prepared in Examples 1 to 5 and Comparative Example 1 at different magnifications are as follows: Figure 5 shown.

[0145] from Figure 5 It can be seen that Figure 5 (a1) and Figure 3(a2) shows hydrogel P0, prepared without phycocyanin. Hydrogel P0 exhibits a relatively compact structure. For hydrogels P1-P5 prepared in Examples 1-5, the voids in the hydrogels increased significantly with increasing phycocyanin concentration. Hydrogel P5, prepared at a phycocyanin concentration of 8 mg / mL, exhibited smaller and more distant voids compared to the previous groups containing phycocyanin. This is consistent with the previously measured results of an initial increase followed by a decrease in permeability and swelling rate. This porous hydrogel maintains a certain level of permeability and swelling, and as a potential medical dressing, this structure may promote wound healing.

[0146] Hydrogel antioxidant properties

[0147] Hydrogel reduction test: Ascorbic acid aqueous solutions with concentrations of 1, 2, 4, 6, and 8 mg / mL were prepared as positive controls and were designated as V1, V2, V3, V4, and V5, respectively. Hydrogels P1, P2, P3, P4, and P5 prepared in Examples 1 to 5 were also prepared as experimental groups. The hydrogels in the experimental groups were fully dissolved in 0.5 mol / L acetic acid solution. 1 mL of the experimental solution and the positive control solution were then taken, and 15 mL of PBS buffer solution (pH = 6.6) and 15 mL of 1% potassium ferricyanide solution were added, respectively. The mixture was reacted in a 50°C environment for 30 min, cooled to room temperature, and 10 mL of 10% trichloroacetic acid solution was added. The mixture was thoroughly shaken and centrifuged at 5000 r / min for 5 min. 1 mL of the supernatant was added to 10 mL of 1% ferric chloride solution and 20 mL of deionized water, mixed thoroughly, and allowed to stand. The absorbance was measured at 700 nm using an ultraviolet spectrophotometer, and the entire operation was performed in the dark. Figure 6 shown.

[0148] Determination of the hydroxyl radical scavenging ability of the hydrogel: Ascorbic acid aqueous solutions with concentrations of 1, 2, 4, 6, and 8 mg / mL were prepared as positive controls, and were denoted as V1, V2, V3, V4, and V5, respectively. The hydrogels P1, P2, P3, P4, and P5 prepared in Examples 1 to 5 were set up as experimental groups, and a deionized water blank control group was also set up. 1 mL of each of the above solutions was added in sequence to 10 mL of 2.5 nmol / L salicylic acid, 10 mL of 5 nmol / L ferrous sulfate solution, and 20 mL of deionized water. Among the three samples, 10 mL of 5 nmol / L hydrogen peroxide solution was continued to be added to the experimental group and the blank control group samples, and the experimental group was denoted as A. x The blank control group was marked as A0, and the positive control group was marked as A without the addition of hydrogen peroxide solution. x0 After reacting in a 36°C water bath for 30 min, the absorbance (A x , A0, A x0The absorbance at 510 nm for different treatments is shown in Figure 2. The entire experimental process was carried out in the dark. The calculation formula for the hydroxyl radical scavenging rate is:

[0149]

[0150] The hydroxyl radical scavenging rate test results of the hydrogel are as follows: Figure 7 shown.

[0151] Figures 6-7 The middle horizontal axis represents the mass concentration of ascorbic acid (Vc) and the mass concentration of phycocyanin (PC) in the mixed solution during the preparation of the hydrogel in Examples 1 to 5.

[0152] from Figure 6 It can be seen that when measuring the reducing property of the hydrogel, with the increase of the concentration of phycocyanin, the reducing property of the prepared hydrogel shows an increasing trend. Although compared with ascorbic acid, the reducing property of the hydrogel is lower than that of ascorbic acid as a whole, the prepared hydrogel still has a certain reducing ability. Figure 7 The hydroxyl radical scavenging ability of the hydrogels was measured and the absorbance was calculated. Similar to the results for reducing activity, the hydroxyl radical scavenging ability of the prepared hydrogels increased with increasing phycocyanin concentration, but it was still lower than that of ascorbic acid at the same concentration. Combining the reducing activity and hydroxyl radical scavenging activity, the hydrogels prepared using transglutaminase (TGase) showed some antioxidant activity, but overall it was lower than that of ascorbic acid.

[0153] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a phycocyanin-collagen composite hydrogel, characterized in that: The following steps are involved: adding fish-derived collagen to acetic acid to obtain a collagen solution; adding phycocyanin to a PBS solution to obtain a phycocyanin solution; The collagen solution and the phycocyanin solution are mixed to obtain a mixed solution, the pH of the mixed solution is adjusted to 6.5, and then transglutaminase is added. After mixing, the pH is further adjusted to neutral, and the mixture is allowed to stand to form a hydrogel, namely, the phycocyanin-collagen composite hydrogel; The concentration of fish-derived collagen in the collagen solution was 8 mg / mL; The phycocyanin concentration in the phycocyanin solution was 8 mg / mL; The concentration of phycocyanin in the mixed solution was 4 mg / mL; The amount of transglutaminase added was 0.3% of the mass of the mixed solution; The concentration of the acetic acid is 0.1 mol / L; The concentration of the PBS solution is 0.01 mol / L; The preparation method of the fish-derived collagen is as follows: The fish skin is placed in an n-butanol aqueous solution for degreasing for 24 to 48 hours, wherein the volume concentration of the n-butanol aqueous solution is 10 to 15%, and the mass volume ratio of the fish skin to the n-butanol aqueous solution is 1 g:20 mL; The defatted fish skin is placed in a 0.1 mol / L sodium hydroxide aqueous solution for 24 to 48 hours to remove non-collagenous components and washed until neutral; In the step of adding acetic acid solution and pepsin in the fish-skin that defatting is complete, the mass volume ratio of the fish-skin that defatting is complete and acetic acid solution is 1g:50mL, and the concentration of acetic acid solution is 0.5mol / L; Adding acetic acid solution and pepsin in the fish-skin that defatting is complete makes the mass concentration of pepsin be 1%; NaCl was added to the supernatant to make the NaCl concentration 0.9 mol / L, and the mixture was centrifuged to collect the precipitate. The precipitate was dissolved in 0.5 mol / L acetic acid solution, and dialyzed against 0.02 mol / L sodium hydrogen phosphate solution for 24-48 hours. The precipitate was collected again by centrifugation. The collected precipitate was dissolved in 0.5 mol / L acetic acid solution, dialyzed again with 0.1 mol / L acetic acid solution for 24 to 48 hours, and then dialyzed with distilled water for 24 to 48 hours to obtain an enzyme-soluble collagen solution; The enzyme-soluble collagen solution is freeze-dried at -20°C to obtain a fish-derived collagen freeze-dried sponge, namely, fish-derived collagen.

2. The method for preparing the phycocyanin-collagen composite hydrogel according to claim 1, wherein: The collagen solution and the phycocyanin solution are mixed to obtain a mixed solution, the pH of the mixed solution is adjusted to 6.5, and then transglutaminase is added. After mixing, the pH is further adjusted to neutral, and the solution is allowed to stand at 34-38° C. for 22-26 hours to form a hydrogel, namely, the phycocyanin-collagen composite hydrogel.

3. The method for preparing the phycocyanin-collagen composite hydrogel according to claim 1, wherein: The collagen solution and the phycocyanin solution are mixed to obtain a mixed solution, the pH of the mixed solution is adjusted to 6.5, and then glutamine transaminase is added. After mixing, the pH is further adjusted to neutral; wherein, 1 mol / L-5 mol / L sodium hydroxide solution and 0.1 mol / L-0.5 mol / L acetic acid solution are used to adjust the pH.

4. A phycocyanin-collagen composite hydrogel, characterized in that: The compound is prepared by the preparation method described in any one of claims 1 to 3.

5. Use of the phycocyanin-collagen composite hydrogel prepared by the preparation method according to any one of claims 1 to 3 or the phycocyanin-collagen composite hydrogel according to claim 4 in the preparation of medical dressings and products with antioxidant effects.

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