Sturgeon swimming bladder collagen peptide for skin repair and preparation method thereof

Abstract

The invention relates to the technical field of bioactive peptides, in particular to sturgeon swimming bladder collagen peptide for skin repair and a preparation method thereof.The sturgeon swimming bladder collagen peptide is obtained through sturgeon swimming bladder enzymolysis directly for the first time, and compared with sturgeon skin or sturgeon bone enzymolysis in the prior art, a new raw material source is developed, and the production cost is lowered. Compared with sturgeon swimming bladder enzymolysis fermentation, the fermentation process is omitted, the cost is reduced, and market popularization is facilitated; the novel collagen peptide is separated, the amino acid sequence of the collagen peptide is GVTGKQGGK, and the collagen peptide can promote human fibroblast proliferation and repair skin injury and can be used in the fields of medical cosmetology and cosmetics.

Description

Technical Field

[0001] The invention relates to the technical field of bioactive peptides, and in particular to a sturgeon swim bladder collagen peptide for skin repair and a preparation method thereof. Background Art

[0002] Sturgeon (Acipenseridae sturgeon), also known as lecherous fish, is a kind of fish with a larger body and longer lifespan among the existing freshwater fish in the world, with a history of more than 200 million years. China is one of the countries with the most sturgeon species, the widest distribution and the richest resources in the world. Due to the high nutritional value and good economic benefits of sturgeon breeding, with the increasing maturity of artificial breeding technology of sturgeon, the total amount of sturgeon breeding in my country continues to grow. The main by-products of sturgeon deep processing - fish skin, fish bones and fish bladder are rich in collagen, so they can be used as raw materials for extracting natural high-quality collagen. At present, relevant research on extracting collagen from sturgeon skin, fish bones and fish bladder as raw materials has been reported, but the structural composition, basic physical and chemical properties of sturgeon fish bladder collagen and its differences from mammalian collagen have not been fully clarified. The existing technology mostly uses acid extraction and acid-enzyme extraction to prepare sturgeon acid-soluble collagen (ASC) and enzyme-soluble collagen (PSC).

[0003] For example, the prior art CN 115191596 B discloses a collagen peptide product with anti-aging effects produced by using sturgeon maw as raw material, blueberry juice as a carrier, Lactobacillus plantarum as a fermentation agent, polysaccharides, polypeptides, vitamins, minerals and pollen extracts as synergistic components, and utilizing protein targeted enzymatic cleavage, precise nutritional regulation and flavor quantitative recombination technology.

[0004] Prior art CN 117384245 A discloses an anti-aging sturgeon roe tetrapeptide, the peptide sequence of which is NLPL. The preparation method specifically comprises the following steps: (1) adding deionized water to sturgeon roe and homogenizing to obtain a sturgeon roe homogenate; (2) adjusting the pH value of the sturgeon roe homogenate, adding alkaline protease, performing enzymolysis, deactivating the enzyme, and cooling to obtain a sturgeon roe enzymolysis solution; (3) centrifuging the sturgeon roe enzymolysis solution and taking the supernatant to obtain the product.

[0005] Prior art CN 114395401 B discloses that the preparation steps of sturgeon small molecule polypeptide nutrient solution include pretreatment, one-step enzymolysis, two-step enzymolysis and three-step enzymolysis.

[0006] Prior art CN 113527467 B discloses a method for extracting collagen polypeptide from sturgeon skin, comprising the following steps: S1: removing foreign proteins, S2: removing fat, S3: spreading the sturgeon skin flat, pre-freezing overnight, and then freeze-drying, S4: expanding the skin, washing it with ultrapure water after the expansion, collecting the sturgeon skin and homogenizing it into a paste for standby use, and S5: enzymolyzing the paste of sturgeon skin with a protease to obtain an enzymolysis product.

[0007] Prior art CN 1101000858 B discloses a method for preparing sturgeon protein peptide powder, comprising: (1) pre-treating sturgeon skin to obtain a crude sturgeon protein extract; (2) adjusting the pH value of the crude sturgeon protein extract to 7.5-8.5, and performing an enzymatic hydrolysis reaction under the action of alkaline protease; the mass percentage of the alkaline protease to the crude sturgeon protein extract is 1.5-3%. The sturgeon protein peptide powder has an auxiliary blood sugar lowering effect for people at all stages of diabetes history.

[0008] Prior art CN 110218756 B discloses a method for extracting selenium-rich sturgeon bone peptide, comprising: (1) crushing sturgeon bone raw material to obtain bone powder; (2) performing a first enzymatic hydrolysis to degrade the fish bone collagen skeleton to obtain a crude enzymatic hydrolysis product; (3) performing a second enzymatic hydrolysis; (4) performing a desalting treatment to obtain a desalted enzymatic hydrolysis product; (5) separating the desalted enzymatic hydrolysis product obtained in step (4) from solid and liquid, and drying the liquid phase to obtain the sturgeon bone peptide. Selenium-rich sturgeon bone peptide has a significant anti-aging effect.

[0009] Prior art CN 108969397 B discloses an active polypeptide facial mask, which has good antioxidant and moisturizing effects. It mainly uses sturgeon skin as a raw material, and obtains active polypeptides through secondary enzymatic hydrolysis and membrane separation and purification. Its application in facial mask cosmetics shows good skin anti-aging effects.

[0010] However, the above-mentioned prior art does not have a technical solution for directly enzymatically hydrolyzing sturgeon maw to obtain fish maw collagen peptides. Therefore, in order to expand the source of sturgeon collagen peptides and simplify the steps, the present application will use sturgeon maw to directly enzymatically hydrolyze to obtain fish maw collagen peptides and verify its corresponding function. Summary of the invention

[0011] In order to solve the above problems, the present invention first provides a sturgeon swim bladder collagen peptide for skin repair, whose amino acid sequence is GVTGKQGGK.

[0012] The present invention also provides a method for preparing the above-mentioned sturgeon maw collagen peptide, the method comprising the step of enzymatic hydrolysis of sturgeon maw; optionally, the method does not comprise the step of fermentation.

[0013] In certain embodiments, the enzymatic digestion uses trypsin

[0014] In some embodiments, the method further comprises a pre-processing step;

[0015] In certain embodiments, the pretreatment step comprises taking the swim bladder of Acipenser schrenckii, washing it, draining it, chopping it with scissors, soaking it in a NaCl solution to remove non-collagenous proteins, filtering to obtain residues, and then soaking the residues in a 30-85wt% isopropanol aqueous solution to remove fat and filtering it.

[0016] In certain embodiments, the pretreatment step precedes the step of enzymatically hydrolyzing the sturgeon swim bladder.

[0017] In certain embodiments, the method further comprises the step of filtering and concentrating using a nanofiltration membrane.

[0018] In certain embodiments, the step of filtering and concentrating using a nanofiltration membrane is performed after the step of enzymatic hydrolysis of the sturgeon swim bladder.

[0019] In certain embodiments, the nanofiltration membrane has a membrane molecular cut-off of 1000 Da.

[0020] The present invention also provides an application of the above method in preparing sturgeon swim bladder collagen peptide.

[0021] The present invention finally provides an application of the sturgeon maw collagen peptide for skin repair, wherein the application is for skin repair and the application is for non-therapeutic purposes; or the application is for preparing a product for skin repair.

[0022] Compared with the prior art, the present invention has at least the following beneficial effects:

[0023] The present invention is the first to directly use sturgeon bladder enzymatic hydrolysis to obtain collagen peptides. Compared with the prior art using sturgeon skin or sturgeon bone enzymatic hydrolysis, it has opened up a new source of raw materials. Compared with the use of sturgeon bladder enzymatic hydrolysis and fermentation, it saves the fermentation process, reduces costs, and is more conducive to market promotion.

[0024] The present invention separates a new collagen peptide, the amino acid sequence of which is GVTGKQGGK. The new collagen peptide can promote the proliferation of human fibroblasts and repair skin damage, and can be used in the fields of medical cosmetology and cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 , S1, S2, and S3 components on the proliferation of HSF.

[0026] Figure 2 , S1-1, S1-2, S1-3, and S1-1 after HPLC purification on the proliferation effects of HSF. DETAILED DESCRIPTION

[0027] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0028] Example 1 Extraction of collagen peptides

[0029] (1) 50 g of Acipenser schrenckii swim bladder was taken, washed, drained, cut into pieces with scissors, immersed in physiological saline to remove non-collagen, filtered to obtain residues, and then immersed in 75 wt % isopropanol aqueous solution to remove fat, and filtered to obtain pretreated sturgeon swim bladder residues;

[0030] (2) Transfer the sturgeon swim bladder residue in step (1) to a large beaker, add 500 mL of deionized water to the large beaker, and then place the beaker in a water bath, stir at 55° C. for 2 h to obtain a mixed solution; after cooling to 45° C., add 5 g of trypsin (120 u / mg), stir and hydrolyze for 3 h; centrifuge at 3000 g for 15 min, and take the supernatant;

[0031] (3) The supernatant of step (2) was filtered and concentrated using a nanofiltration membrane with a membrane molecular cutoff of 1000Da to separate and purify two kinds of polypeptide solutions with molecular weights of B (>1000Da) and S (<1000Da). The S component was collected and desalted using a dialysis bag (100Da), concentrated by rotary evaporation, freeze-dried, collected, and stored in a -20°C refrigerator for later use.

[0032] Example 2 Skin repair effect experiment

[0033] The S component was separated and purified by Sephadex G-25 to obtain three components: S1, S2, and S3. The obtained components were concentrated by rotary evaporation and vacuum freeze-dried. The freeze-dried polypeptide powder was collected and prepared into a certain concentration for skin repair effect experiment. Skin repair effect experiment: Human skin fibroblasts HSF in logarithmic growth phase were taken and 5×10 4 / mL were inoculated on a 96-well plate, with 5 replicate wells in each group. After the cells were iron-walled, DMEM complete medium and CCK-8 solution were mixed at a volume ratio of 100:1 and incubated for 2 hours. The supernatant was aspirated and the absorbance at a wavelength of 450nm was detected with an ELISA reader. The cells in the 96-well plate were washed once with PBS and cultured according to the following groups:

[0034] Experimental group 1: cultured in DMEM complete medium containing 50ug / mL S1;

[0035] Experimental group 2: cultured in DMEM complete medium containing 50ug / mL S2;

[0036] Experimental group 3: cultured in DMEM complete medium containing 50ug / mL S3;

[0037] Positive control group: cultured in DMEM complete medium containing 50ug / mL vitamin C;

[0038] Negative control group: DMEM complete medium was added for culture; the absorbance value at a wavelength of 450nm was measured after 96h of culture. The results are as follows Figure 1 shown.

[0039] from Figure 1 The results show that experimental groups 1-3 have a significant effect on promoting the proliferation of human skin fibroblasts compared with the blank control group (p<0.05), and the promotion effect is also significantly improved compared with the positive control group of vitamin C. Among them, experimental group 1 has the best effect, indicating that the S1 component has the highest effect in promoting the proliferation of human skin fibroblasts.

[0040] Example 3 Skin repair effect experiment

[0041] The S1 component was separated by Sephadex G-25 to obtain three components, S1-1, S1-2, and S1-3. The skin repair effect experiment was repeated and the S1-1 component was purified by HPLC. Figure 2 As shown, the S1-1 fraction has the highest effect on promoting the proliferation of human skin fibroblasts, and the effect of the S1-1 fraction after HPLC purification on promoting the proliferation of human skin fibroblasts is further improved.

[0042] The S1-1 fraction purified by HPLC was subjected to Edman degradation and its amino acid sequence was determined.

[0043] Edman degradation results showed that the amino acid sequence of the active peptide of component S1-1 was GVTGKQGGK.

[0044] Example 4

[0045] Adult male rats weighing about 300g were randomly divided into 3 groups, a blank control group, experimental group 1 (S1-1) and experimental group 2 (S1-1 after HPLC purification), with 10 rats in each group. The rats were anesthetized and their limbs were fixed. After disinfection, a skin wound with a diameter of 5mm was made on the back 2cm away from the left side of the spine and 2.5cm away from the root of the forelimb. The collagen peptide prepared in Example 3 was spray-dried to prepare collagen peptide powder. 20mg of collagen peptide powder was mixed with 20mL of water to prepare a skin repair solution. The experimental group applied 2mL of the skin repair solution of the present invention to the skin wound every day; the blank control group applied 2mL of distilled water every day. The wounds were cleaned and the dressings were changed at the same time every day. After 7d, the wound healing rate of each group was statistically analyzed, and the results are as follows:

[0046] Table 1 Wound healing rate test results

[0047]

[0048]

[0049] Table 1 shows that the collagen peptide of the present invention can effectively promote wound healing and has a good skin repair effect.

[0050] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A sturgeon maw collagen peptide for skin repair, characterized in that: Its amino acid sequence is GVTGKQGGK.

2. A method for preparing sturgeon maw collagen peptide according to claim 1, characterized in that: The method comprises the step of enzymatic hydrolysis using sturgeon swim bladder, and optionally, the method does not comprise the step of fermentation.

3. The method according to claim 2, characterized in that Trypsin was used for the enzymatic digestion.

4. The method according to claim 2, characterized in that: It also includes a preprocessing step.

5. The method according to claim 4, characterized in that The pretreatment step is before the step of enzymatic hydrolysis of the sturgeon swim bladder.

6. The method according to claim 2, characterized in that The method further comprises a step of filtering and concentrating using a nanofiltration membrane.

7. The method according to claim 6, characterized in that The step of filtering and concentrating using a nanofiltration membrane is performed after the step of enzymatic hydrolysis of the sturgeon swim bladder.

8. The preparation method according to claim 6 or 7, characterized in that: The membrane molecular cut-off amount of the nanofiltration membrane is 1000Da.

9. Use of the preparation method according to any one of claims 2 to 8 in the preparation of sturgeon swim bladder collagen peptide.

10. A use of sturgeon maw collagen peptide for skin repair as claimed in claim 1, characterized in that: The application is for repairing the skin for non-therapeutic purposes; or the application is for preparing a product for skin repair.

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