Fish collagen peptide and application thereof in beverage for improving skin such as anti-inflammation, skin repair promotion and like

Abstract

The invention relates to the technical field of fish collagen, in particular to fish collagen peptide of fish skin, a composition of the fish collagen peptide and application of the fish collagen peptide to preparation of a drink for improving skin. The amino acid sequence of the fish collagen peptide is LRGSFLR. The two fish collagen peptides are used as raw materials, and are compounded with three fresh fruits of kumquat, lemon and orange to obtain the composite beverage which is good in stability, yellowish in color, free of browning and uniform in color, and the composite beverage is in a uniform turbid liquid, free of suspended solids and free of layering in tissue state, and is allowed to slightly precipitate after being left to stand for a long time; the taste and smell are sour and sweet, the fruit faint scent of kumquat, lemon and orange is achieved, and the fishy smell and other unpleasant smells are avoided. The composite beverage provided by the invention has the efficacy of improving skin.

Description

Technical Field

[0001] The present invention relates to the technical field of fish collagen, and particularly relates to fish collagen peptides from the skin of Harpadon nehereus, their compositions, and their application in the preparation of beverages for improving skin quality. Background Art

[0002] Harpadon nehereus, scientifically named Harpadon nehereus, is a small and medium-sized fish living in the bottom layer of warm waters. It often inhabits shallow sea areas within 50 meters or coastal sea areas near the shore, and congregates at the estuary during foraging. It is a carnivorous fish, feeding on small fish such as Engraulis japonicus, shrimps, and copepods. Spawning mainly occurs along the coast and at the estuary. The spawning period is from June to August. The egg diameter is about 1 mm, without oil globules, and the yolk has no cracks. Its lifespan is only one year. Harpadon nehereus is a carnivorous fish, rich in nutrition, affordable in price, and has high edible value. In addition, Harpadon nehereus can also be used as medicine, with the effects of tonifying qi and deficiency, strengthening the spleen and kidney, nourishing the liver and stopping bleeding, and promoting diuresis and detumescence.

[0003] Collagen accounts for up to 70% of the skin. If the content of collagen in the skin decreases, the water content will gradually be lost. The decrease in the water content of the skin will lead to the loss of softness and luster of the skin, making the skin rough, thus accelerating the occurrence of skin aging. Collagen molecules contain components such as glycine, which can improve the water storage capacity of tissue cells and keep the skin moisturized. Collagen plays important roles in water retention, moisture retention, and elasticity maintenance of the skin. There has been no report on the development of collagen or collagen peptides from Harpadon nehereus as raw materials for improving skin quality. To deeply explore Harpadon nehereus and utilize its resources, the present invention is thus proposed. Summary of the Invention

[0004] The present invention provides fish collagen peptides, their compositions, and their application in the preparation of beverages for improving skin quality.

[0005] Specifically, the present invention provides a method for preparing Harpadon nehereus skin collagen peptides. The method includes: pre-treating the Harpadon nehereus skin; extracting enzymatically soluble collagen from the pre-treated Harpadon nehereus skin; enzymatically hydrolyzing the enzymatically soluble collagen to obtain an enzymatic hydrolysate; subjecting the enzymatic hydrolysate to ultrafiltration separation and collecting the fraction with a molecular weight less than 1 kD; performing Sephadex G-15 gel chromatography separation on the fraction with a molecular weight less than 1 kD, eluting and collecting the fraction with the highest antioxidant activity; performing automatic high-pressure preparative separation and purification on the fraction with the highest antioxidant activity, and collecting the eluted fraction; separating and identifying the Harpadon nehereus skin collagen peptides from the eluted fraction according to LC-MS / MS analysis.

[0006] Specifically, the steps for pre-treating the fish skin include: soaking the dried fish skin in a 0.1 M alkaline solution at a material-liquid ratio of 1:20 (m / V) and stirring the fish skin for 48 h, changing the solution 4 times in the middle to remove non-collagen substances, and washing with distilled water until neutral; then soaking the fish skin in a 0.5 M disodium ethylenediaminetetraacetate solution and stirring for 48 h for decalcification treatment, changing the solution 4 times midway, and washing several times after the treatment; then stirring the fish skin with a 15% isopropanol solution until the fat in the fish skin is removed, and then washing the isopropanol clean.

[0007] Specifically, the steps for extracting enzyme-soluble collagen include: adding the pre-treated fish skin to a 0.5 M acetic acid solution containing 0.5 wt% pepsin at a material-liquid ratio of 1:30 (m / V), stirring at 4 °C, and filtering with degreased cotton gauze; adding a sodium chloride solution to the filtrate for salting out and continuously stirring until no precipitate is washed out, centrifuging the suspension at 4 °C and 12,000 rpm, redissolving the precipitate at the bottom of the centrifuge tube in a 0.5 M acetic acid solution, and finally loading it into a dialysis bag and dialyzing until neutral, followed by freeze-drying to obtain enzyme-soluble collagen.

[0008] Specifically, the steps for enzymatic hydrolysis include: uniformly mixing 5000 U / g bromelain into distilled water of enzyme-soluble collagen, adjusting the pH to 7.0 and the temperature to 40 °C, reacting for 5 h, boiling in a water bath for 10 min to inactivate, centrifuging at 4 °C and 12,000 rpm to collect the supernatant, and freeze-drying to obtain the enzymatic hydrolysate.

[0009] Specifically, the steps for Sephadex G-15 gel chromatography separation include: preparing a solution of the fraction less than 1 kD collected with ultrapure water at a concentration of 100 mg / mL, filtering with a 0.22 μm filter membrane, statically loading 5 mL, and then eluting with ultrapure water at a flow rate of 0.2 mL / min, collecting the eluate (1 mL / tube), detecting the absorbance at 280 nm, collecting the solutions of different absorption peaks respectively, and freeze-drying and then measuring their antioxidant activities.

[0010] Specifically, the steps for the fully automatic high-pressure preparation, separation and purification include: dissolving the freeze-dried powder of the R2 fraction into a solution of 40 mg / mL, filtering with a 0.22 μm filter membrane and then loading it onto an HP Plus 10D high-pressure preparation and purification system, using a mixture of water and acetonitrile (containing 0.1% trifluoroacetic acid) as the mobile phase, with the elution gradient of the acetonitrile phase being 10% - 95%, the injection volume being 5 mL, the flow rate being 2 mL / min, the elution time being 90 min, the detection wavelength being 280 nm, collecting the elution peak, and freeze-drying.

[0011] Specifically, the steps for LC-MS / MS analysis include:

[0012] Mass spectrometry method: positive ion detection mode, primary resolution of 120,000, AGC set to 3e6, scan range of 300 - 1400 m / z; select 10 ions with the highest intensity from a single MC spectrum for MS / MS analysis, secondary resolution of 15,000, AGC set to 2e4, isolation window of 1.6 m / z;

[0013] Liquid phase method: chromatographic column is Eksigent C18 (3μm, 250mm×75μm), phase A is water + 0.1% formic acid; phase B is acetonitrile + 0.1% formic acid; flow rate is 300 nl / min, injection volume is 4 μL, chromatographic gradient elution for 80 min; elution conditions are: 0 - 16 min, 93% A → 88% A; 16 - 51 min, 88% A → 70% A; 51 - 71 min, 70% A → 58% A; 71 - 72 min, 58% A → 5% A; 72 - 80 min, 5% A;

[0014] Data processing method: the obtained results are retrieved from the NCBI database using PEAKS software; during data processing, first calculate the possible amino acid combinations based on the molecular weight information of the fragments in each MS / MS spectrum, and then compare them to the protein database of the sample source. The sample uses the protein database of the Harpadon nehereus species in NCBI.

[0015] Specifically, the present invention also provides the fish collagen peptide and its composition prepared by the described preparation method, and the amino acid sequence of the fish collagen peptide is LSHGFNK or LRGSFLR.

[0016] Specifically, the present invention also provides a preparation method for a compound drink. The formula of the compound drink includes: any one of 11.2 g / L fish collagen peptide LSHGFNK or LRGSFLR, 40 g / L kumquat juice, 8 g / L lemon juice, 64 g / L orange juice, 10 g / L granulated sugar, 0.2 g / L sodium carboxymethylcellulose, 0.25 g / L low molecular weight sodium hyaluronate, 0.04 g / L ethyl p - hydroxybenzoate.

[0017] The preparation method includes:

[0018] Selection, cleaning and juicing of fruits: Select three fresh fruits, kumquats, lemons and oranges, with normal color, strong fragrance, fully ripe, no mildew, no pests and diseases, and no other obvious adverse changes as processing raw materials. Clean the dust, impurities, microorganisms and pesticide residues on the surfaces of kumquats, lemons and oranges, peel and press the edible pulp part, and store the pressed juice refrigerated.

[0019] Dissolving sugar: Use hot water at 80℃ - 85℃, stir at high speed for 10 min until the granulated sugar is completely dissolved.

[0020] Pretreatment of other raw materials: ethyl paraben is diluted 5 to 10 times with warm water at 30 to 40 degrees Celsius, and filtered with a 200-mesh filter bag; sodium carboxymethyl cellulose, low-molecular sodium hyaluronate and white sugar are dry-mixed, and then dissolved to form a solution;

[0021] Mixing and blending: Except for fish collagen peptide, add materials and stir for 5 minutes. After adding all the materials, continue stirring until the mixture is evenly mixed, add water to make up the volume; then filter through a double filter;

[0022] Vacuum degassing: add fish collagen peptide to the filtrate, mix thoroughly, and then perform vacuum degassing. The degassing conditions are: temperature 60°C, vacuum degree 0.08MPa;

[0023] High-pressure homogenization: The homogenization pressure used is 18MPa~20MPa.

[0024] Specifically, the present invention also provides the use of the fish collagen peptide and the composition thereof in the preparation of a beverage for improving skin.

[0025] The preparation method provided by the present invention comprises the following steps: pretreating fish skin to extract enzyme-soluble collagen from the skin, enzymatically hydrolyzing the enzyme-soluble collagen, performing ultrafiltration separation, coagulation chromatography separation and full-automatic high-pressure preparative chromatography separation and purification on the enzymatic hydrolysate, and performing LC-MS / MS analysis, screening and comparison to obtain two fish collagen peptides LSHGFNK and LRGSFLR.

[0026] The composite beverage prepared by the present invention uses two kinds of fish collagen peptides as raw materials and compounding three kinds of fresh fruits, namely kumquat, lemon and orange, has good stability, is slightly yellow in color, has no browning, has uniform color, and has a uniform turbid liquid structure without suspended matter or stratification, and is allowed to have slight precipitation after standing for a long time; and has a sweet and sour taste, with the fruity fragrance of kumquat, lemon and orange, and has no fishy smell or other unpleasant smells.

[0027] The present invention has found through cytotoxicity experiments that the composite beverage and the test solution provided by the present invention are non-cytotoxic.

[0028] The present invention found through cell experiments that the composite beverage and the test solution provided by the present invention have a protective effect on UVA-stimulated HaCaT cells.

[0029] The in vivo model experiment of the present invention found that the composite drink and the test solution provided by the present invention can promote the wound healing of rats, promote the wound skin to maintain tissue vitality, inhibit the inflammatory expression of IL-1α in the wound skin, and promote the expression of barrier protein in the skin model. That is, the composite drink and the test solution provided by the present invention not only have the function of healing the skin, but also have the effects of anti-inflammatory and improving the skin barrier. Brief Description of the Drawings

[0030] Figure 1 It is the purification diagram of Sephadex G-15 chromatography.

[0031] Figure 2 It is the chromatogram of fully automatic high-pressure preparative separation and purification.

[0032] Figure 3 It is the secondary mass spectrum diagram of LSHGFNK.

[0033] Figure 4 It is the secondary mass spectrum diagram of LRGSFLR.

[0034] Figure 5 It is the result diagram of antioxidant test for intervening HaCaT cells stimulated by UVA with different compound drinks or test samples. "*" indicates P < 0.05 compared with the model group, "**" indicates P < 0.01 compared with the model group, and "#" indicates P < 0.05 compared with both the model group and the control group.

[0035] Figure 6 It is the result diagram of the healing rate and wound skin tissue viability of intervening wound rats with different compound drinks or test samples. "*" indicates P < 0.05 compared with the model group, "**" indicates P < 0.01 compared with the model group, and "#" indicates P < 0.05 compared with both the model group and the control group.

[0036] Figure 7 It is the result diagram of the inflammatory expression and skin model barrier protein of intervening wound rats with different compound drinks or test samples. "*" indicates P < 0.05 compared with the model group, "**" indicates P < 0.01 compared with the model group, and "#" indicates P < 0.05 compared with both the model group and the control group. Detailed Description of the Preferred Embodiments

[0037] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. The reagents not specifically described in detail in the present invention are all conventional reagents and can be obtained from commercial channels; the methods not specifically described in detail are all conventional experimental methods and can be known from the prior art.

[0038] 1. Pretreatment of fish skin

[0039] Take the fish skin, dry it in the sun, stir the fish skin with 0.1 M alkali solution at a material-liquid ratio of 1:20 (m / V) for 48 h, change the solution 4 times in the middle to remove non-collagen substances, and wash it with distilled water until neutral. Then soak the fish skin in 0.5 M disodium ethylenediaminetetraacetate solution and stir for 48 h for decalcification treatment, change the solution 4 times midway, and wash it several times after treatment. Then stir the fish skin with 15% isopropanol solution until the fat in the fish skin is removed, and then wash the isopropanol clean. The whole experimental process is carried out at 4 °C.

[0040] 2. Extraction of enzymatically soluble collagen

[0041] Add 0.5 M acetic acid solution (containing 0.5 wt% pepsin, CAS: 9001-75-6, product number: P6322; purity: >3000 U / mg; Macklin) at a material-liquid ratio of 1:30 (m / V) and stir at 4 °C for one day to extract enzymatically soluble collagen. After the extraction, filter it with degreased cotton gauze, extract it twice repeatedly according to the above method, and combine the filtrates. Add sodium chloride solution to the filtrate for salting out and stir continuously until no precipitate is washed out. Centrifuge the suspension at 4 °C and 12000 rpm. The precipitate at the bottom of the centrifuge tube is redissolved in 0.5 M acetic acid solution, and finally filled into a dialysis bag and dialyzed until neutral, and then freeze-dried to obtain enzymatically soluble collagen.

[0042] 3. Enzymatic hydrolysis

[0043] Take 100 g of enzymatically soluble collagen, add 5 L of distilled water and mix evenly. Add different enzymes at 5000 U / g of enzymatically soluble collagen, and adjust the pH and temperature conditions to the optimal conditions of the corresponding enzymes. Bromelain, CAS: 37189-34-7, brand: Macklin; product number: B832358; purity: BR, 300 U / mg, enzymatic hydrolysis temperature 40 °C, enzymatic hydrolysis pH 7.0. Papain; CAS: 9001-73-4; Ron reagent; product number: R096816; purity: ≥3 units / mg; enzymatic hydrolysis temperature 25 °C, enzymatic hydrolysis pH 6.2. Neutral protease, CAS: 9068-59-1; Macklin; product number: D832687; purity: BR, 50 u / mg, enzymatic hydrolysis temperature 40 °C, enzymatic hydrolysis pH 7.0. Alkaline protease, CAS: 9014-01-1; brand: Ron reagent; product number: R017175; Purity: 200 u / mg, enzymatic hydrolysis temperature 37 °C, enzymatic hydrolysis pH 10.0.

[0044] After reacting for 5 h in the enzymatic hydrolysis reaction system with different enzymes added under their corresponding enzymatic hydrolysis temperature and enzymatic hydrolysis pH conditions, boil it in a water bath for 10 min to inactivate, centrifuge at 4 °C and 12000 rpm to take the supernatant, and freeze-dry to obtain the enzymatically hydrolyzed product.

[0045] The determination of the peptide yield in the hydrolysate was carried out by the Folin-Ciocalteu method. The polypeptide yield % = v × c × 100%; where v is the volume of the enzyme hydrolysate (mL); c is the polypeptide concentration in the enzyme hydrolysate (mg / mL); a is the protein content in the raw material (%). The results showed that the yields of hydrolysis with bromelain, papain, neutral protease, and alkaline protease were 67.3%, 34.8%, 19.3%, and 23.5% in turn.

[0046] 4. Ultrafiltration separation

[0047] After preparing a solution with a concentration of 25 mg / mL with ultrapure water from the hydrolysate obtained by hydrolysis with bromelain, an ultrafiltration tube with a molecular weight cut-off of 1 kDa was used, and centrifuged at 4 °C and 5000 g for 60 min. The fraction with a molecular weight < 1 kDa was collected, freeze-dried, and its antioxidant activity was measured.

[0048] 5. Gel chromatography separation

[0049] Sephadex G-15 gel was packed into a column (column volume 50 mL, column diameter 1.5 cm). The fraction collected with a molecular weight less than 1 kDa was prepared into a solution with a concentration of 100 mg / mL with ultrapure water, filtered through a 0.22 μm filter membrane, statically loaded with 5 mL, and then eluted with ultrapure water at a flow rate of 0.2 mL / min. The eluate was collected (1 mL / tube), and the absorbance was measured at 280 nm. Solutions with different absorption peaks were collected respectively, freeze-dried, and their antioxidant activity was measured.

[0050] DPPH radical scavenging rate activity detection: 100 μL of each tube of sample and 100 μL of DPPH solution (0.2 mM) were added to a 96-well plate, reacted in the dark for 30 min, and then the absorbance was measured at 517 nm. The DPPH radical scavenging rate % = 1(1 - (A sample - A control) / A blank group) × 100%. The group without adding the sample was used as the control group, and the group adding distilled water was used as the blank.

[0051] The fraction with a molecular weight less than 1 kDa was purified by Sephadex G-15 chromatography to obtain 3 absorption peaks, S1 - S3, and their DPPH radical scavenging rates were 28.5%, 43.2%, and 31.% in turn.

[0052] 6. Automatic high-pressure preparation separation and purification

[0053] The R2 component was separated using the HP Plus 10D high-pressure preparation and purification system of LISUI Technology (Suzhou) Co., Ltd. The lyophilized powder of the R2 component was formulated into a 40 mg / mL solution. After filtration through a 0.22 μm filter membrane, a mixed solution of water and acetonitrile (containing 0.1% trifluoroacetic acid) was used as the mobile phase. The elution gradient of the acetonitrile phase was 10% - 95%, the injection volume was 5 mL, the flow rate was 2 mL / min, the elution time was 90 min, the detection wavelength was 280 nm, the elution peaks were collected, and then freeze-dried.

[0054] As Figure 2 shown, a relatively large absorption peak appeared around 52 min. Therefore, the eluted components of this absorption peak were collected multiple times, combined, and then freeze-dried.

[0055] 7. LC-MS / MS Analysis

[0056] The lyophilized sample prepared above was sent to Shanghai Jiyun Biotechnology Co., Ltd. for peptide sequence detection. After dissolving the sample with 0.1% formic acid, centrifuging, desalting with C18 and freeze-drying, mass spectrometry detection was carried out. Mass spectrometry method: positive ion detection mode, the first-order resolution was 120,000, the AGC was set to 3e6, and the scanning range was 50 - 1000 m / z. 10 ions with the highest intensity were selected from a single MC spectrum for MS / MS analysis, the second-order resolution was 15,000, the AGC was set to 2e4, and the isolation window was 1.6 m / z. Liquid phase method: The chromatographic column was Eksigent C18 (3 μm, 250 mm × 75 μm), phase A was water + 0.1% formic acid; phase B was acetonitrile + 0.1% formic acid; the flow rate was 300 nl / min, the injection volume was 4 μL, and the chromatographic gradient elution was for 80 min. The elution conditions were: 0 → 16 min, 93% A → 88% A; 16 → 51 min, 88% A → 70% A; 51 → 71 min, 70% A → 58% A; 71 → 72 min, 58% A → 5% A; 72 → 80 min, 5% A.

[0057] Data processing: The obtained results were retrieved from the NCBI database using PEAKS software. During the data processing, first, the possible amino acid combinations were calculated based on the molecular weight information of the fragments in each MS / MS spectrum, and then compared to the protein database of the sample source. The sample used the protein database of the Harpadon nehereus species in NCBI.

[0058] There were two peptide chains that were completely matched, and the amino acid sequences were LSHGFNK (SEQ ID NO.1) and LRGSFLR (SEQ ID NO.2), which were fish collagen peptides.

[0059] 8. Preparation of the Composite Beverage

[0060] (1) Formulation

[0061] The formula of the composite beverage provided by the present invention comprises: 11.2 g / L of the last one of fish collagen peptide LSHGFNK or LRGSFLR, 40 g / L of kumquat juice, 8 g / L of lemon juice, 64 g / L of orange juice, 10 g / L of white sugar, 0.2 g / L of sodium carboxymethyl cellulose, 0.25 g / L of low molecular weight sodium hyaluronate (molecular weight 1.0 MDa to 1.8 MDa, CAS No.: 9067-32-7, MDL No.: MFCD00131348, McLean), and 0.04 g / L of ethyl parahydroxybenzoate.

[0062] (2) Preparation of compound beverages

[0063] Fruit selection, cleaning and juicing: Select kumquat, lemon and orange fruits with normal color, strong fragrance, full maturity, no mold, no pests and no other obvious adverse changes as processing raw materials. Use running water to clean the dust, impurities, microorganisms and pesticide residues on the surface of kumquat, lemon and orange. Use a knife to remove the peel and seeds, then peel manually, put the edible pulp into the juicer for squeezing, and store the squeezed juice in a refrigerator.

[0064] Dissolving sugar: Use 80℃~85℃ hot water to dissolve a portion of the white sugar, and stir at high speed for 10 minutes until the white sugar is completely dissolved.

[0065] Pretreatment of other raw materials: Ethyl p-hydroxybenzoate is diluted 5 to 10 times with warm water at 30 to 40 degrees Celsius, and filtered with a 200-mesh filter bag. Sodium carboxymethyl cellulose, low-molecular sodium hyaluronate and white sugar are dry-mixed, and then dissolved to form a uniform and stable solution.

[0066] Mixing and blending: The dosage of compound juice is 10%, ethyl paraben is 0.04g / L, and the dosage of other raw materials is determined according to the formula. Add the materials (except fish collagen peptide) and stir, each interval is 5 minutes. After the raw materials are added, continue to stir until the mixture is evenly mixed, add water to make up the volume. Then filter through a double filter.

[0067] Vacuum degassing: add fish collagen peptide to the filtrate, mix thoroughly, and then perform vacuum degassing. The degassing conditions are: temperature 60°C, vacuum degree 0.08MPa.

[0068] High-pressure homogenization: The homogenization pressure used is 18MPa~20MPa, so that the pectin in the juice can be fully dissolved in the juice, the affinity between pectin and juice can be increased, the stability of the juice can be improved, the slurry can be prevented from stratification and precipitation, and the texture can be uniformly viscous with a delicate taste.

[0069] Sterilization, filling and cooling: The prepared fruit juice is sterilized by a UHT ultra-high temperature instantaneous sterilizer, the temperature is rapidly raised to 110 °C and maintained for 15 s, then rapidly cooled to 80 °C after sterilization, filled into a thoroughly washed and sterilized glass bottle with an automatic filling machine and quickly capped. After inverting the bottle for 1 min, it is spray-cooled using a tunnel until it reaches 30 °C.

[0070] Test group 1: Perform the same steps of high-pressure homogenization, sterilization, filling and cooling on a distilled water solution containing 11.2 g / L LSHGFNK as described above.

[0071] Test group 2: Perform the same steps of high-pressure homogenization, sterilization, filling and cooling on a distilled water solution containing 11.2 g / L LRGSFLR as described above.

[0072] Test group 3: Perform the same steps of high-pressure homogenization, sterilization, filling and cooling on a distilled water solution containing 7.8 g / L of LSHGFNK and 4.4 g / L of LRGSFLR as described above.

[0073] (3) Determination of the stability of the compound beverage

[0074] Add 20 mL of the product prepared with different stabilizers to a graduated centrifuge tube, place it in a centrifuge, centrifuge at 3000 r / min for 10 min, quickly aspirate the supernatant, dilute it, and measure the absorbance I after centrifugation at a wavelength of 750 nm using a UV-visible spectrophotometer. Accurately record the absorbance of the beverage before and after centrifugation, measure each sample 3 times, and take the average value. The calculation formula for stability is I = I 后 / I 前 ; where: I - stability, I 前 - absorbance before centrifugation, I 后 is the absorbance after centrifugation. The larger the I value, the better the stability; otherwise, the stability is worse. An I value greater than 95% indicates good stability. Through testing, it was found that the stabilities of the compound beverage (LSHGFNK as a component of fish collagen peptide), test group 1, test group 2, and test group 3 were 96.7%, 100.2%, 100.3%, and 99.8% respectively, indicating that the stabilities of these beverages were good. Moreover, the compound beverage had a slightly yellow color, no browning, and a uniform color; tissue state: it was a uniform turbid liquid, without suspended matter, no stratification, and slight precipitation was allowed after standing for a long time; taste and smell: it had a sour and sweet taste, with the fruity fragrance of kumquat, lemon, and orange, without fishy smell or other unpleasant odors. The solutions of test groups 1 - 3 were transparent and colorless, without fishy smell or other unpleasant odors.

[0075] 9. Cytotoxicity test

[0076] For the evaluation of cytotoxic activity, human gastric mucosal epithelial cells (GMEC, product number PCLH0022-RT, Bihe Biotech) and primary human intestinal mucosal epithelial cells (hIMEC, catalog number Delf-10785, Hefei Wanwu Biology Co., Ltd.) in good logarithmic growth state were selected and seeded in 96-well culture plates at a density of 2×10 4 cells / mL and cultured overnight in an environment of 37°C and 5% CO2 by volume. Sample solutions (compound drink (LSHGFNK as a component of fish collagen peptide), test group 1, test group 2, test group 3) with a final concentration of 10 mg / mL in the wells were added respectively. Seven different mass concentrations were set for each sample, and five parallels were set for each concentration. The cell control group was not treated and cultured for 48 h. 10 μL of CCK-8 solution was added to each well. After culturing for 2.5 h, the absorbance value of each well was measured at 450 nm. Sterile water with the same volume was added as a control. The blank group was the wells without adding any samples.

[0077] The cell survival rate was calculated as = (A sample group - A blank group) / (A control group - A blank group) × 100%. As a result, the cell survival rates in the compound drink, test group 1, test group 2, and test group 3 were not less than 95%, indicating that the compound drink provided by the present invention and the fish collagen peptide do not have toxic effects on gastrointestinal mucosal cells and have high safety.

[0078] 10. Has a protective effect on UVA-stimulated HaCaT cells

[0079] Human HaCaT cells (Shanghai Guyan Industry) in good logarithmic growth phase were counted and seeded in 10-cm culture dishes, with 5×10 6 cells per dish. After culturing overnight at 37°C and 5% CO2 by volume, the culture medium was discarded, and the compound drink (LSHGFNK as a component of fish collagen peptide), test group 1, test group 2, and test group 3 at a concentration of 1 mg / mL were added to act on the cells for 6 h (the model group and the blank control group were added with serum-free DMEM culture medium). Phosphate buffer solution (PBS) (pH = 7.4) was added to just cover the cells. The radiation dose was 7 J / cm 2UVA stimulates cells and irradiates them for 2 h to serve as the model group, while the blank group is not irradiated. Three parallels are made for each sample (the results of each parallel sample are detected three times), and the samples are taken out and placed on ice, washed twice with PBS; 800 μL of lysis buffer is added to lyse the cells, and the cells are centrifuged at 12,000 r / min at 4 °C for 5 min, and the supernatant is taken to obtain the cell lysate supernatant. The detection of the total antioxidant capacity (ABTS method), the activity of glutathione peroxidase (GSH-Px), and the lipid oxidation activity in the samples are all carried out according to the instructions of the kit. Subsequently, the MMP-1 ELISA kit and the CoL-1 ELISA kit are used to detect the contents of MMP-1 and COL-1 in the cells, and protein correction is performed.

[0080] As Figure 5 shown, different compound drinks or test products are used to intervene in UVA-stimulated HaCaT cells. The results show that 1 mg / mL of the compound drink or test product can intervene in UVA-stimulated HaCaT cells, improve the total antioxidant capacity of the cells, reduce the increase in glutathione peroxidase activity and MDA content caused by UVA stimulation. In addition, it can also increase the decrease in the expression levels of MMP-1 and COL-1 caused by UVA stimulation.

[0081] 11. In vivo model experiment

[0082] (1) Experimental animals

[0083] Healthy male Wistar rats, 8 weeks old, weighing 250 ± 20 g, are provided by Beijing Beiyou Biotechnology Co., Ltd.; the animals are fed for 10 d for adaptation before participating in the experiment, fed with ordinary feed, and allowed to drink water freely. The breeding environment temperature is (22 ± 1) °C, and the relative humidity is 50% - 70%.

[0084] (2) Normal rat skin ulcer model

[0085] After 10 d of adaptive feeding, the rats are anesthetized by intraperitoneal injection of 7% chloral hydrate, and the hair on the left side of the dorsal midline skin is removed cleanly using an electric clipper and depilatory cream. According to the full-thickness skin defect standard, a circular skin defect wound with a diameter of 20 mm is marked on the rat skin, and a circular skin defect wound is prepared with surgical scissors, reaching deep to the fascia.

[0086] (3) Drug administration

[0087] After the successful establishment of the model, the rats were singly caged, allowed free access to water and food, and then divided into an experimental group and a drug administration group. Rats in the drug administration group were intragastrically administered 20 mL of the sample test groups (Compound drink (LSHGFNK as a component of fish collagen peptide), Test1, Test2, Test3, and Compound drink1) every day, while rats in the model group were intragastrically administered an equal amount of normal saline. At the same time, a control group was set up, with free diet, to make a skin ulcer model and intragastrically administered an equal amount of normal saline.

[0088] Among them, the formula of Compound drink1 is: 40 g / L of kumquat juice, 8 g / L of lemon juice, 64 g / L of orange juice, 10 g / L of granulated sugar, 0.2 g / L of sodium carboxymethylcellulose, 0.25 g / L of low-molecular-weight sodium hyaluronate, and 0.04 g / L of ethyl p-hydroxybenzoate. Its preparation method is the same as above.

[0089] (4) Wound healing

[0090] After drug intervention on rats in each group, the ulcer wounds of the rats were photographed with a camera on the 7th day, and the wound surface was processed with image software to obtain the wound area. The wound healing rate was calculated using the following formula. Wound healing rate = (original wound area - unhealed wound area) / original wound area × 100%. As Figure 4 shown, after drug intervention on rats in each group, the healing conditions of the wound surfaces of rats in each group were different. Among them, the compound drink, Test Group 1, Test Group 2, and Test Group 3 had better healing conditions.

[0091] (5) Detection of wound skin tissue viability

[0092] Take the wound skin tissue of rats in each group, wash it and transfer it to a 24-well plate. Add 0.3 mL of MTT working solution (1 mg / mL) to each well, and incubate it in an incubator (37 °C, 5% CO2 by volume) for (3 h ± 5 min). After incubation, wash the outer surface of the model with PBS and dry it with a sterile cotton swab, then transfer it to a new 24-well plate. Seal it with a sealing film after adding 2 mL of isopropanol, shake it with a plate shaker for 2 h, pierce the model with a 200 μL pipette tip to make the isopropanol extract flow out from the wound skin tissue, and pipette and mix well. Then, pipette 2 aliquots of 200 μL of the isopropanol extract from each well into a 96-well plate. Use isopropanol as the zero-adjustment well, and read the absorbance (A) at a wavelength of 570 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the relative tissue viability of each test group according to the following formula: Tissue viability = 100% × (A drug administration well - A zero-adjustment well) / (A control well - A zero-adjustment well). As Figure 4 shown, after reculturing the wound skin tissue of rats in each group, the compound drink, Test Group 1, Test Group 2, and Test Group 3 had higher skin tissue viability, and Test Group 3 had the highest skin tissue viability.

[0093] (6) Detection of inflammatory factor expression in skin model

[0094] Collect the culture medium of the wound skin in a centrifuge tube, and then place the samples for ELISA detection in a -80 °C refrigerator for frozen storage. Detection and analysis are carried out according to the operation manuals of the IL-1α ELISA kit, TNFα ELISA kit, and PGE2 ELISA kit. The results are as Figure 6 shown. After drug intervention on rats in each group, the inflammatory expression of IL-1α in the wound skin was inhibited, while there was no obvious inhibitory effect on TNFα and PGE2. In addition, the compound drink, Test Group 1, Test Group 2, and Test Group 3 could significantly promote the inflammatory expression of IL-1α in the wound skin, while the compound drink 1 had no obvious effect.

[0095] (7) Detection of barrier protein expression in skin model

[0096] Collect the culture medium of the wound skin in a centrifuge tube. After collection, fix the model for FLG detection with 4% paraformaldehyde by mass fraction for 24 h, then cut the model off in a circular shape and observe the FLG under a confocal microscope. Scan under a laser confocal microscope (Leica, Germany), the emission wavelength of blue light is 461 nm, the emission wavelength of red light is 658 nm, the resolution is 553×463 pixels, and computer data acquisition is carried out. The relative protein quantification of the positive signal fluorescence is calculated and analyzed with Image J image analysis software. Randomly scan 5 layers under the microscope for 4 samples of each research object, and the computer calculates the ratio of its average area to the integrated optical density as the relative expression level of FLG. The results are as Figure 7 shown. After drug intervention on rats in each group, the compound drink, Test Group 1, Test Group 2, and Test Group 3 could significantly promote the expression of FLG in the wound skin, while the compound drink 1 had no obvious promoting effect.

[0097] As described above, it is only the preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A fish collagen peptide, wherein the amino acid sequence of the fish collagen peptide is LRGSFLR.

2. A compound beverage, the formula of the compound beverage comprising: 11.2 g / L of the fish collagen peptide LRGSFLR described in claim 1, 40 g / L kumquat juice, 8 g / L lemon juice, 64 g / L orange juice, 10 g / L white sugar, 0.2 g / L sodium carboxymethyl cellulose, 0.25 g / L low molecular weight sodium hyaluronate, and 0.04 g / L ethyl p-hydroxybenzoate.

3. The compound beverage according to claim 2, wherein The preparation method comprises: Selection, cleaning and juicing of fruits: Select kumquat, lemon and orange fruits with normal color, strong fragrance, full maturity, no mold, no pests and diseases, and no other obvious adverse changes as processing raw materials, clean the dust, impurities, microorganisms and pesticide residues on the surface of kumquat, lemon and orange, peel and squeeze the edible pulp, and refrigerate and store the squeezed juice; Dissolving sugar: Use 80℃~85℃ hot water, stir at high speed for 10min, and the sugar will be completely dissolved; Pretreatment of other raw materials: ethyl paraben is diluted 5 to 10 times with warm water at 30 to 40 degrees Celsius, and filtered with a 200-mesh filter bag; sodium carboxymethyl cellulose, low-molecular sodium hyaluronate and white sugar are dry-mixed, and then dissolved to form a solution; Mixing and blending: Except for fish collagen peptide, add materials and stir for 5 minutes. After adding all the materials, continue stirring until the mixture is evenly mixed, add water to make up the volume; then filter through a double filter; Vacuum degassing: add fish collagen peptide to the filtrate, mix thoroughly, and then perform vacuum degassing. The degassing conditions are: temperature 60°C, vacuum degree 0.08MPa; High-pressure homogenization: The homogenization pressure used is 18MPa~20MPa.

4. Use of the fish collagen peptide according to claim 1 in preparing a drink for improving skin.

5. The preparation method of the fish skin collagen peptide according to claim 1, characterized in that, include: Pre-treating the fish skin; Extracting enzyme-soluble collagen from the pretreated fish skin; hydrolyzing the enzyme-soluble collagen to obtain an enzymatic hydrolysate; The enzymatic hydrolysate is subjected to ultrafiltration separation to collect components with a molecular weight less than 1 kD; Separating the components with a molecular weight less than 1 kD by Sephadex G-15 gel chromatography, and eluting and collecting the components with the highest antioxidant activity; Performing fully automatic high-pressure preparative separation and purification on the component with the highest antioxidant activity, and collecting the eluted components; The fish skin collagen peptide was separated and identified from the eluted fractions according to LC-MS / MS analysis.

6. The preparation method according to claim 5, characterized in that, The step of pre-treating the fish skin comprises: The dried fish skin was stirred with 0.1M alkaline solution at a material-liquid ratio of 1:20 (m / V) for 48 hours, and the solution was changed 4 times in the middle to remove non-collagenous substances, and washed with distilled water until neutral; Then, the fish bones were immersed in a 0.5M disodium ethylenediaminetetraacetic acid solution and stirred for 48 hours for decalcification. The solution was changed 4 times in the middle and washed several times after treatment. The fish bones were then stirred with a 15% isopropanol solution until the fat in the fish bones was removed, and then the isopropanol was washed clean.

7. The preparation method according to claim 6, characterized in that, The steps of extracting enzymatically soluble collagen include: The pre-treated fish skin was added to a 0.5 M acetic acid solution containing 0.5 wt% pepsin at a material-liquid ratio of 1:30 (m / V), stirred at 4°C, and filtered through degreased cotton gauze. Sodium chloride solution was added to the filtrate for salting out and continuously stirred until no precipitate was washed out. The suspension was centrifuged at 4°C and 12,000 rpm. The precipitate at the bottom of the centrifuge tube was redissolved in 0.5 M acetic acid solution, and finally dialyzed in a dialysis bag until neutral, and freeze-dried to obtain enzymatically soluble collagen.

8. The preparation method according to claim 7, characterized in that, The steps of enzymatic hydrolysis include: 5000 U / g bromelain was uniformly mixed into the enzymatically soluble collagen in distilled water, the pH was adjusted to 7.0, and the temperature was 40°C. After reacting for 5 h, it was inactivated by boiling in a water bath for 10 min, centrifuged at 4°C and 12,000 rpm to obtain the supernatant, and freeze-dried to obtain the enzymatic hydrolysate.

9. The preparation method according to claim 8, characterized in that The steps of Sephadex G-15 gel chromatography separation include: The fraction less than 1 kD collected was prepared into a 100 mg / mL solution with ultrapure water, filtered through a 0.22 μm filter membrane, statically loaded with 5 mL, and then eluted with ultrapure water at a flow rate of 0.2 mL / min. The eluate was collected (1 mL / tube), and the absorbance was detected at 280 nm. The solutions with different absorption peaks were collected respectively, and their antioxidant activities were determined after freeze-drying.

10. The preparation method according to claim 9, characterized in that, The steps of the automatic high-pressure preparation separation and purification include: The freeze-dried powder of the R2 fraction was prepared into a 40 mg / mL solution, filtered through a 0.22 μm filter membrane, and then loaded onto the HP Plus 10D high-pressure preparation purification system. A mixed solution of water and acetonitrile (containing 0.1% trifluoroacetic acid) was used as the mobile phase. The elution gradient of the acetonitrile phase was 10% - 95%, the injection volume was 5 mL, the flow rate was 2 mL / min, the elution time was 90 min, the detection wavelength was 280 nm, the eluate was collected, and freeze-dried.

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