Fish collagen peptide, preparation method and application of fish collagen peptide in preparation of functional drink for improving skin

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 sequences of the fish collagen peptide are LSHGFNK and 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 invention relates to the technical field of fish collagen, and in particular to fish collagen peptides from fish skin and a composition thereof, and application thereof in preparing a drink for improving skin. Background Art

[0002] Harpadon nehereus, scientific name. Harpadon is a small to medium-sized warm-water bottom-dwelling fish that often inhabits shallow seas within 50 meters or nearshore seas. It gathers in estuaries when foraging. It is a carnivorous fish that feeds on small fish such as anchovies, shrimps, and copepods. It mainly lays eggs along the coast and at river mouths. The spawning period is from June to August. The diameter of the eggs is about 1 mm. There are no oil balls and the yolk has no cracks. The lifespan is only one year. Harpadon is a carnivorous fish with rich nutrition, affordable price and high edible value. In addition, Harpadon can also be used as medicine, which has the effects of replenishing qi and deficiency, strengthening the spleen and kidney, nourishing the liver and stopping bleeding, and diuresis and swelling.

[0003] The collagen content in the skin is as high as 70%. If the collagen content in the skin decreases, the moisture content will gradually be lost. The decrease in the moisture content of the skin will cause the skin to lose its softness and luster, and make the skin rough, thereby 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 has important functions for the skin, such as water retention, moisture retention and elasticity maintenance. There is no report on the development of collagen or collagen peptide from fish as a raw material for improving skin. In order to deeply explore fish and utilize its resources, the present invention is proposed. Summary of the invention

[0004] The invention provides fish collagen peptide and a composition thereof and application of the peptide in preparing a drink for improving skin.

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

[0006] Specifically, the step of pretreating the fish skin comprises: stirring the dried fish skin with a 0.1M alkaline solution at a material-liquid ratio of 1:20 (m / V) for 48 hours, changing the solution 4 times in the middle to remove non-collagenous substances, and washing with distilled water until neutral; then immersing the fish skin in a 0.5M disodium ethylenediaminetetraacetic acid solution and stirring for 48 hours for decalcification treatment, changing the solution 4 times in the middle, and washing several times after the treatment; then stirring the fish skin with a 15% isopropanol solution until fat in the fish skin is removed, and then washing the isopropanol clean.

[0007] Specifically, the step of extracting enzyme-soluble collagen comprises: adding the pretreated fish skin to a 0.5M acetic acid solution containing 0.5wt% of pepsin at a material-liquid ratio of 1:30 (m / V), stirring at 4°C, and filtering with absorbent cotton gauze; adding 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 12000rpm, redissolving the precipitate at the bottom of the centrifuge tube in a 0.5M acetic acid solution, and finally putting it into a dialysis bag for dialysis to neutrality, and freeze-drying to obtain enzyme-soluble collagen.

[0008] Specifically, the enzymatic hydrolysis step includes: uniformly mixing 5000U / g bromelain in enzyme-soluble collagen distilled water, adjusting the pH to 7.0 and the temperature to 40°C, reacting for 5 hours, boiling in a water bath for 10 minutes to inactivate, centrifuging at 4°C and 12000rpm to obtain the supernatant, and freeze-drying to obtain the enzymatic hydrolysate.

[0009] Specifically, the steps of Sephadex G-15 gel chromatography separation include: preparing the collected components less than 1 kD into a 100 mg / mL solution with ultrapure water, filtering with a 0.22 μm filter membrane, statically loading 5 mL and 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 solutions with different absorption peaks, and determining their antioxidant activity after freeze-drying.

[0010] Specifically, the steps of the fully automatic high-pressure preparation, separation and purification include: preparing the lyophilized powder of the R2 component into a 40 mg / mL solution, filtering it with a 0.22 μm filter membrane and loading it into 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, the elution gradient of the acetonitrile phase is 10% to 95%, the injection volume is 5 mL, the flow rate is 2 mL / min, the elution time is 90 min, the detection wavelength is 280 nm, the elution peak is collected, and freeze-dried.

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

[0012] Mass spectrometry method: positive ion detection mode, primary resolution of 120000, AGC setting of 3e6, scanning range of 300-1400 m / z; 10 grids with the highest intensity ions were selected from a single MC spectrum for MS / MS analysis, secondary resolution of 15000, AGC setting of 2e4, and separation window of 1.6 m / z;

[0013] 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 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;

[0014] Data processing method: The results were retrieved from the NCBI database using PEAKS software. During the data processing, the possible amino acid combinations were first 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.

[0015] Specifically, the present invention also provides fish collagen peptides prepared by the preparation method and a composition thereof, wherein the amino acid sequence of the fish collagen peptides is LSHGFNK or LRGSFLR.

[0016] Specifically, the present invention also provides a method for preparing a composite drink. The formula of the composite drink includes: 11.2 g / L of either fish collagen peptide LSHGFNK or LRGSFLR, 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 paraben.

[0017] The preparation method comprises:

[0018] 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;

[0019] Dissolving sugar: Use 80℃~85℃ hot water, stir at high speed for 10min, and the sugar will be 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 This is a Sephadex G-15 chromatography purification diagram.

[0031] Figure 2 This is a chromatogram of fully automated high-pressure preparative separation and purification.

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

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

[0034] Figure 5 The results of the antioxidant test of different compound drinks or test products on UVA-stimulated HaCaT cells. "*" 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 The results of the healing rate and wound skin tissue activity of rats intervened by different compound drinks or test products. "*" 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 The results of the intervention of different compound drinks or test products on the inflammatory expression and skin model barrier protein in rats with wounds. "*" 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

[0037] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The reagents not described separately in detail in the present invention are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained 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 0.1M alkaline solution at a material-liquid ratio of 1:20 (m / V) for 48 hours, change the solution 4 times in the middle to remove non-collagenous substances, and wash it with distilled water until it is neutral. Then soak the fish skin in a 0.5M disodium ethylenediaminetetraacetic acid solution and stir it for 48 hours for decalcification. Change the solution 4 times in the middle and wash it several times after the treatment. Then stir the fish skin with 15% isopropanol solution until the fat in the fish skin is removed, and then clean the isopropanol. The entire experimental process is carried out at 4°C.

[0040] 2. Extraction of enzyme-soluble collagen

[0041] Add 0.5M acetic acid solution (containing 0.5wt% pepsin, CAS: 9001-75-6, product number: P6322; purity: >3000U / mg; McLean) at a solid-liquid ratio of 1:30 (m / V) and stir at 4°C for one day to extract enzyme-soluble collagen. After the extraction, filter with absorbent cotton gauze, repeat the extraction twice according to the above method, and combine the filtrate. 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 12000rpm, and redissolve the precipitate at the bottom of the centrifuge tube in 0.5M acetic acid solution. Finally, put it into a dialysis bag and dialyze it to neutrality, and freeze-dry it to obtain enzyme-soluble collagen.

[0042] 3. Enzymatic hydrolysis

[0043] Take 100g of enzyme-soluble collagen, add 5L of distilled water and mix evenly, add different enzymes of 5000U / g enzyme-soluble collagen respectively, and adjust the pH and temperature conditions to the optimal conditions of the corresponding enzymes. Bromelain, CAS: 37189-34-7, brand: McLean; product number: B832358; purity: BR, 300U / mg, enzymatic hydrolysis temperature 40℃, enzymatic hydrolysis pH7.0. Papain; CAS: 9001-73-4; Ron's reagent; product number: R096816; purity: ≥3units / mg; enzymatic hydrolysis temperature 25℃, enzymatic hydrolysis pH6.2. Neutral protease, CAS: 9068-59-1; McLean; product number: D832687; purity: BR, 50u / mg, enzymatic hydrolysis temperature 40℃, enzymatic hydrolysis pH7.0. Alkaline protease, CAS: 9014-01-1; Brand: Ron's Reagent; Product No.: R017175; Purity: 200u / mg, enzymatic hydrolysis temperature 37℃, enzymatic hydrolysis pH 10.0.

[0044] According to the enzymatic hydrolysis reaction system with different enzymes added, the reaction was carried out for 5 hours under the corresponding enzymatic hydrolysis temperature and enzymatic hydrolysis pH conditions, and then the reaction was inactivated by boiling in a water bath for 10 minutes, and the supernatant was obtained by centrifugation at 4°C and 12000 rpm, and the enzymatic hydrolysate was obtained by freeze-drying.

[0045] The peptide yield in the hydrolysate was determined by the Folin phenol method. Peptide yield % = v × c × 100%; where v is the volume of the hydrolysate (mL); c is the peptide concentration of the hydrolysate (mg / mL); a is the protein content in the raw material (%). The results show that the yields of bromelain, papain, neutral protease, and alkaline protease are 67.3%, 34.8%, 19.3%, and 23.5%, respectively.

[0046] 4. Ultrafiltration separation

[0047] The enzymatic hydrolysate obtained by using bromelain was prepared into a solution with a concentration of 25 mg / mL using ultrapure water, and then centrifuged at 4°C and 5000g for 60 minutes using a 1 kD ultrafiltration tube to collect the components with a molecular weight of less than 1 kD. The components were freeze-dried and their antioxidant activity was determined.

[0048] 5. Gel chromatography separation

[0049] Sephadex G-15 gel was filled into a column (column volume of 50 mL, column diameter of 1.5 cm). The collected components less than 1 kD were prepared into a 100 mg / mL solution with ultrapure water, filtered with a 0.22 μm filter membrane, and then 5 mL was statically loaded and eluted with ultrapure water at a flow rate of 0.2 mL / min. The eluate was collected (1 mL / tube), the absorbance was detected at 280 nm, and the solutions with different absorption peaks were collected respectively, and their antioxidant activity was determined after freeze-drying.

[0050] DPPH free radical scavenging activity test: Take 100 μL of sample and 100 μL of DPPH solution (0.2 mM) from each tube and add them to a 96-well plate. After reacting in the dark for 30 minutes, measure the absorbance at 517 nm. DPPH free radical scavenging rate % = 1 (1-(A sample-A control) / A blank group) × 100%. The control group was taken without adding sample, and the blank group was taken with adding distilled water.

[0051] The fraction less than 1 kD was purified by Sephadex G-15 chromatography to obtain three absorption peaks, S1~S3, whose DPPH free radical scavenging rates were 28.5%, 43.2% and 31.% respectively.

[0052] 6. Fully 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 prepared into a 40 mg / mL solution, filtered with a 0.22 μm filter membrane, and a mixture of water and acetonitrile (containing 0.1% trifluoroacetic acid) was used as the mobile phase, the elution gradient of the acetonitrile phase was 10% to 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, and the elution peak was collected and freeze-dried.

[0054] like Figure 2 As shown, a large absorption peak appeared at about 52 minutes, so the elution components of the absorption peak were collected multiple times, combined and freeze-dried.

[0055] 7. LC-MS / MS analysis

[0056] The freeze-dried samples prepared above were sent to Shanghai Jiyun Biotechnology Co., Ltd. for peptide sequence detection. After the samples were dissolved with 0.1% formic acid, they were centrifuged, desalted and freeze-dried by C18 for mass spectrometry detection. Mass spectrometry method: positive ion detection mode, primary resolution of 120000, AGC set to 3e6, scanning range of 50-1000m / z. The ions with the highest intensity in 10 grids were selected from a single MC spectrum for MS / MS analysis, with a secondary resolution of 15000, AGC set to 2e4, and a separation window of 1.6m / z. Liquid phase method: The chromatographic column was Eksigent C18 (3μm, 250mm×75μm), phase A was water + 0.1% formic acid; phase B was acetonitrile + 0.1% formic acid; the flow rate was 300nl / min, the injection volume was 4μL, and the chromatographic gradient elution was 80min. The elution conditions are: 0→16min, 93%A→88%A; 16→51min, 88%A→70%A; 51→71min, 70%A→58%A; 71→72min, 58%A→5%A; 72→80min, 5%A.

[0057] Data processing: The results were retrieved from the NCBI database using PEAKS software. During the data processing, the possible amino acid combinations were first 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] The results showed that there were two completely matched peptide chains, with amino acid sequences of LSHGFNK (SEQ ID NO.1) and LRGSFLR (SEQ ID NO.2), which were fish collagen peptides.

[0059] 8. Preparation of compound drinks

[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: Use UHT ultra-high temperature instantaneous sterilizer to sterilize the prepared juice, quickly raise the temperature to 110℃, maintain it for 15s, and quickly cool it to 80℃ after sterilization. Use automatic filling machine to fill it into fully cleaned and sterilized glass bottles and quickly seal them. After turning the bottles over for 1 minute, use tunnel to spray cool them until the temperature reaches 30℃.

[0070] Test Group 1: The distilled water solution containing 11.2 g / L LSHGFNK was subjected to the same high pressure homogenization, sterilization, filling and cooling steps as above.

[0071] Test Group 2: A distilled water solution containing 11.2 g / L LRGSFLR was subjected to the same high pressure homogenization, sterilization, filling and cooling steps as above.

[0072] Test Group 3: A distilled water solution containing 7.8 g / L of LSHGFNK and 4.4 g / L of LRGSFLR was subjected to the same high-pressure homogenization, sterilization, filling and cooling steps as above.

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

[0074] Add 20 mL of the product prepared with different stabilizers to the graduated centrifuge tubes, put them into the centrifuge, centrifuge at 3000 r / min for 10 minutes, quickly draw the supernatant, dilute it, and measure the absorbance 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 worse the stability. An I value greater than 95% indicates good stability. After testing, it was found that the stability of the composite 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 these beverages have good stability. In addition, the color of the composite beverage is slightly yellow, without browning, and the color is uniform; tissue state: a uniform turbid liquid, no suspended matter, no stratification, and a slight precipitation is allowed after standing for a long time; taste and smell: the taste is sweet and sour, with the fruit fragrance of kumquat, lemon, and orange, without fishy smell or other bad smells. The solutions of test groups 1 to 3 are transparent and colorless, without fishy smell or other bad smells.

[0075] 9. Cytotoxicity test

[0076] For cytotoxic activity evaluation, human gastric mucosal epithelial cells (GMEC, product number PCLH0022-RT, Bihe Biotechnology) and primary human intestinal mucosal epithelial cells (hIMEC, product number Delf-10785, Hefei Wanwu Biotechnology Co., Ltd.) with good logarithmic growth were selected and cultured at 2×10 4 The cells were inoculated at a density of 100 / mL in a 96-well culture plate and incubated at 37°C with 5% CO 2 Incubate overnight in an environment. Sample solutions with a final concentration of 10 mg / mL were added to the wells respectively (compound drink (LSHGFNK as a component of fish collagen peptide), test group 1, test group 2, test group 3), 7 different mass concentrations were made for each sample, and 5 parallels were made for each concentration. The cell control group was not treated and cultured for 48 hours. 10 μL of CCK-8 solution was added to each well. After culturing for 2.5 hours, the absorbance of each well was measured at 450 nm. The same volume of sterile water was added as a control. The blank group was the well without any sample added.

[0077] Calculate cell survival rate = (A sample group - A blank group) / (A control group - A blank group) × 100%. As a result, the cell survival rates in the composite drink, test group 1, test group 2, and test group 3 were not less than 95%, indicating that the composite drink provided by the present invention and the fish collagen peptide had no toxic effects on gastrointestinal mucosal cells and had high safety.

[0078] 10. Protective effect on UVA-stimulated HaCaT cells

[0079] Human HaCaT cells (Shanghai Guyan Industrial) in good logarithmic growth phase were counted and inoculated into 10 cm culture dishes, with 5×10 cells per dish. 6 cells. At 37°C, 5% CO 2 After culturing overnight under the environment, the culture medium was discarded, and 1 mg / mL of compound drink (LSHGFNK as a component of fish collagen peptide) was added to the cells for 6 hours (serum-free DMEM culture medium was added to the model group and the blank control group), and phosphate buffered saline (PBS) (pH = 7.4) was added until the cells were just covered. The radiation dose was 7 J / cm 2UVA stimulated cells and irradiated them for 2 hours as the model group, while the blank group was not irradiated. Each sample was tested in triplicate (each parallel sample was tested three times), taken out and placed on ice, washed twice with PBS; 800uL of lysis buffer was added to lyse the cells, centrifuged at 4°C and 12,000r / min for 5 minutes, and the supernatant was obtained to obtain the cell lysis supernatant. The total antioxidant capacity detection (ABTS method), glutathione peroxidase (GSH-Px) activity, and lipid oxidation activity detection in the samples were performed according to the instructions of the kit. Subsequently, the intracellular MMP-1 and COL-1 contents were detected using the MMP-1 ELISA kit and the CoL-1 ELISA kit, respectively, and protein correction was used.

[0080] like Figure 5 As shown, different compound drinks or test products were used to intervene in UVA-stimulated HaCaT cells. The results showed that 1 mg / mL of compound drinks or test products intervened in UVA-stimulated HaCaT cells, which could improve the total antioxidant capacity of cells, reduce the increase in glutathione peroxidase activity and MDA content caused by UVA stimulation, and also increase the decrease in MMP-1 and COL-1 expression caused by UVA stimulation.

[0081] 11. In vivo model experiments

[0082] (1) Experimental animals

[0083] Healthy male Wistar rats, 8 weeks old, weighing 250±20g, were provided by Beijing Beiyou Biotechnology Co., Ltd. The animals were fed with ordinary feed and had free access to drinking water. The temperature of the breeding environment was (22±1)℃ and the relative humidity was 50%-70%.

[0084] (2) Normal rat skin ulcer model

[0085] After 10 days of adaptive feeding, the rats were anesthetized with 7% chloral hydrate intraperitoneal injection, and the hair on the left side of the dorsal midline was removed using electric clippers and depilatory cream. According to the full skin defect standard, a modeling area with a diameter of 20 mm was marked on the mouse skin, and a circular skin defect was prepared with surgical scissors, deep to the fascia.

[0086] (3) Administration

[0087] After the model was successfully established, the rats were kept in a single cage, allowed to drink water and eat freely, and were divided into a model group and a drug-treated group. The drug-treated group was gavaged with 20 mL of the sample test group (Compound drink (LSHGFNK as a component of fish collagen peptide), Test1, Test2, Test3 and Compound drink1) every day, and the model group was gavaged with an equal amount of normal saline. At the same time, a control group was set up to eat freely, a skin ulcer model was made, and the same amount of normal saline was gavaged.

[0088] The formula of Compound drink 1 is: 40g / L kumquat juice, 8g / L lemon juice, 64g / L orange juice, 10g / L white sugar, 0.2g / L sodium carboxymethyl cellulose, 0.25g / L low molecular weight sodium hyaluronate, and 0.04g / L ethyl paraben. Its preparation method is the same as above.

[0089] (4) Wound healing

[0090] After drug intervention, the ulcer wounds of the rats were photographed with a camera on the 7th day, and the wounds were 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%. Figure 4 As shown, after drug intervention in each group of rats, the healing conditions of the wounds in each group of rats were different. Among them, the healing conditions of the compound drink, test group 1, test group 2 and test group 3 were better.

[0091] (5) Wound skin tissue vitality detection

[0092] The wound skin tissues of rats in each group were collected, washed and transferred to a 24-well plate. 0.3 mL of MTT working solution (1 mg / mL) was added to each well and placed in an incubator (37°C, 5% CO 2 ) incubate (3h±5min). At the end of incubation, clean the outer surface of the model with PBS and wipe it dry with a sterile cotton swab, and transfer it to a new 24-well plate. Add 2mL of isopropanol sealing film to seal, shake the well plate on a shaker for 2h, pierce the model with a 200μL pipette tip to allow the isopropanol extract to flow out of the wound skin tissue, and mix it by blowing. Then, draw 2 portions of 200μL of isopropanol extract from each well into a 96-well plate, use isopropanol as the zeroing well, read the absorbance (A) at a wavelength of 570nm on the microplate reader, and calculate the relative tissue viability of each test group according to the following formula: Tissue viability = 100% × (A dosing well - A zeroing well) / (A control well - A zeroing well). Figure 4 As shown, the wound skin tissues of rats in each group were recultured, and the skin tissues obtained from the composite beverage, test group 1, test group 2 and test group 3 had higher vitality, and the skin tissue obtained from test group 3 had the highest vitality.

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

[0094] The culture fluid of the wound skin was collected in a centrifuge tube, and then the samples for ELISA detection were placed in a -80℃ refrigerator for freezing and stored. The detection and analysis were performed according to the operating instructions of the IL-1α ELISA kit, TNFα ELISA kit, and PGE2 ELISA kit. Figure 6 As shown, after drug intervention in each group of rats, the inflammatory expression of IL-1α in the wound skin was inhibited, while TNFα and PGE2 had no significant inhibitory effect. In addition, the compound drink, test group 1, test group 2 and test group 3 were able to significantly promote the inflammatory expression of IL-1α in the wound skin, while the compound drink 1 had no significant effect.

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

[0096] The culture fluid of the wound skin was collected in a centrifuge tube. After collection, the model used for FLG detection was fixed with 4% paraformaldehyde for 24 hours, and then the model was removed by ring cutting for confocal microscopy observation of FLG. Scanning was performed under a laser confocal microscope (Leica, Germany), with a blue light emission wavelength of 461nm, a red light emission wavelength of 658nm, and a resolution of 553×463 pixels. Computer data was collected, and Image J image analysis software was used to perform relative quantitative calculation and analysis of protein for positive signal fluorescence. Five levels of four samples of each research object were randomly scanned under a microscope, and the computer calculated the ratio of the average area to the integrated optical density as the relative expression of FLG. The results are as follows: Figure 7 As shown, after drug intervention in each group of rats, the compound drink, test group 1, test group 2 and test group 3 could significantly promote the FLG expression in the wound skin, while the compound drink 1 had no significant promoting effect.

[0097] The above description is only a 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 conceived by any technician familiar with the technical field 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 LSHGFNK and LRGSFLR.

2. A compound beverage, the formula of which comprises: A mixture of 11.2g / L fish collagen peptides LSHGFNK and LRGSFLR, 40g / L kumquat juice, 8g / L lemon juice, 64g / L orange juice, 10g / L white sugar, 0.2g / L sodium carboxymethyl cellulose, 0.25g / L low molecular weight sodium hyaluronate, and 0.04g / L ethyl paraben.

3. The composite beverage according to claim 2, characterized in that: 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 method for preparing 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 enzyme-soluble collagen include: The pretreated fish skin is added into a 0.5 M acetic acid solution containing 0.5 wt % of pepsin at a material-liquid ratio of 1:30 (m / V), stirred at 4° C., and filtered through absorbent cotton gauze; Sodium chloride solution was added to the filtrate for salting out and the mixture was stirred continuously until no precipitate was washed out. The suspension was centrifuged at 4°C and 12000rpm. The precipitate at the bottom of the centrifuge tube was redissolved in 0.5M acetic acid solution and finally placed in a dialysis bag for dialyzation to neutrality and freeze-dried to obtain enzyme-soluble collagen.

8. The preparation method according to claim 7, characterized in that: The enzymatic hydrolysis step comprises: Evenly mix 5000U / g bromelain in enzyme-soluble collagen distilled water, adjust pH7.0 and temperature to 40°C, react for 5 hours, boil in a water bath for 10 minutes to inactivate, centrifuge at 4°C and 12000rpm to obtain the supernatant, and lyophilize 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 collected components less than 1 kD were prepared into a 100 mg / mL solution with ultrapure water, filtered with a 0.22 μm filter membrane, and then 5 mL was statically loaded and 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. Solutions with different absorption peaks were collected separately, and their antioxidant activity was determined after lyophilization.

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

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