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

Abstract

The present application relates to fish collagen technology field, specifically to fish collagen peptide of the first fish skin and its composition and application in preparing the drink for improving the skin. The amino acid sequence of the fish collagen peptide is LSHGFNK and LRGSFLR. The two kinds of fish collagen peptides are used as raw materials, and the compound drink obtained by compounding gold orange, lemon and orange three kinds of fresh fruits has good stability, yellow color, no browning, uniform color, and the organization state is uniform turbidity liquid, no suspended matter, no stratification, and a little sedimentation is allowed after long time standing; taste and smell: sweet and sour taste, fruit fragrance of gold orange, lemon and orange, no fishy smell and other bad smell. The compound drink provided by the present application has the effect of improving the skin.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fish collagen, in particular to a fish collagen peptide from the skin of Harpadon nehereus and a composition thereof and application thereof in preparing a drink for improving skin. BACKGROUND

[0002] Harpadon nehereus, also known as the scad, is a small-sized fish of warm water and bottom layer, which usually inhabits in the shallow sea area within 50 meters or the offshore sea area and gathers in estuary when foraging. The scad is a carnivorous fish, which feeds on small fish such as anchovy, shrimps and copepods. The scad mainly spawns in the coastal and estuary areas, and the spawning period is from June to August. The diameter of the egg is about 1 mm, and the egg yolk has no oil globule and no fissure. The scad has a life span of only one year. The scad is a carnivorous fish with rich nutrition and high edible value. In addition, the scad can also be used as medicine, which has the effects of benefiting qi, tonifying deficiency, invigorating the spleen and kidney, nourishing the liver to stop bleeding and diuresis and edema.

[0003] The content of collagen in the skin is as high as 70%. If the content of collagen in the skin decreases, the water content will gradually be lost, and the decrease of the water content in the skin will cause the skin to lose softness and luster, become rough, and thus accelerate the occurrence of skin aging. The collagen molecule contains glycine and other components, which can improve the water storage capacity of tissue cells and keep the skin moisturized. The collagen has important effects on the skin, such as water retention, moisturization and elasticity maintenance. There is no report on the development of collagen or collagen peptide from the scad as a raw material for improving the skin. Therefore, the present application is proposed to deeply exploit the scad and utilize its resources. SUMMARY

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

[0005] Specifically, the present application provides a preparation method of a collagen peptide from the skin of Harpadon nehereus. The method comprises the following steps: pretreating the skin of Harpadon nehereus; extracting enzyme-soluble collagen from the pretreated skin of Harpadon nehereus; subjecting the enzyme-soluble collagen to enzymolysis to obtain an enzymolysis product; subjecting the enzymolysis product to ultrafiltration separation to collect a component with a molecular weight less than 1 kD; subjecting the component with a molecular weight less than 1 kD to Sephadex G-15 gel chromatography separation to elute and collect a component with the highest antioxidant activity; subjecting the component with the highest antioxidant activity to automatic high-pressure preparation separation and purification to collect an elution component; and separating and identifying the collagen peptide from the skin of Harpadon nehereus from the elution component according to LC-MS / MS analysis.

[0006] Specifically, the step of pretreating the first fish skin includes: stirring the dried first fish skin with 0.1M alkali solution at a solid-liquid ratio of 1:20 (m / V) for 48h, with liquid replacement for 4 times in the middle, and washing with distilled water to neutral; then, soaking the fish skin in 0.5M ethylenediaminetetraacetic acid disodium salt solution for 48h for decalcification treatment, with liquid replacement for 4 times in the middle, and washing several times after the treatment; then, stirring the fish skin with 15% isopropanol solution until the fat in the fish skin is removed, and then washing the isopropanol clean.

[0007] Specifically, the step of extracting enzyme-soluble collagen includes: adding the pretreated first fish skin into 0.5M acetic acid solution containing 0.5wt% pepsin at a solid-liquid ratio of 1:30 (m / V), stirring at 4℃, and filtering with defatted 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 12000rpm at 4℃, resuspending the precipitate at the bottom of the centrifuge tube in 0.5M acetic acid solution, and finally loading the dialysis bag for dialysis to neutral, freeze-drying, and obtaining enzyme-soluble collagen.

[0008] Specifically, the step of enzymolysis includes: uniformly mixing 5000U / g bromelain into the enzyme-soluble collagen in distilled water, adjusting pH to 7.0 and temperature to 40℃, reacting for 5h, inactivating by boiling in water bath for 10min, centrifuging at 12000rpm at 4℃ to obtain the supernatant, and freeze-drying to obtain the enzymolysis product.

[0009] Specifically, the step of Sephadex G-15 gel chromatography separation includes: configuring the collected component less than 1kD into a 100mg / mL solution with ultrapure water, filtering with a 0.22μm filter membrane, and then statically loading 5mL and eluting with ultrapure water at a flow rate of 0.2mL / min, collecting the eluate (1mL / tube), detecting the absorbance at 280nm, and collecting the solutions of different absorption peaks, and freeze-drying and determining the antioxidant activity.

[0010] Specifically, the step of full-automatic high-pressure preparation separation and purification includes: configuring the freeze-dried powder of the R2 component into a 40mg / mL solution, filtering with a 0.22μm filter membrane, and then loading the HP Plus 10D high-pressure preparation purification system, using a mixture of water and acetonitrile (containing 0.1% trifluoroacetic acid) as the mobile phase, setting the elution gradient of acetonitrile phase to 10% to 95%, setting the injection amount to 5mL, setting the flow rate to 2mL / min, setting the elution time to 90min, setting the detection wavelength to 280nm, collecting the elution peaks, and freeze-drying.

[0011] Specifically, the step of LC-MS / MS analysis includes:

[0012] Mass spectrometry method: positive ion detection mode, first order resolution is 120000, AGC is set to 3e6, the scanning range is 300-1400 m / z; 10 grid intensity highest ions are selected from a single MC spectrum for MS / MS analysis, the second order resolution is 15000, AGC is set to 2e4, and the separation window is 1.6 m / z;

[0013] Liquid phase method: the chromatographic column is Eksigent C18 (3 μm, 250 mm*75 μm), the A phase is water+0.1% formic acid; the B phase is acetonitrile+0.1% formic acid; the flow rate is 300 nl / min, the injection volume is 4 μL, and the gradient elution is 80 min; the elution condition is as follows: 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 subjected to NCBI database retrieval by using PEAKS software; in the data processing process, first, the possible amino acid combination is calculated according to the molecular weight information of the fragments in each MS / MS spectrum, and then the amino acid combination is compared to the protein database of the sample source, and the protein database of the Harpadon nehereus species in NCBI is used as the sample source.

[0015] Specifically, the application further provides fish collagen peptides and compositions thereof prepared by the preparation method, and the amino acid sequence of the fish collagen peptides is LSHGFNK or LRGSFLR.

[0016] Specifically, the application further provides a preparation method of a composite beverage.

[0017] The preparation method comprises the following steps:

[0018] Fruit selection, cleaning and juicing: select three kinds of fresh fruits of gold orange, lemon and orange with normal color, strong aroma, full maturity, no mildew, no disease and insect damage, and no other obvious adverse changes as processing raw materials, clean the dust, impurities, microorganisms and pesticide residues on the surface of the gold orange, lemon and orange, peel and press the edible pulp part, and store the pressed fruit juice in cold storage;

[0019] Dissolving sugar: use 80-85 DEG C hot water, high-speed stirring for 10 min, and the white sugar is completely dissolved;

[0020] Other raw material pretreatment: ethyl p-hydroxybenzoate is first diluted 5-10 times with 30-40 DEG C warm water, and filtered with a 200 mesh filter bag; carboxymethyl cellulose sodium, low molecular weight sodium hyaluronate and white granulated sugar are dry mixed, and then dissolved to form a solution;

[0021] Mixing and blending: in addition to fish collagen peptides, the ingredients are added and stirred, with an interval of 5 minutes each time. After the addition of all the ingredients, stirring is continued until the mixture is uniform. Water is then added to make up the volume. The mixture is then filtered through a double filter;

[0022] Vacuum degassing: fish collagen peptides are added to the filtrate, and after thorough mixing, vacuum degassing is performed at a temperature of 60 DEG C and a vacuum degree of 0.08 MPa;

[0023] High-pressure homogenization: the homogenization pressure is 18-20 MPa.

[0024] Specifically, the application also provides the use of the fish collagen peptides and the composition thereof in the preparation of a drink for improving the skin.

[0025] The preparation method provided by the application extracts enzyme-soluble collagen from fish skin through pretreatment, and subjects the enzyme-soluble collagen to enzymolysis. The enzymolysis product is subjected to ultrafiltration separation, size-exclusion chromatography separation and full-automatic high-pressure preparative chromatography separation and purification. Through LC-MS / MS analysis screening and comparison, two fish collagen peptides, LSHGFNK and LRGSFLR, are obtained.

[0026] The composite drink prepared by using the two fish collagen peptides as raw materials and compounding gold orange, lemon and orange three kinds of fresh fruits has good stability, a yellowish color, no browning, uniform color, and a uniform and consistent state of organization, i.e., a uniform turbidity without suspended matter or stratification, and a small amount of sedimentation after long-term standing. The taste and smell are sweet and sour, with the fruit fragrance of gold orange, lemon and orange, no fishy smell and other unpleasant odors.

[0027] The composite drink and the test solution provided by the application are found to have no cytotoxicity through a cytotoxicity experiment.

[0028] The composite drink and the test solution provided by the application are found to have a protective effect on UVA-stimulated HaCaT cells through a cell experiment.

[0029] The composite drink and the test solution provided by the application are found to be able to promote and accelerate the healing of a wound in a rat, promote the maintenance of tissue vitality of the skin of the wound, inhibit the expression of IL-1 alpha inflammation of the skin of the wound, and promote the expression of barrier proteins of the skin model. That is, the composite drink and the test solution provided by the application not only have the function of healing the skin, but also have the effects of anti-inflammation and improvement of the skin barrier. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is a chromatogram of Sephadex G-15 purification.

[0031] Figure 2 is a chromatogram of full-automatic high-pressure preparation separation and purification.

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

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

[0034] Figure 5 is an antioxidant test result diagram of UVA stimulated HaCaT cells intervened by different composite drinks or test products. "*" indicates P<0.05 compared with the model group, "**" indicates P<0.01 compared with the model group, "#" indicates P<0.05 compared with the model group and the control group.

[0035] Figure 6 is a wound healing rate and skin tissue vitality result diagram of wound rats intervened by different composite drinks or test products. "*" indicates P<0.05 compared with the model group, "**" indicates P<0.01 compared with the model group, "#" indicates P<0.05 compared with the model group and the control group.

[0036] Figure 7 is an inflammation expression and skin model barrier protein result diagram of wound rats intervened by different composite drinks or test products. "*" indicates P<0.05 compared with the model group, "**" indicates P<0.01 compared with the model group, "#" indicates P<0.05 compared with the model group and the control group. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application. The reagents not described in detail in the present application are conventional reagents, and can be obtained from commercial channels; the methods not described in detail are conventional experimental methods, and can be known from the prior art.

[0038] 1. Pretreatment of fish skin

[0039] Take the fish skin, dry, according to 1:20 (m / V) of the ratio of liquid to 0.1 M alkali solution to stir fish skin 48 h, the middle of the liquid to remove non-collagenous substances, distilled water to wash to neutral. Then the fish skin soaked in a concentration of 0.5 M ethylenediaminetetraacetic acid disodium salt solution to stir 48 h for decalcification treatment, midway to change liquid 4 times, after treatment clean several times. Then use 15% of isopropanol solution to stir fish skin until the fish skin fat, and then isopropanol clean. The whole process is carried out at 4 DEG C.

[0040] 2, enzyme soluble collagen extraction

[0041] According to 1:30 (m / V) of the ratio of liquid to 0.5 M acetic acid solution (containing 0.5 wt% pepsin, CAS: 9001-75-6, product number: P6322; purity: > 3000 U / mg; McLean) to stir at 4 DEG C for one day to extract enzyme soluble collagen, after extraction with filter paper filter, according to the above method repeatedly extracted twice, combined filtrate. To the filtrate, add sodium chloride solution for salting out and continue to stir until no precipitate wash out, the suspension in the centrifuge tube bottom of the precipitate redissolved in 0.5 M acetic acid solution, finally into dialysis bag dialysis to neutral, freeze drying, to prepare enzyme soluble collagen.

[0042] 3, enzyme

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

[0044] According to the enzyme hydrolysis reaction system of adding different enzymes at its corresponding enzyme hydrolysis temperature and enzyme hydrolysis pH conditions, after 5 h of reaction, water bath boiling 10 min inactivation, 4 DEG C, 12000 rpm centrifugation to take supernatant, freeze drying to obtain enzyme hydrolysate.

[0045] The yield of peptides in the enzymatic hydrolysate was determined by the Folin-phenol method. Polypeptide yield % = v x c x 100%; wherein, v is the volume of the enzymatic hydrolysate (mL); c is the polypeptide concentration of the enzymatic hydrolysate (mg / mL); and a is the protein content of the raw material (%). The results show that the yields of enzymatic hydrolysis using 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 sequentially subjected to centrifugation at 4°C and 5000 g for 60 min using a 1 kD ultrafiltration tube, so as to collect components with a molecular weight of <1 kD, which were then lyophilized and determined for antioxidant activity.

[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 collected components with a molecular weight of <1 kD were prepared into a solution with a concentration of 100 mg / mL using ultrapure water, filtered using a 0.22 μm filter membrane, and then subjected to static sample loading of 5 mL, elution with ultrapure water at a flow rate of 0.2 mL / min, collection of eluate (1 mL / tube), detection of absorbance at 280 nm, collection of solutions of different absorption peaks, and lyophilization and determination of antioxidant activity.

[0050] DPPH free radical scavenging rate activity detection: 100 μL of each sample and 100 μL of DPPH solution (0.2 mM) were added to a 96-well plate, which was placed in the dark for reaction for 30 min, and then the absorbance was measured at 517 nm. DPPH free radical scavenging rate % = 1 (1-(A sample - A control) / A blank group) x 100%. Distilled water was added as a control group, and distilled water was added as a blank.

[0051] The components with a molecular weight of <1 kD were subjected to Sephadex G-15 chromatography purification to obtain three absorption peaks, S1-S3, and the DPPH free radical scavenging rates thereof were 28.5%, 43.2% and 31%, respectively.

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

[0053] The R2 component was separated by HP Plus 10D high pressure preparation purification system of Lissui Technology (Suzhou) Co., Ltd. The freeze-dried powder of the R2 component was prepared into a 40 mg / mL solution, filtered by a 0.22 μm filter, and then eluted by a mixture of water and acetonitrile (containing 0.1% trifluoroacetic acid) as the mobile phase, with an elution gradient of acetonitrile of 10%-95%, an injection amount of 5 mL, a flow rate of 2 mL / min, an elution time of 90 min, and a detection wavelength of 280 nm. The elution peak was collected and freeze-dried.

[0054] As shown in FIG. 2, a larger absorption peak appeared at about 52 min, so the elution components of the absorption peak were collected several times and then freeze-dried after being combined. Figure 2

[0055] 7. LC-MS / MS analysis

[0056] The freeze-dried sample prepared above was sent to Shanghai Jiyun Biotechnology Co., Ltd. for peptide sequence detection. After the sample was dissolved in 0.1% formic acid, centrifuged, and desalted by C18 freeze-drying, mass spectrometry detection was performed. Mass spectrometry method: positive ion detection mode, first-order resolution of 120000, AGC setting of 3e6, and scanning range of 50-1000 m / z. From a single MC spectrum, 10 grid ions with the highest intensity were selected for MS / MS analysis, with a second-order resolution of 15000, an AGC setting of 2e4, and a separation window of 1.6 m / z. Liquid chromatography method: the chromatographic column was Eksigent C18 (3 μm, 250 mm x 75 μm), A was water + 0.1% formic acid, B was acetonitrile + 0.1% formic acid, the flow rate was 300 nl / min, the injection volume was 4 μL, and the chromatography gradient elution was 80 min. The elution conditions were as follows: 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 subjected to NCBI database retrieval by PEAKS software. During data processing, the possible amino acid combinations were first calculated according to the molecular weight information of the fragments in each MS / MS spectrum, and then compared to the protein database of the sample source. The protein database of the Harpadon nehereus species in NCBI was used as the sample source.

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

[0059] 8. Preparation of a compound beverage ​

[0060] (1) Formulation

[0061] The formulation of the compound beverage provided by the present application comprises: 11.2 g / L of fish collagen peptide LSHGFNK or the last of LRGSFLR, 40 g / L of gold orange juice, 8 g / L of lemon juice, 64 g / L of orange juice, 10 g / L of white granulated sugar, 0.2 g / L of sodium carboxymethyl cellulose, 0.25 g / L of low molecular weight sodium hyaluronate (molecular weight 1.0 MDa-1.8 MDa, CAS number: 9067-32-7, MDL number: MFCD00131348, Mcllvaine), 0.04 g / L of ethyl p-hydroxybenzoate.

[0062] (2) Preparation of the compound beverage

[0063] Fruit selection, washing and juicing: Select three kinds of fresh fruits of gold orange, lemon and orange with normal color, strong aroma, full maturity, no mold, no disease and insect damage, and no other obvious adverse changes as raw materials. Wash the surface of the gold orange, lemon and orange with flowing water to remove dust, impurities, microorganisms and pesticide residues. Remove the peel and seeds with a knife, then manually peel the edible pulp part and put it into a juicer for pressing. The pressed juice is stored in a refrigerator.

[0064] Sugar dissolution: Stir a part of white granulated sugar with 80-85°C hot water, high-speed stir for 10 min until the white granulated sugar is completely dissolved.

[0065] Other raw material pretreatment: Dilute ethyl p-hydroxybenzoate with 30-40°C warm water by 5-10 times, and filter with a 200 mesh filter bag. Dry mix sodium carboxymethyl cellulose, low molecular weight sodium hyaluronate and white granulated sugar, then dissolve to form a uniform and stable solution.

[0066] Mixing and blending: The amount of compound juice is 10%, ethyl p-hydroxybenzoate is 0.04 g / L, and the amount of other raw materials is determined according to the formulation amount. Stir the ingredients (except fish collagen peptide) at intervals of 5 min. After all the raw materials are added, continue to stir until they are evenly mixed, and then add water to the desired volume. Then filter through a double filter.

[0067] Vacuum degassing: Add fish collagen peptide to the filtrate, mix thoroughly, and then perform vacuum degassing at a temperature of 60°C and a vacuum degree of 0.08 MPa.

[0068] High-pressure homogenization: The homogenization pressure is 18-20 MPa, so that the pectin in the juice is fully dissolved in the juice, the affinity between pectin and juice is increased, the stability of the juice is improved, the separation and precipitation of the serum are prevented, and the organization is uniform and viscous, with a delicate taste.

[0069] Sterilization, filling and cooling: the prepared juice was sterilized by UHT ultra-high temperature instantaneous sterilizer, so that the temperature rapidly rose to 110°C, and maintained for 15s. After sterilization, the juice was rapidly cooled to 80°C, and then filled into the glass bottles which were washed and sterilized. The bottles were sealed, and then inverted after 1 min. The bottles were cooled by spraying in a tunnel until the temperature reached 30°C.

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

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

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

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

[0074] Into a graduated centrifuge tube, 20mL of the product prepared by different stabilizers was added, and then placed into a centrifuge. After centrifugation at 3000r / min for 10min, the supernatant was rapidly absorbed, diluted, and then the absorbance I after centrifugation was measured by UV-visible spectrophotometer at a wavelength of 750nm. The absorbance of the beverage before and after centrifugation was accurately recorded, and each sample was measured 3 times to take the average value. The calculation formula of stability is I = I 后 / I 前 ; in the formula, I is the stability, I 前 is the absorbance before centrifugation, and I 后 is the absorbance after centrifugation. The greater the value of I, the better the stability, otherwise, the worse the stability. The value of I greater than 95% indicates good stability. It was found by testing that the stability of the compound beverage (LSHGFNK as a component of fish collagen peptide), test group 1, test group 2, test group 3 was 96.7%, 100.2%, 100.3%, 99.8% respectively, indicating that the stability of these beverages was good. In addition, the compound beverage had a yellowish color without browning, and the color was uniform and consistent; the organization state was a uniform turbid liquid without suspended matter and stratification, and a little sediment was allowed after long-term standing; the taste and smell were sweet and sour, with the fruit fragrance of gold orange, lemon and orange, without fishy smell and other bad smell. The solutions of test groups 1-3 had transparent color without fishy smell and other bad smell.

[0075] 9. Cytotoxicity test

[0076] Cytotoxic activity was evaluated using human gastric mucosal epithelial cells (GMEC, product number PCLH0022-RT, Bihe Biotechnology) and primary human intestinal mucosal epithelial cells (hIMEC, catalog number Delf-10785, Hefei Wanwu Biotechnology Co., Ltd.) in good logarithmic growth state. Cytotoxic activity was evaluated using 2×10⁻⁶ cells of each cell. 4 Cells were seeded at a density of [number] cells / mL in 96-well plates and incubated overnight at 37°C with 5% CO2. Sample solutions (compound beverage (LSHGFNK as a component of fish collagen peptides), test group 1, test group 2, and test group 3) were added to each well to a final concentration of 10 mg / mL, with seven different concentrations for each sample and five replicates for each concentration. The cell control group received no treatment. Cells were incubated for 48 h. 10 μL of CCK-8 solution was added to each well, and after 2.5 h of incubation, the absorbance of each well was measured at 450 nm. A control group was prepared by adding the same volume of sterile water. The blank group consisted of wells without any sample.

[0077] Cell survival rate was calculated as (Sample Group A - Blank Group A) / (Control Group A - Blank Group A) × 100%. The results showed that the cell survival rates in the compound beverage, test group 1, test group 2, and test group 3 were all no less than 95%, indicating that the compound beverage and fish collagen peptides provided by this invention have no toxic effects on gastrointestinal mucosal cells and possess high safety.

[0078] 10. It has a protective effect against UVA-stimulated HaCaT cells.

[0079] Human HaCaT cells (Shanghai Guyan Industrial Co., Ltd.) in good logarithmic growth phase were counted and seeded into 10cm culture dishes, with 5 × 10⁶ cells per dish. 6 Cells were cultured overnight at 37°C with 5% CO2 (v / v), then the culture medium was discarded. 1 mg / mL of a compound beverage (LSHGFNK as a component of fish collagen peptides) was added to each of the three groups (test group 1, test group 2, and test group 3) for 6 hours. (The model group and blank control group were treated with serum-free DMEM). Phosphate-buffered saline (PBS) (pH = 7.4) was added until the cells were just covered. Radiation at 7 J / cm² was applied. 2UVA stimulated cells, irradiation 2h, as a model group, blank group without irradiation. Each sample was made 3 parallel, (each parallel sample detection 3 results), take out on ice, PBS wash 2 times; add 800uL lysis buffer lysis cells, 4℃, 12 000r / min centrifugal 5min, take supernatant to get cell lysis supernatant. The total antioxidant capacity detection (ABTS method), glutathione peroxidase (GSH-Px) activity, lipid oxidation activity detection in sample were carried out according to the instructions of the kit. Subsequently, MMP-1 ELISA kit, CoL-1 ELISA kit were used to detect the content of MMP-1 and COL-1 in cells, and protein correction was used.

[0080] As shown in Figure 5 , by different composite drinks or test products on UVA stimulated HaCaT cells intervention, the results found that 1mg / mL of composite drinks or test products can intervene UVA stimulated HaCaT cells, can improve the total antioxidant capacity of cells, reduce the increase of glutathione peroxidase activity and MDA content caused by UVA stimulation, in addition, can also improve the expression 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±20g, provided by Beijing Beiyou Biotechnology Co., Ltd.; animals were adaptively fed for 10d before participating in the experiment, fed with ordinary feed, free water, the temperature of the feeding environment was (22±1)℃, the relative humidity was 50%~70%.

[0084] (2) Normal rat skin ulcer model

[0085] After the rats were adaptively fed for 10d, they were anesthetized by intraperitoneal injection of 7% chloral hydrate, and the hair on the left side of the dorsal midline skin was removed completely using an electric clipper and depilatory cream. According to the whole skin defect standard, a 20mm diameter modeling area was marked on the rat skin, and a circular skin defect wound was prepared by surgical scissors, which reached the fascia.

[0086] (3) Drug administration

[0087] After the model is successfully established, the rats are single-caged, free-fed and free-drinked, and are grouped for experiment, into a model group and a dosing group. The rats in the dosing group are dosed with 20 mL of sample test group (Compound drink (LSHGFNK as a component of fish collagen peptide), Test 1, Test 2, Test 3 and Compound drink 1) per rat per day, and the rats in the model group are dosed with an equal amount of normal saline. Meanwhile, a control group is set up, free-fed, and the skin ulcer model is prepared, and is dosed with an equal amount of normal saline.

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

[0089] (4) Wound healing

[0090] After the drug intervention on the rats in each group, the ulcer wound of each rat is photographed by a camera on the 7th day, the wound is processed by image software, and the wound area is obtained. The wound healing rate is calculated by the following formula: Wound healing rate = (original wound area - unhealed wound area) / original wound area x 100%. As shown in Table 1, the healing of the wounds of the rats in each group is different after the drug intervention, among which, the healing of the wounds of the rats in the compound drink group, the test group 1, the test group 2 and the test group 3 is better. Figure 4

[0091] (5) Wound skin tissue vitality detection

[0092] The wound skin tissue of the rats in each group is taken, washed and transferred to a 24-well plate, 0.3 mL of MTT working solution (1 mg / mL) is added to each well, and the plate is incubated in an incubator (37°C, 5% CO2) for (3 h ± 5 min). After incubation, the outer surface of the model is washed with PBS and dried with a sterile cotton swab, and is transferred to a new 24-well plate. 2 mL of isopropanol sealing film is added to seal the plate, the plate shaker is shaken for 2 h, a 200 μL pipette gun head is used to pierce the model, and the isopropanol extract is allowed to flow out of the wound skin tissue, and is mixed by blowing. Then 2 portions of 200 μL isopropanol extract are taken from each well to a 96-well plate, isopropanol is used as a zero-adjustment well, and the absorbance (A) is read at 570 nm by an enzyme marker. The relative tissue vitality of each test group is calculated according to the following formula: Tissue vitality = 100% x (A dosing well - A zero-adjustment well) / (A control well - A zero-adjustment well). As shown in Table 2, after the wound skin tissue of the rats in each group is recultured, the skin tissue vitality obtained by the rats in the compound drink group, the test group 1, the test group 2 and the test group 3 is higher, and the skin tissue vitality obtained by the rats in the test group 3 is the highest. Figure 4 ​​

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

[0094] The culture solution of the skin of the wound was collected in a centrifuge tube, and then the sample for ELISA detection was stored in a-80°C refrigerator, and detection analysis was performed according to the operation instructions of the IL-1α ELISA kit, the TNFα ELISA kit, and the PGE2 ELISA kit. The results are shown in Table 1. Figure 6 After drug intervention on the rats in each group, the IL-1α inflammatory expression of the skin of the wound was inhibited, but the TNFα and PGE2 had no obvious inhibitory effect. In addition, the compound beverage, test group 1, test group 2, and test group 3 could significantly promote the IL-1α inflammatory expression of the skin of the wound, but the compound beverage 1 had no obvious effect.

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

[0096] The culture solution of the skin of the wound was collected in a centrifuge tube, and after the collection was completed, the model for FLG detection was fixed with 4% polyformaldehyde for 24h, and then the model ring was cut off and taken down, and confocal microscope observation of FLG was performed. Scanning was performed under a laser confocal microscope (Germany leica), the emission wavelength of blue light was 461nm, the emission wavelength of red light was 658nm, the resolution was 553x463 pixels, computer data acquisition was performed, and Image J image analysis software was used for protein relative quantification calculation and analysis of positive signal fluorescence. Four samples of each research object were randomly scanned under a microscope for 5 layers, and the computer calculated the average area and product optical density ratio, which was used as the relative expression amount of FLG. The results are shown in Table 2. Figure 7 After drug intervention on the rats in each group, the compound beverage, test group 1, test group 2, and test group 3 could significantly promote the FLG expression of the skin of the wound, but the compound beverage 1 had no obvious promoting effect.

[0097] The above is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A fish collagen peptide, the amino acid sequence of which is LSHGFNK and LRGSFLR.

2. Use of the fish collagen peptide of claim 1 in the preparation of a health care beverage for assisting in improving the skin.

3. The method of claim 1, wherein the fish collagen peptide is prepared by the steps of: Comprising: ​ pretreating a first fish skin; extracting enzyme-soluble collagen from the pretreated first fish skin; subjecting the enzyme-soluble collagen to enzymolysis to obtain an enzymolysate; subjecting the enzymolysate to ultrafiltration separation to collect a component with a molecular weight less than 1 kD; subjecting the component with a molecular weight less than 1 kD to Sephadex G-15 gel chromatography separation to elute and collect a component with the highest antioxidant activity; subjecting the component with the highest antioxidant activity to automatic high-pressure preparation separation and purification to collect an eluted component; separating and identifying the first fish skin collagen peptide from the eluted component according to LC-MS / MS analysis; the step of pretreating the first fish skin comprises: stirring the sun-dried first fish skin with a 0.1 M alkali solution at a solid-liquid ratio of 1:20 (m / V) for 48 h, with the solution being replaced 4 times in the middle to remove non-collagen substances, and then washing with distilled water until neutral; then, immersing the fish bone in a 0.5 M ethylenediaminetetraacetic acid disodium salt solution for 48 h to perform decalcification treatment, with the solution being replaced 4 times in the middle, and then washing several times after the treatment; then, stirring the fish bone with a 15% isopropanol solution until the fat in the fish bone is removed, and then washing the isopropanol clean; the step of extracting enzyme-soluble collagen comprises: adding the pretreated first fish skin to a 0.5 M acetic acid solution containing 0.5 wt% pepsin at a solid-liquid ratio of 1:30 (m / V), stirring at 4°C, and filtering with a cotton gauze; adding a sodium chloride solution to the filtrate to perform salting-out and continuously stirring until no precipitate is washed out, centrifuging the suspension at 4°C and 12000 rpm, resuspending the precipitate at the bottom of the centrifuge tube in a 0.5 M acetic acid solution, finally placing the solution in a dialysis bag to dialyze to neutral, and freeze-drying to obtain enzyme-soluble collagen; the step of enzymolysis comprises: adding 5000 U / g of bromelain to the enzyme-soluble collagen in distilled water, adjusting the pH to 7.0 and the temperature to 40°C, reacting for 5 h, inactivating by boiling in a water bath for 10 min, centrifuging at 4°C and 12000 rpm to obtain the supernatant, and freeze-drying to obtain the enzymolysate.

4. The production method according to claim 3, characterized by, the step of Sephadex G-15 gel chromatography separation comprises: configuring the collected component with a molecular weight 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, eluting with ultrapure water at a flow rate of 0.2 mL / min, collecting 1 mL / tube of eluate, detecting the absorbance at 280 nm, collecting different absorption peak solutions, freeze-drying, and determining the antioxidant activity.

5. The production method according to claim 4, characterized by, The step of the full-automatic high-pressure preparation separation and purification comprises the following steps: preparing a 40 mg / mL solution of the freeze-dried powder of the R2 component, filtering the solution through a 0.22 μm filter membrane, and then loading the solution into a HP Plus 10D high-pressure preparation purification system, using a mixture of water and acetonitrile containing 0.1% trifluoroacetic acid as the mobile phase, setting the elution gradient of the acetonitrile phase to 10%-95%, setting the injection amount to 5 mL, setting the flow rate to 2 mL / min, setting the elution time to 90 min, setting the detection wavelength to 280 nm, collecting the eluate, and freeze-drying.

Images