A method for preparing zinc ion chelates of tuna skin collagen peptides

Abstract

This invention provides a method for preparing tuna skin collagen peptide zinc ion chelate, belonging to the field of marine biological functional product processing. The method mainly involves hydrolyzing tuna skin using a complex protease to obtain collagen active peptides, and then chelating these active peptides with a zinc ion solution to finally obtain the tuna skin collagen peptide zinc ion chelate. The preparation method provided by this invention is simple and convenient to operate, low in cost, and has good safety. The yield of the chelate prepared by this invention is as high as 70% or more, the chelation rate is over 85%, and the molecular weight distribution of the chelate has a certain regularity, with the mass percentage of molecules with a molecular weight distribution of 3-5 kDa exceeding 49%. The chelate prepared by this invention, with active peptides chelated with zinc ions, can exert a synergistic effect, not only enhancing the biological activity of the original polypeptide components but also greatly reducing the toxic side effects caused by direct zinc ion supplementation.

Description

Technical Field

[0001] This invention relates to a method for preparing zinc ion chelates of tuna skin collagen peptides, belonging to the field of marine biological functional product processing. Background Technology

[0002] Tuna belongs to the order Perciformes, family Scombridae, and comprises 61 species, widely distributed in major river basins worldwide. Due to its high metabolism, tuna meat is low in fat and high in protein, rich in nutrients such as DHA, EPA, and methionine, giving it high nutritional and economic value. Statistics show that annual tuna processing volume exceeds 4 million tons, accounting for 60% of total high seas fisheries production. Byproducts from tuna processing, such as scales, skin, and bones, account for approximately 50-70% of the total mass, containing abundant protein. Except for a small portion used in fish bait and feed, the vast majority are not rationally utilized, resulting in significant resource waste. Collagen, due to its unique physiological activity and wide range of applications, has become a hot research topic. However, current research focuses primarily on the properties of collagen, such as its structure and stability, molecular weight, applicable industries, and functional properties like water absorption, moisturizing properties, and emulsification stability. As a hydrolysate of collagen, collagen peptides have attracted considerable attention due to their excellent biological activity. Tuna, as a deep-sea fish, has thick skin free of drug residues, making it an excellent source of collagen. Therefore, hydrolyzing tuna skin to obtain bioactive peptides with different functions has broad development and utilization potential.

[0003] Skin is one of the largest organs in the human body. Due to the combined effects of external environment and endogenous genes, skin gradually exhibits signs of aging such as sagging and pigmentation. In particular, long-term exposure to ultraviolet radiation alters skin structure, leading to decreased elasticity, moisture, and suppleness. The main histological characteristic of aging skin is the alteration of the extracellular matrix composition, with reduced collagen synthesis and increased degradation, resulting in a gradual decrease in collagen content. Studies have shown that collagen peptides can enhance the activity of antioxidant enzymes, scavenge excess free radicals in the body, and reduce oxidative stress. The tyrosine residues in collagen peptides can bind to the active site of tyrosinase, preventing tyrosinase from catalyzing the conversion of tyrosine to melanin, thereby improving the condition of photo-aged skin and having a good restorative effect on skin tissue disorders.

[0004] Trace elements play a vital role in maintaining normal human life activities. In recent years, polypeptide-metal ion chelates have gradually attracted attention and become a research hotspot due to their high absorption and utilization rate, significant efficacy, and minimal side effects. Currently, polypeptides from various sources have been successfully isolated from organisms and chelated with calcium. Zinc is an essential trace metal element for the human body, playing a crucial role in human growth and development, reproduction and heredity, maintaining the normal morphology of epithelial cells, protein synthesis, and metabolism. However, different methods of preparing polypeptide-metal ion chelates yield products with different characteristics and significantly different functional effects. Chinese patent document CN101225102A (application number: 200810046739.0) discloses a method for preparing tilapia skin polypeptide chelated zinc salt, and Chinese patent document CN110684814A (application number: 201910744737.7) discloses a method for preparing tuna skin collagen peptide iron chelate. The existing patent documents' methods for preparing fish skin polypeptides and metal ion chelation technologies are significantly different from this invention. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for preparing zinc ion chelates of tuna skin collagen peptides.

[0006] The technical solution of this invention is as follows:

[0007] A method for preparing a zinc ion chelate of tuna skin collagen peptides includes the following steps:

[0008] (1) After thawing the tuna skin, add Tris-HCl at a mass-volume ratio of 1:(5-15) and soak it. Then, sonicate for 5-30 minutes to obtain sonicated fish skin.

[0009] (2) Take the fish skin that has been ultrasonically treated in step (1), add a compound protease to hydrolyze for 15-90 minutes, separate the solid and liquid, and freeze-dry the liquid to obtain collagen active peptides.

[0010] (3) Dissolve the collagen active peptides prepared in step (2) in water, add zinc ion solution at a volume ratio of 1:(2-8), adjust the pH to 6-8, and chelate for 20-80 min to obtain the reaction solution;

[0011] (4) Add anhydrous ethanol to the reaction solution obtained in step (3) and let it stand for 8-24 hours. Separate the solid and liquid. Load the solid onto an propylene dextran gel column and elute with 4-8 times the volume of NaCl solution. Collect the middle and lower eluent fractions, concentrate and freeze dry to obtain tuna skin collagen peptide zinc ion chelate.

[0012] According to a preferred embodiment of the present invention, in step (1), the concentration of the Tris-HCl solution is 8 to 20 mmol / L and the ultrasonic frequency is 30 to 80 kHz.

[0013] More preferably, in step (1), the concentration of the Tris-HCl solution is 6 to 15 mmol / L, and the ultrasonic frequency is 40 to 60 kHz.

[0014] More preferably, the concentration of the Tris-HCl solution is 10 mmol / L and the ultrasonic frequency is 50 kHz.

[0015] According to a preferred embodiment of the present invention, in step (2), the solid-liquid separation method is centrifugation at 3000-6000 rpm / min for 10-30 min, and the supernatant is freeze-dried to obtain collagen active peptides.

[0016] According to a preferred embodiment of the present invention, in step (2), the complex protease is a neutral protease and a trypsin. For each gram of fish skin raw material, the enzyme activity of the neutral protease is 3000-5500 U / g, and the enzyme activity of the trypsin is 2500-4500 U / g; the enzymatic hydrolysis temperature is 30-50℃.

[0017] Further preferred, for each gram of fish skin raw material, the enzyme activity of neutral protease is 4000-5000 U / g, and the enzyme activity of trypsin is 3000-4000 U / g; the enzymatic hydrolysis temperature is 35-45℃.

[0018] More preferably, for each gram of fish skin raw material, the enzyme activity of neutral protease is 4500 U / g, and the enzyme activity of trypsin is 3500 U / g; the enzymatic hydrolysis temperature is 40℃.

[0019] According to a preferred embodiment of the present invention, in step (3), the concentration of the active peptide dissolved in water is 3-6 mg / mL; the zinc ion solution can be zinc gluconate, zinc citrate, or zinc acetate, with a concentration of 20-80 mmol / L; and the chelation temperature is 40-70℃.

[0020] More preferably, in step (3), the concentration of the active peptide dissolved in water is 4-5 mg / mL; the zinc ion solution is zinc gluconate or zinc citrate with a concentration of 30-60 mmol / L; and the chelation temperature is 45-60℃.

[0021] More preferably, the concentration of the active peptide dissolved in water is 4.5 mg / mL; the zinc ion solution is zinc gluconate with a concentration of 50 mmol / L; and the chelation temperature is 50 °C.

[0022] According to a preferred embodiment of the present invention, in step (4), the volume ratio of the reaction solution to anhydrous ethanol is 1:(4-8); and the concentration of the NaCl eluent is 0.15-0.6 mol / L.

[0023] More preferably, in step (4), the volume ratio of the reaction solution to anhydrous ethanol is 1:(5-7); and the concentration of the NaCl eluent is 0.3-0.5 mol / L.

[0024] More preferably, the volume ratio of the reaction solution to anhydrous ethanol is 1:6; and the concentration of the NaCl eluent is 0.4 mol / L.

[0025] According to a preferred embodiment of the present invention, the solid-liquid separation method in step (4) is to centrifuge at 3000-6000 rpm / min for 10-30 min, and then load the precipitate onto an propylene dextran gel column.

[0026] According to a preferred embodiment of the present invention, in step (4), the mid-to-late stage elution fraction is collected, specifically by eluting 4 to 8 column volumes with NaCl solution at a flow rate of 2 BV / h, and collecting the elution fraction after 30 to 60 minutes.

[0027] According to a preferred embodiment of the present invention, the tuna skin collagen peptide zinc ion chelate prepared by the above method has a chelate yield of over 70%, a chelation rate of over 85%, and a mass percentage of molecular weights distributed in the range of 3-5 kDa of over 49%.

[0028] The above method is used as an active ingredient in the preparation of functional foods, health foods or daily chemical products.

[0029] The application of the tuna skin collagen peptide zinc ion chelate prepared by the above method as an active ingredient in the preparation of pharmaceuticals.

[0030] According to a preferred embodiment of the present invention, the tuna skin collagen peptide zinc ion chelate prepared by the above method is used as an active ingredient in the preparation of antioxidants, tyrosinase inhibitors, skin care products, and antihypertensive drugs.

[0031] Beneficial effects

[0032] 1. The method for preparing tuna skin collagen peptide zinc ion chelate provided by the present invention is simple and convenient to operate, low in cost and safe.

[0033] 2. The tuna skin collagen peptide zinc ion chelate prepared by this invention has a yield of over 70% and a chelation rate of over 85%. Furthermore, the molecular weight distribution of the chelate exhibits a certain regularity, with over 49% of the mass having a molecular weight distribution of 3-5 kDa. The chelate prepared by this invention allows the active peptides and zinc ions to synergistically enhance each other, not only improving the bioactivity of the original polypeptide components but also significantly reducing the toxic side effects caused by direct zinc ion supplementation. It can be used as a raw material in industrial production as an antioxidant, food additive, and daily chemical additive, possessing excellent biological efficacy and significant commercial value. Attached Figure Description

[0034] Figure 1 Bar chart showing the free radical scavenging ability of ABTS, a zinc ion chelate of tuna skin collagen peptides.

[0035] Figure 2 Line graph showing the total reducing power of zinc ion chelates of tuna skin collagen peptides. Detailed Implementation

[0036] The technical solution of the present invention will be further described below through specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0037] Unless otherwise specified, the reagents and raw materials involved in the examples are all commercially available products; unless otherwise specified, the operating steps and methods involved in the examples are all conventional operations in the art.

[0038] The neutral protease (1:50, i.e., 50 U / mg) and trypsin (1:250, i.e., 250 U / mg) used in the examples were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; the flavor protease (1:30, i.e., 30 U / mg) was purchased from Beijing Solarbio Technology Co., Ltd.

[0039] Zinc gluconate, zinc citrate, and zinc acetate were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0040] Example 1

[0041] A method for preparing zinc ion chelates of tuna skin collagen peptides, the specific steps of which are as follows:

[0042] (1) After thawing 1 kg of tuna skin, add 10 mmol / L Tris-HCl at a mass-volume ratio of 1:10 g / mL to fully soak the skin, then transfer it to an ultrasonic extractor and sonicate at a frequency of 50 kHz for 15 min to obtain ultrasonically treated fish skin.

[0043] (2) Take the fish skin that has been ultrasonically treated in step (1), add compound protease, and for every gram of fish skin raw material, the enzyme activity of neutral protease is 4500U / g and the enzyme activity of trypsin is 3500U / g. Hydrolyze at 40℃ for 60min, centrifuge at 4000rpm / min for 15min, and freeze-dry the supernatant to obtain collagen active peptides.

[0044] (3) Take the dried active peptide powder, add distilled water to fully dissolve and adjust the concentration to 4.5 mg / mL, add 50 mmol / L zinc gluconate solution at a volume ratio of 1:5, adjust the pH to 7, chelate at 50℃ for 40 min to obtain the reaction solution.

[0045] (4) Add anhydrous ethanol to the reaction solution at a volume ratio of 1:6 and let it stand for 16 h. Centrifuge at 4000 rpm / min for 15 min. Load the precipitate onto an propylene dextran gel column and elute with 6 times the volume of 0.4 mol / L NaCl solution at a flow rate of 2 BV / h. Collect the elution fraction after 45 min, concentrate and freeze dry to obtain 341 g of chelate.

[0046] Example 2

[0047] A method for preparing a zinc ion chelate of tuna skin collagen peptides includes the following steps:

[0048] (1) After thawing 1 kg of tuna skin, add 15 mmol / L Tris-HCl at a mass-volume ratio of 1:5 g / mL to fully soak the skin, then transfer it into an ultrasonic extractor and sonicate at a frequency of 60 kHz for 10 min to obtain ultrasonically treated fish skin.

[0049] (3) Take the fish skin that has been ultrasonically treated in step (1), add compound protease, and for every gram of fish skin raw material, the enzyme activity of neutral protease is 5000U / g and the enzyme activity of trypsin is 3000U / g. Hydrolyze at 45℃ for 90min, centrifuge at 6000rpm / min for 10min, and freeze-dry the supernatant to obtain collagen active peptides.

[0050] (3) Take the dried active peptide powder, add distilled water to fully dissolve and adjust the concentration to 5 mg / mL, add 60 mmol / L zinc citrate solution at a volume ratio of 1:8, adjust the pH to 8, chelate at 60℃ for 30 min to obtain the reaction solution.

[0051] (4) Add anhydrous ethanol to the reaction solution at a volume ratio of 1:5 and let it stand for 24 hours. Centrifuge at 6000 rpm / min for 10 minutes. Load the precipitate onto an propylene dextran gel column and elute with 0.3 mol / L NaCl solution at a flow rate of 2 BV / h for 8 column volumes. Collect the elution fraction after 60 minutes, concentrate and freeze dry to obtain 327 g of chelate.

[0052] Example 3

[0053] A method for preparing a zinc ion chelate of tuna skin collagen peptides includes the following steps:

[0054] (1) After thawing 1 kg of tuna skin, add 8 mmol / L Tris-HCl at a mass-volume ratio of 1:15 g / mL to fully soak the skin, then transfer it to an ultrasonic extractor and sonicate at a frequency of 40 kHz for 25 min.

[0055] (2) Take the ultrasonicated tuna skin, add compound protease, the amount of neutral protease activity per gram of fish skin raw material is 3000 U / g, the amount of trypsin activity is 4000 U / g, hydrolyze at 35℃ for 30 min, centrifuge at 5000 rpm for 30 min, and freeze-dry the supernatant to obtain collagen active peptides.

[0056] (3) Take the dried active peptide powder, add distilled water to fully dissolve and adjust the concentration to 3 mg / mL, add 30 mmol / L zinc acetate solution at a volume ratio of 1:2, adjust the pH to 6, chelate at 40℃ for 60 min to obtain the reaction solution.

[0057] (4) Add anhydrous ethanol to the reaction solution at a volume ratio of 1:8 and let it stand for 10 h. Centrifuge at 5000 rpm / min for 30 min. Load the precipitate onto an propylene dextran gel column and elute with 0.6 mol / L NaCl solution at a flow rate of 2 BV / h for 4 column volumes. Collect the elution fraction after 30 min, concentrate and freeze dry to obtain 321 g of chelate.

[0058] Comparative Example 1

[0059] A method for preparing a zinc ion chelate of tuna skin collagen peptides differs from Example 1 in that:

[0060] (1) The zinc ion solution added during the chelation process was 50 mmol / L zinc sulfate.

[0061] (2) Other steps are the same as in Example 1, and 307g of chelate is obtained.

[0062] Comparative Example 2

[0063] A method for preparing a zinc ion chelate of tuna skin collagen peptides differs from Example 1 in that:

[0064] (1) The enzymatic hydrolysis process uses 4000 U / g trypsin;

[0065] (2) Other steps are the same as in Example 1, and 264g of chelate is obtained.

[0066] Comparative Example 3

[0067] A method for preparing a zinc ion chelate of tuna skin collagen peptides differs from Example 1 in that:

[0068] (1) The enzymatic hydrolysis process uses a compound protease. For each gram of fish skin raw material, the enzyme activity of neutral protease is 4500 U / g, the enzyme activity of trypsin is 3500 U / g and the enzyme activity of flavor protease is 2400 U / g. The temperature is 55℃ and the hydrolysis time is 120 min.

[0069] (2) Other steps are the same as in Example 1, and 316g of chelate is obtained.

[0070] Comparative Example 4

[0071] A method for preparing a tuna skin collagen peptide calcium ion chelate differs from Example 1 in that:

[0072] (1) Add 50 mmol / L calcium gluconate solution during the chelation process.

[0073] (2) The steps are the same as in Example 1, and 332g of chelate is obtained.

[0074] Comparative Example 5

[0075] A method for preparing tuna skin collagen peptides using traditional protease hydrolysis technology:

[0076] The difference from Example 1 is that the chelation reaction in steps (3) and (4) is not performed, but all other steps are the same, and 412g of collagen active peptides are obtained.

[0077] Comparative Example 6

[0078] A method for preparing soybean peptide zinc ion chelates, the specific steps of which are as follows:

[0079] 1 kg of soybeans were crushed, and the enzymatic hydrolysis and chelation reaction steps were the same as in Example 1, yielding 257 g of chelate.

[0080] Application Test Example 1

[0081] (1) Molecular weight distribution determination: The molecular weight distribution of collagen peptide-zinc ion chelates was determined by gel permeation chromatography (GPC). The chromatographic conditions were: Shodex OHpak SB-806M HQ size exclusion column; mobile phase: water + 0.1% trifluoroacetic acid; flow rate: 1 mL / min; column temperature: 40℃; detection wavelength: 205 nm. Standards were: cytochrome C (12384 Da), aprotinin (6511 Da), bacitracin (1450 Da), oxidized glutathione (651 Da), and Gly-Gly-Gly (189 Da). The distribution ratio of chelates with different molecular weights was calculated using the area normalization method based on the standard curve. The results are shown in Table 1.

[0082] (2) Water solubility test: Take 0.1g of the sample to be tested, gradually increase the volume of water, stir and mix for 10min, and then observe with the naked eye whether there is any undissolved sample. The test results are shown in Table 2.

[0083] Table 1

[0084]

[0085] Table 2

[0086]

[0087]

[0088] As shown in Table 1, the molecular weights of the collagen peptide zinc ion chelates prepared according to the method of the present invention are mostly distributed in the range of 3–5 kDa. The test results of Comparative Examples 1, 2, and 3 show that using inorganic zinc as the chelating agent or using different enzymatic hydrolysis methods to prepare active peptides will affect the molecular weight distribution of the final product, resulting in a decrease in the proportion of products with a molecular weight of 3–5 kDa.

[0089] As shown in Table 2, the water solubility of the chelate prepared by the method of this invention is significantly higher than that of the peptide sample obtained by the same enzymatic hydrolysis process in Comparative Example 5. Water solubility is a prerequisite for oral administration of drugs and a necessary condition for drug transport in vivo. Therefore, chelation of collagen peptides with zinc ions can effectively compensate for the corresponding deficiencies and improve absorption and utilization.

[0090] Application Test Example 2

[0091] (1) Calculation of chelate yield: Chelation yield (%) = (W1 / W2) × 100%

[0092] W1 represents the mass of the peptide zinc complex, and W2 represents the total mass of the initial reactants.

[0093] (2) Chelation rate determination: After dissolving the peptide zinc complex in 20 mL of NH3-NH4Cl buffer solution, add Eriochrome Black T indicator, titrate with 0.05 mol / L disodium ethylenediaminetetraacetate until light blue, record the volume of disodium ethylenediaminetetraacetate consumed, calculate the zinc chelation rate, and the test results are shown in Table 3.

[0094] Y(%) = (a × X / b) × 100%

[0095] Y represents the zinc chelation rate, X represents the mass fraction of zinc in the zinc peptide complex, a represents the mass of the zinc peptide complex, and b represents the amount of zinc added.

[0096] Table 3

[0097]

[0098] As shown in Table 3, the yield of the tuna skin collagen peptide zinc chelate prepared by this invention can reach more than 70%, and the chelation rate can reach more than 85%, which has the dual advantages of large amount of target component obtained and high chelation rate.

[0099] Comparative Example 1 used inorganic salts as chelating agents for the complex. Compared with Examples 1-3, the zinc ion chelation rate decreased significantly, indicating that the organic zinc salt chelation method used in this invention is more conducive to the reaction with tuna skin collagen peptides, reflecting the unique characteristics of this technology.

[0100] Comparative Example 2, compared to the technology of this invention, uses a single enzymatic hydrolysis preparation method. Due to the reduced enzymatic hydrolysis efficiency, the collagen peptides from tuna skin are not completely hydrolyzed, ultimately affecting the yield of the peptide zinc chelate product.

[0101] Comparative Example 3 used three compound enzymes to prepare tuna skin collagen peptides. Due to the influence of different enzymatic hydrolysis products on the reaction process, its zinc ion chelation rate was slightly lower than that of the product of this invention.

[0102] Comparative Example 6 attempted to prepare soybean peptide zinc ion chelates using the technology of this invention. It was found that the yield and chelation rate of the target component were much lower than those of the sample group in the example, indicating that the active peptide enzymatic hydrolysis-chelation technology established in this invention is specific for the preparation of tuna skin collagen peptide zinc ion chelates.

[0103] Application Test Example 3

[0104] Antioxidant experiment of tuna skin collagen peptide zinc chelate

[0105] (1) Mix 10 mL of 5 mmol / L ABTS solution and 0.5 mL of 150 mmol / L potassium persulfate solution thoroughly, and dilute with 0.05 mol / L pH 7.4 phosphate buffer until the absorbance at 734 nm is 0.68–0.72. Add 1 mL of sample solutions of different concentrations to 4 mL of ABTS solution and react in a water bath at 35 °C for 10 min. Measure the absorbance at 734 nm and calculate the ABTS radical scavenging rate.

[0106] ABTS radical scavenging rate (%) = 1 - (A1 - A2) / A0

[0107] A1: Absorbance after adding the sample; A2: Absorbance when water is used instead of ABTS solution; A0: Absorbance when water is used instead of the sample.

[0108] (2) Place 0.2 mL of samples of different concentrations in test tubes, add 1.0 mL of 0.2 mol / L calcium phosphate buffer and 1.0 mL of 1% potassium ferricyanide, react in a water bath at 55℃ for 20 min, then add 1.0 mL of 5% trichloroacetic acid solution to terminate the reaction, and centrifuge at 3000 r / min for 10 min. Take 2.0 mL of the supernatant, add 2.0 mL of distilled water and 0.5 mL of 0.5% FeCl3 solution, mix well and let stand for 15 min, measure the absorbance value A1 at 700 nm wavelength, and measure the absorbance value A2 by replacing the sample solution with water, and calculate the total reducing power.

[0109] Total reducing power = A1 - A2; (A1: absorbance of the sample group, A2: absorbance of the water-substituted sample group)

[0110] (3) Forty-eight six-week-old male Kunming mice were selected and acclimatized for 7 days in an animal room with an ambient temperature of 20–25℃ and a relative humidity of 50%–60%. Subsequently, six mice in each group were administered the drug. The model control group was administered 0.5 mL of physiological saline by gavage, while the example and comparative groups were administered their respective samples by gavage at a dose of 50 mg / kg. After four weeks of continuous gavage, each group of mice was administered 10 mL / kg of 50% ethanol solution by gavage to establish a mouse model of ethanol oxidative damage. Six hours after ethanol gavage, blood was collected from the orbital sinus of the mice. Serum was separated by centrifugation at 3000 rpm / min for 15 min at 4℃. The activity indicators of ALT, AST, and AKP were measured according to the kit requirements. The results are shown in Table 4.

[0111] Table 4

[0112]

[0113]

[0114] * Compared with the model group, P < 0.05; ** Compared with the model group, P < 0.01

[0115] from Figure 1 and Figure 2In vitro experiments showed that the ABTS scavenging rate and total reducing capacity of the samples gradually increased with increasing concentration. At the same concentration, the antioxidant capacity of the example samples was consistently higher than that of the comparative samples, but the overall difference was not significant. Table 4 shows that the in vivo experiments at the tested concentrations significantly affected the activities of ALT, AST, and AKP in mouse serum (P<0.01), exhibiting statistical differences. The experimental results confirm that the antioxidant activity of collagen active peptides can be enhanced after zinc ion chelation, and the enhanced activity is closely related to the degree of chelation. The in vivo detection effect of the examples was better than that of the in vitro experiments, possibly because the peptide zinc chelate has higher bioavailability in vivo. The products prepared by comparative examples 1-3 and comparative example 5 showed weaker improvements in ALT, AST, and AKP than the example group, indicating that the chelate prepared in this invention has a superior effect.

[0116] Application Test Example 4

[0117] (1) Tyrosinase inhibition experiment

[0118] Prepare 0.2 mol / L PBS solution and 100 U / mL tyrosinase solution. Using a 96-well plate as a carrier, set up a sample group, a sample background control group, a blank group, and a blank control group. Add 20 μL of 50 μg / mL sample solution and 10 μL of tyrosinase solution to the 96-well plate sequentially, incubate at 37°C for 10 min, then add 40 μL of L-DOPA solution, and continue incubation at 37°C for 5 min. Measure the absorbance at 475 nm to obtain the absorbance value of the sample group (A1). Replace 10 μL of 10 μL of tyrosinase solution with 10 μL of PBS to obtain the absorbance value of the sample background control group (A2). Replace 20 μL of sample solution with 20 μL of PBS to obtain the absorbance value of the blank group (A3). Replace 20 μL of sample solution and 10 μL of tyrosinase solution with 30 μL of PBS to obtain the absorbance value of the blank control group (A4).

[0119] Tyrosinase inhibition rate (%) = [1 - (A1 - A2) / (A3 - A4)] × 100%

[0120] (2) Anti-aging effect on H2O2-induced HaCaT cells

[0121] HaCaT cells were seeded in 6-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h. Control, H2O2 model, and drug-treated groups were established. The control and H2O2 model groups were cultured in 2.5 mL of 1% serum-containing DMEM medium for 24 h, followed by treatment with 200 μmol / L H2O2 for 4 h. The drug-treated groups were pretreated with 50 μg / mL sample for 24 h, followed by treatment with 200 μmol / L H2O2 for 4 h. Cells were collected, and the levels of SOD, GSH, and MDA were measured according to the kit instructions. The results are shown in Table 5.

[0122] Table 5

[0123]

[0124] As shown in Table 5, the sample from the examples exhibits high tyrosinase inhibitory activity and shows an increasing trend in SOD and GSH activity and a decreasing trend in MDA level at the cellular level, demonstrating superior effects compared to the comparative groups. This indicates that the chelates prepared in this invention have potential applications in skin care and anti-aging. Compared to the examples, Comparative Examples 1, 2, and 3 show some differences in the composition of the final chelate products due to adjustments in the chelation and enzymatic hydrolysis preparation processes, thus affecting their experimental results. Comparative Example 4, the prepared peptide-calcium chelate product, while showing strong tyrosinase inhibitory activity, does not exhibit prominent HaCaT cell protective activity, differing somewhat from the activity characteristics of the peptide-zinc chelate. Comparative Example 5 uses a traditional proteolytic hydrolysis process, and Comparative Example 6 uses plant-derived peptides for zinc ion chelation; both showed weaker activity than the example groups at the tested concentrations. The peptide-zinc chelate prepared in this invention can exert excellent skin protective effects even at low concentrations, significantly improving the quality of existing collagen products, further proving the superior efficacy of the chelates prepared in this invention.

[0125] Application Test Example 5

[0126] ACE inhibition assay of tuna skin collagen peptide zinc chelate

[0127] (1) Take 50 μL of 2.5 mmol / L Hip-His-Leu and add 50 μL of samples of different concentrations respectively. Preheat in a water bath at 37℃ for 5 min. Then add 40 μL of 10 mg / mL ACE solution and shake well. Continue to react in a water bath at 37℃ for 30 min. Add 200 μL of 0.5 mol / L HCl to stop the reaction.

[0128] (2) Add 1 mL of ethyl acetate to the above reaction solution, shake to mix and separate hippuric acid. Centrifuge at 4000 r / min for 15 min, take 750 μL of ester layer and dry in an oven at 120 °C. Add 3 mL of distilled water and shake to dissolve. Use 50 μL of boric acid buffer solution to replace the sample as a blank. Measure the absorbance at a UV wavelength of 228 nm and calculate the ACE inhibition rate.

[0129] ACE inhibition rate (%) = (BA) / (B-B0) × 100

[0130] In the formula: A is the absorbance when the sample is added; B is the absorbance when the sample is not added; B0 is the absorbance of the blank reaction.

[0131] Table 5 ACE inhibitory activity

[0132]

[0133] Normal blood pressure is closely related to the renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS). Angiotensin-converting enzyme (ACE) plays an important role in both systems and is widely present in human tissues and plasma, mainly in vascular endothelial cells. ACE inhibitors have a greater affinity for the active ACE site than angiotensin I and bradykinin for ACE, and once bound to the active ACE site, they are difficult to release. This inhibits the two biochemical reactions catalyzed by ACE—the hydrolysis of angiotensin I to angiotensin II and the hydrolysis of bradykinin to inactive fragments—thus lowering blood pressure.

[0134] The ACE inhibitory activity of the example group increased with increasing concentration, exhibiting a dose-dependent effect, although the increase in inhibition rate was slightly slower at high concentrations. In contrast, the bioactive peptide obtained by the conventional enzymatic hydrolysis method in Comparative Example 5 showed significantly weaker ACE inhibitory activity than the peptide zinc chelate product of the examples. Therefore, the peptide zinc ion chelate prepared in this invention enhances the efficacy of the original active peptide product. Similarly, due to changes in the preparation process, Comparative Examples 1, 2, and 3 produced final products with different molecular weights and degrees of chelation; experiments showed that their ACE inhibitory activity was weaker than that of the chelate prepared in this invention.

[0135] In summary, the preparation method described in this invention can obtain tuna skin collagen peptide zinc ion chelate products, and has the dual advantages of large target component acquisition and high chelation rate. The chelate yield can reach over 70%, and the chelation rate can reach over 85%. The molecular weight distribution of the peptide zinc chelate product is over 49% by mass, with a molecular weight distribution of 3-5 kDa. This process is simple and convenient to operate, low in cost, and has good safety, and can be scaled up for mass production. The obtained peptide zinc chelate products have good biological activity, and can significantly reduce the activity of ALT, AST, and AKP in the serum of mice damaged by ethanol oxidation; increase the SOD and GSH levels in HaCaT cells damaged by H2O2; and inhibit the catalytic reaction process of ACE enzyme. It has the characteristics of simple operation process and excellent activity, which greatly improves the quality of bioactive peptide products. It has good application prospects in scavenging free radicals in the body, inhibiting tyrosinase, protecting skin keratinocytes, and regulating blood pressure. It has antioxidant, skin protection, and blood pressure lowering effects. It can be used as an active ingredient in the preparation of food, cosmetics, and even pharmaceuticals, and is easily accepted by consumers.

Claims

1. A method for preparing a tuna skin collagen peptide-zinc ion chelate, characterized by, It comprises the following steps: (1) After the tuna skin is thawed, Tris-HCl is added at a mass volume ratio g / mL of 1:(5-15) for infiltration, followed by ultrasonic treatment for 5-30 min to obtain ultrasonic-treated fish skin; (2) The ultrasonic-treated fish skin obtained in step (1) is added with complex protease for hydrolysis for 15-90 min, and then solid-liquid separation is performed, and the liquid is freeze-dried to obtain collagen active peptide; (3) The collagen active peptide prepared in step (2) is dissolved in water, zinc ion solution is added at a volume ratio of 1:(2-8), the pH is adjusted to 6-8, and chelation is performed for 20-80 min to obtain a reaction solution; (4) The reaction solution obtained in step (3) is added with anhydrous ethanol and left to stand for 8-24 h, then solid-liquid separation is performed, and the solid is loaded on a propylene dextran gel column, eluted with 4-8 times the volume of NaCl solution, and the middle section of the elution fraction is collected, concentrated and freeze-dried to obtain a tuna skin collagen peptide-zinc ion chelate; In step (1), the concentration of the Tris-HCl solution is 8-20 mmol / L, and the ultrasonic frequency is 30-80 KHz. In step (2), the complex protease is neutral protease and trypsin, the enzyme activity of the neutral protease is 3000-5500 U / g per gram of fish skin raw material, and the enzyme activity of the trypsin is 2500-4500 U / g per gram of fish skin raw material; the enzymolysis temperature is 30-50℃. In step (3), the concentration of the active peptide dissolved in water is 3-6 mg / mL; the zinc ion solution is zinc gluconate, zinc citrate or zinc acetate, and the concentration is 20-80 mmol / L; the chelation temperature is 40-70℃. The tuna skin collagen peptide-zinc ion chelate prepared in step (4) has a chelate yield of more than 70%, a chelation rate of more than 85%, and a mass percentage of molecular weight distribution of 3 kDa-5 kDa of more than 49%.

2. The method of claim 1, wherein, In step (1), the concentration of the Tris-HCl solution is 6-15 mmol / L, and the ultrasonic frequency is 40-60 KHz.

3. The method of claim 2, wherein, The concentration of the Tris-HCl solution is 10 mmol / L, and the ultrasonic frequency is 50 KHz.

4. The method of claim 1, wherein, In step (2), the solid-liquid separation method is centrifugation at 3000-6000 rpm / min for 10-30 min, and the supernatant is freeze-dried to obtain collagen active peptide.

5. The method of claim 1, wherein, In step (2), the enzyme activity of the neutral protease is 4000-5000 U / g per gram of fish skin raw material, and the enzyme activity of the trypsin is 3000-4000 U / g per gram of fish skin raw material; the enzymolysis temperature is 35-45℃.

6. The method of claim 5, wherein, In step (2), the enzyme activity of the neutral protease is 4500 U / g per gram of fish skin raw material, and the enzyme activity of the trypsin is 3500 U / g per gram of fish skin raw material; the enzymolysis temperature is 40℃.

7. The method of claim 1, wherein, In step (3), the concentration of the active peptide dissolved in water is 4-5 mg / mL; the zinc ion solution is zinc gluconate or zinc citrate, and the concentration is 30-60 mmol / L; the chelation temperature is 45-60℃.

8. The method of claim 7, wherein, In step (3), the concentration of the active peptide dissolved in water is 4.5 mg / mL; the zinc ion solution is zinc gluconate, and the concentration is 50 mmol / L; and the chelation temperature is 50°C.

9. The method of claim 1, wherein, In step (4), the volume ratio of the reaction solution to anhydrous ethanol is 1: (4-8); and the concentration of the NaCl eluent is 0.15-0.6 mol / L.

10. The method of claim 9, wherein, In step (4), the volume ratio of the reaction solution to anhydrous ethanol is 1: (5-7); and the concentration of the NaCl eluent is 0.3-0.5 mol / L.

11. The method of claim 10, wherein, In step (4), the volume ratio of the reaction solution to anhydrous ethanol is 1:6; and the concentration of the NaCl eluent is 0.4 mol / L.

12. The method of claim 1, wherein, In step (4), the solid-liquid separation is performed by centrifugation at 3000-6000 rpm / min for 10-30 min, and the precipitate is loaded onto a Sephadex column; In step (4), the elution fraction in the later stage is collected, specifically, the column is eluted with a NaCl solution at a flow rate of 2 BV / h for 4-8 times the column volume, and the elution fraction collected in 30-60 min is collected.

13. The application of the tuna skin collagen peptide-zinc ion chelate prepared by the method of any one of claims 1-12 as an effective component in the preparation of daily-use products or health-care food with antioxidant function.

14. The application of the tuna skin collagen peptide-zinc ion chelate prepared by the method of any one of claims 1-12 as an effective component in the preparation of an antioxidant, skin care product, or blood pressure-lowering functional drug.

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