Fish collagen peptide with liver protection and antioxidant activity as well as preparation method and application of fish collagen peptide

Abstract

The invention particularly relates to fish collagen peptide with liver protection and antioxidant activity as well as a preparation method and application thereof, and belongs to the technical field of collagen peptide preparation. The preparation method comprises the following steps: taking fish skin or fish scale as a raw material, and extracting fish collagen through an alkali extraction and acid precipitation method; carrying out enzymolysis on the obtained fish collagen by using chitosan microsphere immobilized alkaline protease, wherein the addition amount of the enzyme is 800-1000U per gram of substrate; and carrying out high-temperature inactivation on the enzymatic hydrolysate, and sequentially carrying out ultrafiltration and DEAE-52 ion exchange chromatography purification to obtain a target product. The immobilized enzyme technology and the combined purification technology are combined, the extraction efficiency and product purity of the collagen peptide are remarkably improved, and the obtained peptide fragment is small in molecular weight and high in characteristic amino acid content, has excellent liver protection and antioxidant activity and can be used for preparing various products such as health care products for dispelling the effects of alcohol and protecting the liver, anti-aging cosmetics and the like.

Description

Technical Field

[0001] This invention belongs to the field of collagen peptide preparation technology, specifically relating to a fish collagen peptide with hepatoprotective and antioxidant activities, its preparation method, and its applications. Background Technology

[0002] Fish resources, as an important source of protein, generate numerous byproducts during aquatic product processing, such as fish skin, scales, and bones, which are rich in collagen. Traditional fish collagen is mostly a large molecular structure, typically with a molecular weight of 100–300 kDa or higher. Its poor solubility and instability result in low absorption and utilization efficiency in the human body, limiting its functional performance. In contrast, enzymatic hydrolysis of fish collagen into smaller peptides can significantly improve its solubility and bioactivity. Studies have shown that oligopeptides with a molecular weight of 1–5 kDa are more easily translocated through the small intestinal mucosa into the bloodstream, significantly increasing bioavailability.

[0003] In recent years, fish collagen peptides have shown excellent functional properties in anti-oxidation, anti-fatigue, blood pressure reduction, and liver and skin protection, especially in free radical scavenging and liver protection, where they have potential application value. Existing patent CN120309716A discloses an anti-glycation and anti-aging cod collagen peptide, its preparation method, and its applications. This method utilizes ultrasonic-assisted nano-grinding pretreatment, combined with stepwise directional enzymatic hydrolysis and three-stage ultrafiltration membrane precision separation technology, significantly improving the yield and functional properties of active peptides. However, the step-by-step directional enzymatic hydrolysis process has strict temperature, pH, and time control requirements and is complex, placing high demands on equipment and control systems. Existing patent CN120309714A discloses a fish collagen peptide with skin-brightening and spot-removing effects, its preparation method, and its applications. Using fish skin and / or scales as raw materials, after degreasing and deodorizing, the peptides undergo sequential heat treatment, homogenization, enzymatic hydrolysis, and enzyme inactivation purification to obtain fish collagen peptides. The fish collagen peptides prepared by this invention contain over 90% polypeptides of 189-3000 Da and a protein content of over 99.23%, exhibiting excellent skin-brightening and blemish-removing effects. However, this process involves numerous influencing factors, including temperature treatment, homogenization (high-speed shearing, emulsification, homogenization, and blending), and degreasing and deodorizing treatment (soaking in oxidants and acids or alkalis), making the process complex. Furthermore, the use of free complex enzymes makes recycling and reuse difficult, resulting in high production costs and hindering large-scale application. Summary of the Invention

[0004] To address the problems of low extraction rate, complex processing conditions, and high cost in existing fish collagen peptide technologies, there is an urgent need to develop a simple and reasonable preparation process for fish collagen peptides that achieves high extraction rate, low cost, and hepatoprotective and antioxidant activities, as well as a method for their preparation. The purpose of this invention is to provide a fish collagen peptide with hepatoprotective and antioxidant activities, its preparation method, and its applications. This method utilizes a chitosan microsphere immobilization enzymatic hydrolysis process to prepare fish collagen peptides, combined with ultrafiltration separation and ion exchange purification technology. This not only yields high-purity, high-activity low-molecular-weight peptides but also meets food safety and industrialization requirements, possessing significant scientific research and application value.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention discloses a method for preparing fish collagen peptides with hepatoprotective and anti-aging activities, comprising the following steps:

[0007] (1) Fish collagen was extracted using the alkaline extraction and acid precipitation method;

[0008] (2) Fish collagen peptides were extracted by microsphere immobilization of alkaline protease to obtain enzymatic hydrolysate;

[0009] (3) High-temperature inactivation of the enzyme hydrolysate;

[0010] (4) The inactivated enzyme hydrolysate is subjected to ultrafiltration and ion exchange chromatography to obtain fish collagen peptides.

[0011] Specifically, the microspheres are selected from chitosan microspheres.

[0012] Chitosan, derived from the shells of crustaceans, possesses excellent biocompatibility, biodegradability, and food safety. Its surface amino groups facilitate binding with enzyme molecules, making it particularly suitable as a carrier for enzyme immobilization in the food and health product industries. Immobilizing alkaline proteases using chitosan microspheres not only improves enzyme stability and tolerance but also enables enzyme reuse, reduces costs, and ensures the entire process meets food-grade standards.

[0013] Specifically, the amount of alkaline protease immobilized on the microspheres is 800-1000 units of enzyme activity per gram of substrate.

[0014] In some embodiments, the specific steps of the alkali extraction and acid precipitation method are as follows:

[0015] The raw materials were dried in a 60℃ drying oven to constant weight and then ground into powder to obtain fish collagen powder. The fish collagen powder was dissolved in distilled water and ultrasonically broken for 20 min to obtain fish collagen solution. The pH of the fish collagen solution was adjusted to 11.0 with 0.1 mol / L NaOH solution, and after extraction for 2 h, it was centrifuged at 5000 rpm for 10 min. The supernatant was collected, and the pH of the supernatant was adjusted to the isoelectric point with 0.1 mol / L HCl. Then, it was allowed to settle for 2 h. After the precipitate was formed, it was centrifuged again at 5000 rpm for 10 min to obtain fish collagen precipitate.

[0016] In some embodiments, the method of immobilizing alkaline protease on microspheres includes:

[0017] (1) Dissolve chitosan in acetic acid solution to prepare a 2-3% chitosan solution, then add liquid paraffin and emulsifier dropwise and stir magnetically to form an oil-in-water emulsion; add glutaraldehyde dropwise at 50-60℃ for crosslinking, and stir for 2-3 hours to generate chitosan microspheres with a particle size of 50-150 μm.

[0018] (2) After washing the chitosan microspheres obtained in step (1), disperse them in PBS buffer at pH 7.4, add alkaline protease solution (concentration 10 mg / mL), and gently shake at 4°C for 24 h to immobilize the enzyme on the surface of the chitosan microspheres through the interaction of amino and aldehyde groups.

[0019] (3) Centrifuge to collect immobilized enzyme microspheres, wash with PBS to remove unbound enzymes, and freeze dry for later use.

[0020] Specifically, the conditions for enzymatic hydrolysis include: a hydrolysis temperature of 35-55℃ and a hydrolysis time of 1-3 hours.

[0021] Specifically, the conditions for high-temperature inactivation in step (3) include: a temperature of 80-90℃ and a time of 20-30min.

[0022] Specifically, the raw material is preferably fish skin or fish scales, but may also be other parts rich in collagen.

[0023] Specifically, the ultrafiltration process includes: ultrafiltration of the inactivated enzyme hydrolysate using an ultrafiltration membrane with a molecular weight cutoff of 1-8 kDa under the conditions of a membrane pressure of 1.5-4 Bar, a feed temperature of 25-35°C, a flow rate of 3-7 LMH, and a pH of 6-8, and collection of the permeate or retentate.

[0024] The ion exchange chromatography process employed DEAE-52 cellulose ion exchange chromatography. The specific steps included: uniformly loading fully swollen DEAE-52 cellulose packing material into the chromatography column, avoiding the generation of air bubbles; observing the gel bed's condition until it became smooth after the column bed stabilized and the interface became flat; passing an appropriate amount of sample solution through a 0.22 μm filter membrane; adding ultrapure water to the equilibrated chromatography column for elution at a flow rate of 2 mL / min; monitoring the UV absorption at 249 nm; and collecting the eluted fraction by volume or time.

[0025] Secondly, this invention discloses a fish collagen peptide with hepatoprotective and antioxidant activities prepared by the aforementioned method.

[0026] The fish collagen peptide contains more than 80% small molecule peptides, and its total content of glycine (Gly), proline (Pro), and hydroxyproline (Hyp) accounts for more than 50% of the total mass of amino acids.

[0027] Thirdly, this invention discloses the application of the aforementioned fish collagen peptides in the preparation of products with hepatoprotective and antioxidant activities.

[0028] Specifically, this includes: products for preventing or assisting in the treatment of chemically induced liver damage, especially products for relieving hangovers and protecting the liver; cosmetics or skincare products that eliminate free radicals and combat skin aging; and health supplements or functional foods that enhance the body's antioxidant capacity.

[0029] The products include tablets, capsules, oral liquids, beverages, powders, serums, lotions, or creams.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The "alkali extraction and acid precipitation method" combined with the "chitosan microsphere immobilization and enzymatic hydrolysis" technology significantly improved the extraction rate of fish collagen and the efficiency of subsequent enzymatic hydrolysis. The immobilized enzyme can be reused, reducing enzyme consumption, simplifying subsequent separation steps, and lowering production costs. Moreover, the entire process is mild, avoiding the destruction of peptide activity by strong acids, strong alkalis or high temperatures, which meets the requirements of green environmental protection.

[0032] (2) Combining ultrafiltration with DEAE-52 cellulose ion exchange chromatography, high-purity small molecule peptides with molecular weights concentrated in the range of 1-8 kDa (small molecule peptides account for more than 80%) can be accurately obtained. The product is rich in characteristic amino acids such as glycine, proline and hydroxyproline, which give it good liver protection (such as increasing SOD and ADH activity and reducing MDA content) and antioxidant bioactivity;

[0033] (3) The enzymatic hydrolysis process is stable and controllable and easy to industrialize: Using chitosan microspheres as an immobilization carrier significantly improves the thermal stability and operational stability of alkaline protease, making the enzymatic hydrolysis reaction conditions (such as temperature and pH) easier to control, and the process has good reproducibility, laying a solid foundation for large-scale industrial continuous production.

[0034] (4) The fish collagen peptides obtained have clear liver protection (hemostasis and liver protection) and antioxidant (anti-skin aging) effects. They can be widely used in functional foods, health foods, cosmetics and other fields. The products are diverse and meet different market demands. They have important scientific research value and broad commercial application prospects. Detailed Implementation

[0035] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0036] The following examples illustrate the preparation method of chitosan microspheres immobilized with alkaline protease, specifically including:

[0037] (1) Chitosan was dissolved in 1% acetic acid solution to prepare 2% chitosan solution, and then liquid paraffin and a small amount of emulsifier were added dropwise to form an oil-in-water emulsion under magnetic stirring; glutaraldehyde was added dropwise at 50°C for crosslinking, and after stirring for 2 hours, chitosan microspheres with a particle size of 50–150 μm were generated.

[0038] (2) After washing the chitosan microspheres obtained in step (1), disperse them in PBS buffer at pH 7.4, add alkaline protease solution (concentration 10 mg / mL), and gently shake at 4°C for 24 h to immobilize the enzyme on the surface of the chitosan microspheres through the interaction of amino and aldehyde groups.

[0039] (3) Centrifuge to collect immobilized enzyme microspheres, wash with PBS to remove unbound enzymes, and freeze dry for later use.

[0040] Example 1

[0041] A method for preparing fish collagen peptides with hepatoprotective and antioxidant activities, comprising the following steps:

[0042] (1) Fish skin or scales were dried in a 60℃ drying oven to constant weight and ground into powder to obtain fish collagen powder; fish collagen powder was dissolved in distilled water and ultrasonically crushed for 20 min to obtain fish collagen solution; the pH of the fish collagen solution was adjusted to 11.0 with 0.1 mol / L NaOH solution, and after extraction treatment for 2 h, it was centrifuged at 5000 rpm for 10 min; the supernatant was taken out, and the pH of the supernatant was adjusted to the isoelectric point with 0.1 mol / L HCl, and then settled for 2 h; after the precipitate was precipitated, it was centrifuged at 5000 rpm for 10 min again to obtain fish collagen precipitate, and the fish collagen precipitate was adjusted to neutral and then freeze-dried to obtain fish collagen powder, which was stored in a desiccator.

[0043] (2) Dissolve fish collagen powder in distilled water at a ratio of 1:40 at 50°C for 60 min, adjust the pH of the fish collagen solution to 11 with 0.1 mol / L NaOH solution, add 800 U / g chitosan microspheres to immobilize alkaline protease and enzymatically hydrolyze at 45°C for 2 h, then heat to 90°C to inactivate for 30 min.

[0044] (3) The enzymatic hydrolysate obtained in step (2) was subjected to ultrafiltration using ultrafiltration membranes with molecular weight cutoffs of 8kDa, 4kDa, and 1kDa under the conditions of membrane pressure of 2 Bar, feed temperature of 25℃, flow rate of 5LMH, and pH of 7. Components with different molecular weight ranges were collected. Components with molecular weight less than 1kDa were identified as target fish collagen peptides. The target fish collagen peptides were freeze-dried to prepare fish collagen peptide freeze-dried powder for subsequent use.

[0045] (4) Prepare a 50 mg / mL solution of fish collagen peptide lyophilized powder with ultrapure water. Pack the fully swollen DEAE-52 cellulose packing material into the chromatography column at a uniform speed, avoiding the generation of air bubbles. After the column bed stabilizes and the interface is smooth, observe the state of the gel bed until it is smooth. Take an appropriate amount of sample solution and filter it through a 0.22 μm filter membrane. Add ultrapure water to the equilibrated chromatography column for elution at a flow rate of 2 mL / min. Monitor the UV absorption at a wavelength of 249 nm and collect the eluted fraction by volume or time. The eluted fraction is the purified fish collagen peptide.

[0046] Example 2

[0047] The difference between this embodiment and embodiment 1 is that the specific parameters involved in steps (2) and (3) are different, while the others are the same.

[0048] (2) Dissolve fish collagen powder in distilled water at a ratio of 1:40 at 50°C for 60 min, adjust the pH of the fish collagen solution to 11 with 0.1 mol / L NaOH solution, add 850 U / g chitosan microspheres to immobilize alkaline protease and enzymatically hydrolyze at 35°C for 1.5 h, then heat to 85°C to inactivate for 20 min.

[0049] (3) The enzymatic hydrolysate obtained in step (2) was subjected to ultrafiltration using ultrafiltration membranes with molecular weight cutoffs of 8kDa, 4kDa, and 1kDa under the conditions of membrane pressure of 3 Bar, feed temperature of 28℃, flow rate of 4LMH, and pH of 6.5. Components with different molecular weight ranges were collected. Components with molecular weight less than 1kDa were identified as target fish collagen peptides. The target fish collagen peptides were freeze-dried to prepare fish collagen peptide freeze-dried powder for subsequent use.

[0050] Example 3

[0051] The difference between this embodiment and embodiment 1 is that the specific parameters involved in steps (2) and (3) are different, while the others are the same.

[0052] (2) Dissolve fish collagen powder in distilled water at a ratio of 1:40 at 50°C for 60 min, adjust the pH of the fish collagen solution to 11 with 0.1 mol / L NaOH solution, add 900 U / g chitosan microspheres to immobilize alkaline protease and hydrolyze at 55°C for 2.5 h, then heat to 90°C to inactivate for 25 min.

[0053] (3) The enzymatic hydrolysate obtained in step (2) was subjected to ultrafiltration using ultrafiltration membranes with molecular weight cutoffs of 8kDa, 4kDa, and 1kDa under the conditions of membrane pressure of 4 Bar, feed temperature of 30℃, flow rate of 6LMH, and pH of 7.5. Components with different molecular weight ranges were collected. Components with molecular weight less than 1kDa were identified as target fish collagen peptides. The target fish collagen peptides were freeze-dried to prepare fish collagen peptide freeze-dried powder for subsequent use.

[0054] Example 4

[0055] The difference between this embodiment and embodiment 1 is that the specific parameters involved in steps (2) and (3) are different, while the others are the same.

[0056] (2) Dissolve fish collagen powder in distilled water at a ratio of 1:40 at 50°C for 60 min, adjust the pH of the fish collagen solution to 11 with 0.1 mol / L NaOH solution, add 820 U / g chitosan microspheres to immobilize alkaline protease and hydrolyze at 40°C for 1.8 h, then heat to 88°C to inactivate for 30 min.

[0057] (3) The enzymatic hydrolysate obtained in step (2) was subjected to ultrafiltration using ultrafiltration membranes with molecular weight cutoffs of 8kDa, 4kDa, and 1kDa under the conditions of membrane pressure of 1.5 Bar, feed temperature of 26℃, flow rate of 3LMH, and pH of 6.8. Components with different molecular weight ranges were collected. Components with molecular weight less than 1kDa were identified as target fish collagen peptides. The target fish collagen peptides were freeze-dried to prepare fish collagen peptide freeze-dried powder for subsequent use.

[0058] Example 5

[0059] The difference between this embodiment and embodiment 1 is that the specific parameters involved in steps (2) and (3) are different, while the others are the same.

[0060] (2) Dissolve fish collagen powder in distilled water at a ratio of 1:40 at 50°C for 60 min, adjust the pH of the fish collagen solution to 11 with 0.1 mol / L NaOH solution, add 830 U / g chitosan microspheres to immobilize alkaline protease and hydrolyze at 50°C for 2 h, then heat to 90°C to inactivate for 30 min.

[0061] (3) The enzymatic hydrolysate obtained in step (2) was subjected to ultrafiltration using ultrafiltration membranes with molecular weight cutoffs of 8kDa, 4kDa, and 1kDa under the conditions of membrane pressure of 1.5 Bar, feed temperature of 27℃, flow rate of 6LMH, and pH of 7. Components with different molecular weight ranges were collected. Components with molecular weight less than 1kDa were identified as target fish collagen peptides. The target fish collagen peptides were freeze-dried to prepare fish collagen peptide freeze-dried powder for subsequent use.

[0062] Example 6

[0063] The difference between this embodiment and embodiment 1 is that the specific parameters involved in steps (2) and (3) are different, while the others are the same.

[0064] (2) Dissolve fish collagen powder in distilled water at a ratio of 1:40 at 50°C for 60 min, adjust the pH of the fish collagen solution to 11 with 0.1 mol / L NaOH solution, add 810 U / g chitosan microspheres to immobilize alkaline protease and enzymatically hydrolyze at 40°C for 2 h, then heat to 90°C to inactivate for 30 min.

[0065] (3) The enzymatic hydrolysate obtained in step (2) was subjected to ultrafiltration using ultrafiltration membranes with molecular weight cutoffs of 8kDa, 4kDa, and 1kDa under the conditions of membrane pressure of 1.5 Bar, feed temperature of 30℃, flow rate of 5LMH, and pH of 7.2. Components with different molecular weight ranges were collected. Components with molecular weight less than 1kDa were identified as target fish collagen peptides. The target fish collagen peptides were freeze-dried to prepare fish collagen peptide freeze-dried powder for subsequent use.

[0066] Comparative Example 1

[0067] The difference between this comparative example and Example 1 is that step (2) uses free alkaline protease for enzymatic hydrolysis, while the rest is the same as in Example 1.

[0068] (2) Dissolve fish collagen powder in distilled water at a ratio of 1:40 at 50°C for 60 min, adjust the pH of the fish collagen solution to 11 with 0.1 mol / L NaOH solution, add 800 U / g free alkaline protease and hydrolyze at 45°C for 2 h, then heat to 90°C to inactivate for 30 min.

[0069] Comparative Example 2

[0070] The difference between this comparative example and Example 1 is that step (2) uses sodium alginate microspheres to immobilize enzymes for enzymatic hydrolysis, while the rest is the same as in Example 1.

[0071] Preparation method of enzyme immobilized by sodium alginate microspheres: Prepare a 2% sodium alginate solution (dissolved in ultrapure water), add it dropwise to a 2% CaCl2 solution under magnetic stirring to form microspheres, let it stand for 2 hours to solidify, and collect the microspheres by filtration; disperse the microspheres in PBS buffer at pH 7.4, add alkaline protease solution (concentration 10 mg / mL), gently shake at 4℃ for 24 hours, then crosslink with 0.5% glutaraldehyde solution for 1 hour, wash with PBS to remove unbound enzymes, and freeze dry for storage.

[0072] (2) Dissolve fish collagen powder in distilled water at a ratio of 1:40 at 50°C for 60 min, adjust the pH of the fish collagen solution to 11 with 0.1 mol / L NaOH solution, add 800 U / g sodium alginate microspheres to immobilize alkaline protease and hydrolyze at 45°C for 2 h, then heat to 90°C to inactivate for 30 min.

[0073] Comparative Example 3

[0074] The difference between this comparative example and Example 1 is that step (2) uses enzyme immobilized with magnetic Fe3O4 nanoparticles for enzymatic hydrolysis, while the rest is the same as in Example 1.

[0075] Preparation method of alkaline protease immobilized on magnetic Fe3O4 nanoparticles: Magnetic Fe3O4 nanoparticles were prepared by co-precipitation and modified with amino groups using 3-aminopropyltriethoxysilane (APTES). The modified Fe3O4 nanoparticles were dispersed in PBS buffer at pH 7.4, and alkaline protease solution (concentration 10 mg / mL) was added. The mixture was gently shaken at 4℃ for 24 h, cross-linked with glutaraldehyde, and the immobilized enzyme was magnetically separated and collected. Unbound enzyme was removed by washing with PBS and then lyophilized for storage.

[0076] (2) Dissolve fish collagen powder in distilled water at a ratio of 1:40 at 50°C for 60 min, adjust the pH of the fish collagen solution to 11 with 0.1 mol / L NaOH solution, add 800 U / g magnetic Fe3O4 nanoparticles to immobilize alkaline protease and hydrolyze at 45°C for 2 h, then heat to 90°C to inactivate for 30 min.

[0077] Comparative Example 4

[0078] The difference between this comparative example and Example 1 is that step (2) uses commercially available immobilized alkaline protease (brand: Novozymes, model: (Immobilized), otherwise the same as in Example 1.

[0079] (2) Dissolve fish collagen powder in distilled water at a ratio of 1:40 at 50°C for 60 min, adjust the pH of the fish collagen solution to 11 with 0.1 mol / L NaOH solution, add 800 U / g of commercially available immobilized alkaline protease and hydrolyze at 45°C for 2 h, then heat to 90°C to inactivate for 30 min.

[0080] Example 1: Antioxidant assay of fish collagen peptides

[0081] The DPPH free radical scavenging rate of the fish collagen peptides prepared in the above examples and comparative examples was detected.

[0082] DPPH free radical scavenging test: Take 2 mL of 12 mM sample methanol solution, add 2 mL of 0.2 mM DPPH solution prepared with anhydrous methanol, mix thoroughly, and place in a dark room for about 30 min. Measure the absorbance of the sample at a wavelength of 517 nm (A). 样品 Subsequently, 2 mL of methanol was used to replace the sample methanol solution, and 2 mL of the above DPPH solution was added. After thorough mixing, the solution was placed in a dark room for about 30 minutes, and the absorbance of the blank solution was measured at a wavelength of 517 nm (A). 空白 Take another 2 mL of the sample methanol solution, add 2 mL of anhydrous methanol, mix thoroughly, and place in a dark room for about 30 min. Measure the absorbance of the drug solution itself at a wavelength of 517 nm (A). 对照 The clearance rate formula is as follows:

[0083]

[0084] The DPPH free radical scavenging results are shown in Table 1. The fish collagen peptides prepared in the embodiments of this invention have significantly higher antioxidant activity, and their DPPH free radical scavenging rates (73.57%-88.22%) are significantly higher than those of all four comparative examples (59.76%-68.75%). This indicates that the complete process scheme of "chitosan microsphere immobilized alkaline protease hydrolysis + ultrafiltration + ion exchange chromatography" adopted in this invention is efficient and superior for preparing fish collagen peptides with high antioxidant activity. By comparing Example 1 with Comparative Examples 1-4, it can be seen that chitosan, as an immobilization carrier, is more effective than sodium alginate, Fe3O4, and commercially available immobilized alkaline proteases, respectively. This may be due to the better biocompatibility, more stable binding mode, and lower mass transfer resistance between chitosan and enzymes and substrates.

[0085] Table 1 Comparison of DPPH free radical scavenging rates of fish collagen peptides in each example and the comparative example.

[0086]

[0087] Example 2: Performance Determination of Immobilized Enzyme

[0088] The enzymatic hydrolysis efficiency, number of times the enzyme can be reused, and storage stability of the immobilized enzymes used to prepare fish collagen peptides in the above embodiments and comparative examples were tested.

[0089] (1) Enzymatic hydrolysis efficiency:

[0090] Degree of hydrolysis (DH) determination: The ninhydrin colorimetric method was used. 1 mL of the enzymatic hydrolysate was added to 0.2 mL of citrate buffer (pH 5.4), followed by 0.2 mL of ninhydrin reagent. The mixture was heated in a boiling water bath for 15 min, cooled, and then 5 mL of 60% ethanol was added. The absorbance was measured at 570 nm. The free amino acid content was calculated based on the standard curve. The formula for calculating the degree of hydrolysis is as follows:

[0091] DH(%) = (Free amino acid content after enzymatic hydrolysis / Total amino acid content of protein before enzymatic hydrolysis) × 100%.

[0092] Target peptide yield determination: After ultrafiltration separation, the fraction with a molecular weight <1000Da is collected, weighed, and its proportion of the total peptide powder is calculated, which is the target peptide yield.

[0093] Peptide purity determination: The Kjeldahl method was used to determine the peptide content of the sample according to national standard GB5009.5-2016. Peptides decompose in a digester to produce ammonia, which is then absorbed by boric acid in the sample via a Kjeldahl nitrogen analyzer. The peptide content is determined by hydrochloric acid titration. Peptide purity calculation formula:

[0094] X={(△V×C×0.014) / M}×F×100;

[0095] In the formula: X is the protein content, in %; ΔV is the volume of hydrochloric acid standard titrant consumed by the sample, in mL; C is the hydrochloric acid concentration, in mol / L; 0.014 is the mass of nitrogen element equivalent to 1.0 mL of hydrochloric acid (1.0 mol / L) standard titrant, in g; M is the sample mass, in g; F is the conversion factor, and this study selects the conversion factor F = 5.9.

[0096] Table 2 shows the degree of hydrolysis, peptide purity, and percentage of peptides <1000Da for each example and comparative example.

[0097]

[0098] As shown in Table 2, the degree of hydrolysis (DH, 27.8%-32.8%) of all embodiments of the present invention was significantly higher than that of all comparative examples (22.3%-26.2%). This indicates that immobilizing alkaline protease with chitosan microspheres can more effectively cleave the peptide bonds of collagen, and its enzymatic hydrolysis efficiency is superior to that of free enzymes (Comparative Example 1) and other carrier-immobilized enzymes (Comparative Examples 2, 3), and even commercially available products (Comparative Example 4). The peptide purity and the proportion of small molecule peptides obtained in the embodiments of the present invention are generally higher than those in the comparative examples. This proves the effectiveness of the entire preparation process system of the present invention, which can better remove impurities and other contaminants, efficiently enrich small molecule peptides with higher biological activity, and obtain purer collagen peptide products.

[0099] (2) Number of times the enzyme can be reused:

[0100] Each immobilized enzyme (Example 1, Comparative Examples 2-4) was used for enzymatic hydrolysis of fish collagen. After each hydrolysis, the immobilized enzyme was collected, washed three times with PBS buffer, and used again for the next hydrolysis, for a total of 10 repetitions. The relative enzyme activity after each hydrolysis was measured, with the enzyme activity of the first hydrolysis taken as 100%.

[0101] (3) Storage stability test:

[0102] Each immobilized enzyme (Example 1, Comparative Examples 2-4) was stored in a refrigerator at 4°C. Samples were taken at 0, 5, 10, 15, 20, 25 and 30 days to determine enzyme activity. The enzyme activity at 0 days was taken as 100%, and the relative enzyme activity at each time point was calculated.

[0103] Table 3. Relative enzyme activity after different numbers of uses.

[0104]

[0105] Table 4. Relative enzyme activities after different storage times

[0106]

[0107] Combining Tables 3 and 4, it can be seen that Example 1 exhibits the best performance in terms of reusability and storage stability. With the same initial enzyme activity (100%), all samples showed a significant decreasing trend in enzyme activity with increasing usage and storage time, but Example 1 showed the smallest decrease. Therefore, the immobilized enzyme in this invention exhibits the best performance.

[0108] Example 3: Determination of the amino acid composition of fish collagen peptides

[0109] Based on the above antioxidant detection and immobilized enzyme performance detection, Example 5, which has the best antioxidant activity and immobilized enzyme performance, was selected for the following experiments.

[0110] Detection method: The fish collagen peptide prepared in Example 5 was subjected to acid hydrolysis (6 mol / L HCl, 110℃ for 22 h) using an automatic amino acid analyzer (model: Hitachi L-8900), and the amino acid composition and content were determined by the analyzer.

[0111] Table 5. Amino acid composition of fish collagen peptides

[0112]

[0113] The amino acid composition results are shown in Table 5. The amino acid composition of this fish collagen peptide conforms to the typical characteristics of collagen, is rich in glycine, proline, and hydroxyproline, and has strong hydrophilicity, making it suitable for the development of bioactive products. Specifically, the total content of hydrophobic amino acids (Gly, Pro, Hyp, Ala, Val, Leu, Ile, Met, Phe, Tyr) is 71.34 g / 100 g peptide, and the total content of antioxidant-related amino acids (Gly, Pro, Hyp, Tyr, Phe) is 54.78 g / 100 g peptide.

[0114] Application examples

[0115] To illustrate the positive effects of fish collagen peptides in preventing alcoholic liver injury, this invention further demonstrates this through animal experimental data.

[0116] Experimental animals: ICR grade male mice, weighing 18–20 g, provided by Wu's Mouse Animal Co., Ltd. Mice were housed in independently ventilated positive and negative pressure cages, with controlled temperature (23±2℃) and humidity (50±15%), maintaining a 12-hour light / dark cycle, and free access to food and water. Experiments began after the mice acclimatized to the environment.

[0117] Drug management: Fish collagen peptides and biphenyl diester droplets prepared in Example 5 (positive control drug).

[0118] Animal grouping and administration: Mice were randomly divided into 6 groups (n=10 per group) according to body weight: blank control group, model group, positive control group, low-dose fish collagen peptide group, medium-dose fish collagen peptide group, and high-dose fish collagen peptide group. The blank control group and model group were administered 10 mL / kg of physiological saline by gavage; the positive control group was administered 200 mg / kg bw of biphenyl diester droplet solution by gavage; the low-, medium-, and high-dose fish collagen peptide groups were administered 100 mg / kg bw, 200 mg / kg bw, and 400 mg / kg bw of fish collagen peptide by gavage, respectively.

[0119] Experimental Methods: Mice were administered 56° Red Star Erguotou (a type of Chinese liquor) via gavage for 4 consecutive weeks. Five hours after the last administration, all groups except the control group were given 12 mL / kg of 56° Red Star Erguotou to establish an acute alcoholic liver injury model. Twelve hours after modeling (during which time the mice were fasted but not watered), blood was collected from the eyeballs, and the mice were euthanized by cervical dislocation. The livers were dissected and placed in liquid nitrogen for relevant index testing.

[0120] Detection indicators: Liver function peroxidation indicators include superoxide dismutase (SOD), malondialdehyde (MDA), and aldehyde dehydrogenase (ADH). Among them: SOD is an important antioxidant enzyme in the body, which can reflect the state of free radical metabolism; MDA is a lipid peroxidation product, and its content reflects the degree of cell damage; ADH is a key enzyme in liver metabolism of alcohol, and its activity reflects the ability to metabolize alcohol.

[0121] Table 6 Results of Liver Function Peroxidation Indicators

[0122]

[0123] In Table 6, "**" means "compared with the blank group, p<0.01, i.e., the difference is highly significant", and "##" means "compared with the model group, p<0.01, i.e., the difference is highly significant".

[0124] The results are shown in Table 6. Compared with the blank group, the SOD and ADH activities in the liver of mice in the model group were significantly reduced (p<0.01), and the MDA content was significantly increased (p<0.01), indicating that the alcoholic liver injury model was successfully established. Compared with the model group, the SOD and ADH activities in the positive control group and each fish collagen peptide dosage group were significantly increased (p<0.01), and the MDA content was significantly reduced (p<0.01), indicating that fish collagen peptides can alleviate alcohol-induced liver damage and have a good hepatoprotective effect. Its mechanism of action is likely related to enhancing the body's antioxidant capacity (increasing SOD), reducing lipid peroxidation damage (reducing MDA), and improving metabolic function (increasing ADH). In terms of reducing MDA and increasing ADH, the high-dose fish collagen peptides were even more effective than the positive control drug, indicating that it has great potential for development and application.

[0125] The above description is only a part of the embodiments of the present invention and does not limit the scope of protection of the present invention. Any equivalent device or equivalent process transformation made using the present invention specification, or directly / indirectly applied to other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for preparing fish collagen peptide having hepatoprotective and anti-aging activities, characterized in that, The method comprises extracting fish collagen peptides by using microsphere immobilized alkaline protease for enzymolysis.

2. The production method according to claim 1, characterized by, The microsphere is selected from chitosan microspheres.

3. The production method according to claim 1 or 2, characterized by, The microsphere immobilized alkaline protease is added in an amount of 800-1000 units of enzyme activity per gram of substrate.

4. The production method according to any one of claims 1 to 3, characterized by, The method for preparing the microsphere immobilized alkaline protease comprises: (1) dissolving chitosan with an acetic acid solution to prepare a 2-3% chitosan solution, then adding liquid paraffin and an emulsifier under magnetic stirring to form an oil-in-water emulsion, and then adding glutaraldehyde under the condition of 50-60℃ for crosslinking, and stirring for 2-3h to generate chitosan microspheres with a particle size of 50-150μm; (2) dispersing the chitosan microspheres obtained in step (1) in a PBS buffer solution with a pH of 7.4, adding an alkaline protease solution (concentration of 10mg / mL), and gently oscillating at 4℃ for 24h to immobilize the enzyme on the surface of the chitosan microspheres through the action of amino groups and aldehyde groups; (3) centrifuging to collect the immobilized enzyme microspheres, washing with PBS to remove unbound enzymes, and freeze-drying for preservation.

5. The preparation method according to claim 1, characterized in that, The enzymolysis conditions comprise an enzymolysis temperature of 35-55℃ and an enzymolysis time of 1-3h.

6. The method of claim 1, wherein, The preparation method further comprises: extracting fish collagen by using an alkali extraction and acid precipitation method; high-temperature inactivating the enzymolysis solution; and treating the inactivated enzymolysis solution by ultrafiltration and ion exchange chromatography to obtain fish collagen peptides; wherein the high-temperature inactivation conditions comprise a temperature of 80-90℃ and a time of 20-30min.

7. The production method according to claim 6, characterized by, The raw material is preferably fish skin or fish scales.

8. The preparation method according to claim 6, characterized in that, The specific steps of the ultrafiltration comprise: under the conditions of a membrane pressure of 1.5-4Bar, a feed liquid temperature of 25-35℃, a flow rate of 3-7LMH, and a pH of 6-8, using an ultrafiltration membrane with a molecular weight cut-off of 1-8kDa to perform ultrafiltration on the inactivated enzymolysis solution, and collecting the permeate or the retentate. 9.A fish collagen peptide with hepatoprotective and antioxidant activities, which is prepared by the method of any one of claims 1-8. 10.The use of the fish collagen peptide of claim 9 in the preparation of a product with hepatoprotective and antioxidant activities.

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