A method for preparing crocodile liver peptides to improve liver damage

Abstract

This application discloses a method for preparing crocodile liver peptides to improve liver damage, relating to the field of bioactive peptide extraction technology, including the following steps: Crocodile liver pretreatment: Fresh crocodile liver is cut into pieces and homogenized with deionized water, then a zirconium phosphate molecular sieve is added to obtain a crocodile liver homogenate; Enzymatic hydrolysis: The pH of the homogenate is adjusted, and a compound enzyme preparation is added for enzymatic hydrolysis; Enzyme inactivation treatment: After heating and cooling, the supernatant is collected by centrifugation; Ultrafiltration separation: The supernatant is filtered through an ultrafiltration membrane to collect a small molecule peptide solution; Purification and refining: Purification is achieved by dextran gel column chromatography, followed by freeze-drying to obtain the crocodile liver peptide product. By constructing a dual-size zirconium phosphate molecular sieve and cooperating with a compound enzyme preparation for enzymatic hydrolysis, combined with phosphorylation modification to enhance electron transfer, this method overcomes metal-mediated inhibition of enzyme activity, insufficient cleavage of hidden peptide segments, and oxidative damage. Simultaneously, the application of this crocodile liver peptide in improving liver function is explored, providing a new option for the prevention and treatment of liver diseases and liver health maintenance.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide extraction technology, and in particular to a method for preparing crocodile liver peptides that improve liver damage. Background Technology

[0002] As a vital metabolic and detoxification organ, the liver is susceptible to various factors such as viral infections, drug damage, alcohol consumption, and unhealthy lifestyle habits, leading to impaired liver function and various liver diseases. In today's society, with changes in lifestyle and dietary structure, liver health issues are becoming increasingly prominent.

[0003] Currently, clinical treatment for improving liver function mainly relies on drug therapy. However, some drugs have significant side effects and limited efficacy. Therefore, it is of great significance to find safe and effective substances for improving liver function from natural biological resources. GSH levels are related to liver function.

[0004] Crocodiles contain a variety of substances with unique biological activities. Studies have found that bioactive peptides extracted from crocodile tissue have multiple benefits, such as boosting immunity, anti-oxidation, and anti-inflammation. However, current research on peptides extracted from crocodile liver that improve liver function is limited, especially peptides containing glutamate and related to liver detoxification. Successful extraction of such peptides would provide a new source of natural drugs and raw materials for health products aimed at improving liver function. Summary of the Invention

[0005] This application provides a method for preparing crocodile liver peptides that improve liver damage. This method can extract glutathione-rich peptides and two high-purity short peptides from crocodile liver, which have a significant effect on improving liver function. By constructing a dual-size zirconium phosphate molecular sieve in conjunction with Bacillus subtilis protease and combining phosphorylation modification to enhance electron transfer, the method overcomes the problems of metalloinhibitory enzyme activity, insufficient cleavage of hidden peptide segments, and oxidative damage during the extraction process. At the same time, the application of this crocodile liver peptide in improving liver function is explored, providing a new option for the prevention and treatment of liver diseases and liver health care.

[0006] This application provides a method for preparing crocodile liver peptides that improve liver damage, including the following steps:

[0007] (1) Crocodile liver pretreatment: Fresh crocodile liver is cleaned, fascia and fat tissue are removed, and it is cut into pieces and mixed with deionized water at a mass ratio of 1:2 to form a homogenate. During the homogenization process, zirconium phosphate molecular sieve is added at a mass ratio of 0.1%-0.3% of the homogenate mass. The zirconium phosphate molecular sieve has a particle size of 100-200 nm to obtain crocodile liver homogenate.

[0008] (2) Enzymatic hydrolysis: Adjust the pH of the homogenate to 7.5-8.0, add a compound enzyme preparation, which is composed of alkaline protease, trypsin and subtilis protease in a mass ratio of 2:1:(0.2-0.5), and add 0.5%-1.5% of the mass of the homogenate. Enzymatic hydrolysis is carried out at pH 8.0-9.0 and 45-55℃ for 2-4 hours.

[0009] (3) Enzyme inactivation treatment: Heat to 95-100℃ and maintain for 10-15 minutes, then cool and centrifuge to collect the supernatant;

[0010] (4) Ultrafiltration separation: The supernatant is filtered through a 1000-2000 Da ultrafiltration membrane to collect the small molecule peptide solution;

[0011] (5) Purification and refining: The product is obtained by lyophilization after purification by dextran gel column chromatography.

[0012] Furthermore, the zirconium phosphate molecular sieve is composed of small particles with a diameter of 50±5 nm and large particles with a diameter of 300±20 nm, with a mass ratio of 1:2.

[0013] Furthermore, the large-particle-size zirconium phosphate molecular sieve has a core-shell structure:

[0014] The core is a 300±20 nm zirconium phosphate molecular sieve;

[0015] The outer shell is made of 20 nm thick mesoporous silica with a pore size of 8 nm;

[0016] Enzymes accounting for 40% of the total protease in Bacillus subtilis are immobilized within the shell pores.

[0017] Furthermore, the preparation of the core-shell structure includes:

[0018] Zirconium phosphate molecular sieves were dispersed in an ethanol / ammonia mixture, and tetraethyl orthosilicate was added. The mixture was reacted at 50°C for 6 hours to form a mesoporous silica shell.

[0019] Immerse in a solution of Bacillus subtilis protease with a concentration of 8 mg / mL and a pH of 7.0, and allow to adsorb by shaking at 25°C for 12 hours, followed by freeze fixation.

[0020] Furthermore, the enzymatic hydrolysis in step (2) is divided into two stages:

[0021] First stage: Add proteinase K, 0.8%-1.0%, and enzymatically hydrolyze at 56-60℃ for 4-5 hours;

[0022] Second stage: Add compound enzyme preparation, containing Bacillus subtilis protease, and enzymatically hydrolyze at 52-55℃ until the degree of hydrolysis is 25%-30%.

[0023] Furthermore, step (5) also includes phosphorylation modification:

[0024] Dissolve the purified peptide in 0.02 mol / L Tris-HCl buffer at pH 7.0 to prepare an 8%-10% peptide solution;

[0025] Add a compound phosphate solution, wherein sodium hexametaphosphate:sodium tripolyphosphate = 2:1, concentration 0.15-0.20 mol / L, and the volume ratio of peptide solution to phosphate solution is 1:1;

[0026] Adjust the pH to 4.0-4.2 and react for 30-60 minutes, then spray dry.

[0027] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages:

[0028] This invention employs a compound enzyme preparation for enzymatic hydrolysis. By optimizing the type, ratio, dosage, and hydrolysis conditions of the enzymes, the extraction rate and activity of peptides are improved. Compared with single enzymatic hydrolysis, compound enzymatic hydrolysis can more comprehensively break down protein peptide chains, releasing more active peptides related to liver detoxification function. It also increases the extraction yield of glutamate-containing peptides and is rich in glutamate and proline, which are beneficial for alcohol metabolism. The entire preparation process is green and environmentally friendly, using no toxic or harmful chemical reagents and having no impact on the environment or product safety. Furthermore, the process is simple to operate, easy to scale up for industrial production, and reduces production costs.

[0029] Crocodile liver peptides are rich in glutamic acid and polypeptides related to liver detoxification. Glutamic acid, an important amino acid in liver metabolism, participates in the urea cycle, promotes ammonia metabolism, and reduces the liver's detoxification burden. Related polypeptides can enhance the liver's antioxidant capacity and reduce free radical damage to hepatocytes by regulating the activity of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px); they can also regulate metabolic pathways in hepatocytes, promoting the repair and regeneration of damaged hepatocytes, thereby effectively improving liver function. The short peptides are high in glutamic acid and proline, and their structure is beneficial to alcohol metabolism; therefore, crocodile liver peptides can intervene to improve alcohol-induced liver damage.

[0030] By introducing subtilisin and zirconium phosphate molecular sieve, subtilisin releases liver repair active substances by targeting key peptide segments for enzymatic hydrolysis, while ZrP-MS blocks the oxidation chain reaction and protects enzyme activity through atomic-level metal capture. The integration of the two in the homogenization stage forms a synergistic mechanism of enzyme conformation activation and metal decoupling, thereby improving the antioxidant properties, liver damage repair efficiency and product purity of crocodile liver peptides.

[0031] By introducing dual-particle-size ZrP-MS, the complementary scale effect allows for the focused atomic-level metal removal of small-particle-size (50 nm) particles, which blocks the oxidation chain reaction; while the large-particle-size (300 nm) particles construct an enzymatic microreactor to enhance the release of targeted peptides. The two particles work synergistically with Bacillus subtilis protease to remove, protect, and activate the peptides, ultimately leading to further improvements in the yield, purity, and function of the active peptides. Detailed Implementation

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to limit the invention; the term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0033] Example 1: A method for preparing crocodile liver peptides to improve liver damage, specifically including the following steps:

[0034] 1. Crocodile liver pretreatment: Select fresh artificially farmed crocodile livers, rinse the surface blood and impurities with physiological saline, remove fascia and fat tissue, cut the treated crocodile livers into small pieces, put them into a homogenizer, add an appropriate amount of deionized water, homogenize to obtain crocodile liver homogenate.

[0035] 2. Enzymatic hydrolysis reaction: Adjust the pH of the system to 7.5-8.0 using NaHCO3. Add a compound enzyme preparation to the above-mentioned crocodile liver homogenate. The compound enzyme preparation consists of alkaline protease and trypsin in a mass ratio of 2:1. The amount of compound enzyme preparation added is 0.5%-1.5% of the mass of the crocodile liver homogenate. Adjust the pH of the reaction system to 8.0-9.0. Under the condition of 45-55℃, carry out the enzymatic hydrolysis reaction for 2-4 hours, stirring continuously during the process to ensure that the enzymatic hydrolysis reaction is fully carried out.

[0036] 3. Enzyme inactivation treatment: After the enzymatic hydrolysis reaction is completed, the reaction solution is rapidly heated to 95-100℃ and maintained for 10-15 minutes to inactivate the enzyme and terminate the enzymatic hydrolysis reaction. Then the reaction solution is cooled to room temperature.

[0037] 4. Centrifugation: Transfer the cooled reaction solution to a centrifuge and centrifuge for 15-20 minutes at a speed of 5000-8000 rpm to remove the precipitate and obtain the supernatant.

[0038] 5. Ultrafiltration separation: The supernatant is passed through an ultrafiltration membrane with a molecular weight cutoff of 1000-2000 Da for ultrafiltration separation. The small molecule peptide solution that permeates through the ultrafiltration membrane is collected to obtain the crude crocodile liver peptide solution.

[0039] 6. Purification and Refining: The crude crocodile liver peptide solution was further purified by gel filtration chromatography. The crude solution was loaded into a dextran gel column and eluted with phosphate buffer. The fractions corresponding to the elution peaks were collected to obtain the refined crocodile liver peptide solution. The refined solution was then pulse-dried into powder using pulse drying technology to obtain a high-purity crocodile liver peptide product. The product contains two short peptide components from crocodile liver, with high contents of glutamic acid and proline, which are beneficial to alcohol metabolism.

[0040] Experiments were conducted on the technical solutions of the above embodiments:

[0041] Experimental Group 1: Crocodile Liver Pretreatment: Take 1000g of fresh, artificially farmed crocodile liver, rinse three times with physiological saline to remove surface blood and impurities, and carefully peel off the fascia and adipose tissue. Cut the liver into 1-2cm pieces. 3 Place small pieces of crocodile liver into a homogenizer, add 2000ml of deionized water, and homogenize for 5 minutes to obtain a uniform and delicate crocodile liver homogenate.

[0042] Enzymatic hydrolysis: Add 10g of compound enzyme preparation (alkaline protease and trypsin mass ratio 2:1) to the crocodile liver homogenate, adjust the pH value to 8.5 with sodium hydroxide solution, and hydrolyze for 3 hours at 200 rpm in a constant temperature water bath at 50℃.

[0043] Enzyme inactivation treatment: After the enzymatic hydrolysis is completed, the reaction solution is quickly poured into a large beaker containing boiling water, so that the temperature of the reaction solution rises to 98°C within 1 minute and is maintained for 12 minutes. Then, the large beaker is placed in cold water to cool rapidly to room temperature.

[0044] Centrifugation: Transfer the cooled reaction solution to a 5000ml centrifuge tube, place it in a centrifuge, and centrifuge at 6000 rpm for 18 minutes. Carefully aspirate the supernatant and discard the precipitate.

[0045] Ultrafiltration separation: The supernatant is passed through an ultrafiltration membrane with a molecular weight cutoff of 2000 Da and ultrafiltration is performed at a pressure of 0.1 MPa. The small molecule peptide solution that permeates through the ultrafiltration membrane is collected.

[0046] Purification and refining: The crude crocodile liver peptide solution was loaded onto a dextran gel G-25 column (1.6 cm × 60 cm), and eluted with 0.05 mol / L phosphate buffer (pH 7.4) at a flow rate of 0.5 ml / min. The elution peaks were detected at 280 nm using a UV detector, and the fractions corresponding to the main elution peaks were collected. The collected fractions were placed in a pulse dryer and freeze-dried at 60 °C and 10 Pa for 24 hours to obtain a white powder of high-purity crocodile liver peptide product.

[0047] Experimental Group 2: Crocodile Liver Pretreatment: Take 800g of fresh artificially farmed crocodile liver and repeat the pretreatment steps in Experimental Group 1 to obtain crocodile liver homogenate.

[0048] Enzymatic hydrolysis: Add 8g of compound enzyme preparation (alkaline protease and trypsin mass ratio 2:1) to the homogenate, adjust the pH to 8.0, and stir at 150 rpm for 4 hours at 45℃.

[0049] Subsequent steps: Follow the enzyme inactivation, centrifugation, ultrafiltration, and purification steps in Experimental Group 1 to obtain high-purity crocodile liver peptide product.

[0050] Comparative Experiment 1: Crocodile liver pretreatment: same as experimental group 1.

[0051] Enzymatic hydrolysis: 10g of alkaline protease was added to the crocodile liver homogenate, the pH was adjusted to 8.5, and the mixture was hydrolyzed for 3 hours at 200 rpm in a 50℃ constant temperature water bath. Subsequent enzyme inactivation, centrifugation, ultrafiltration, and purification steps were the same as in experimental group 1.

[0052] The yield of crocodile liver peptides was determined using the initial fresh crocodile liver weight as a baseline (Experimental Group 1: 1000g; Experimental Group 2: 800g; Control Experiment 1: 1000g). All livers were washed, defascial fat removed, and weighed. The crocodile liver peptide powders obtained by freeze-drying in each experimental group were weighed using an analytical balance with an accuracy of 0.001g.

[0053] Yield calculation formula: Crocodile liver peptide yield (%) = mass of freeze-dried crocodile liver peptide (g) / initial crocodile liver mass (g) × 100%;

[0054] Each experiment was repeated 3 times, and the results were taken as mean ± standard deviation.

[0055] Results: The yield of crocodile liver peptides in experimental group 1 was 7.82±0.15%, the yield of crocodile liver peptides in experimental group 2 was 7.85±0.15%, and the yield of crocodile liver peptides in comparative experiment 1 was 6.20±0.18%. The content of specific peptides related to liver detoxification function in the peptides extracted from comparative experiment 1 was lower than that in experimental group 1, and the proportion of glutamic acid peptides was also lower. Under the same animal experimental conditions, the effect of crocodile liver peptides on improving liver function in liver injury model animals was not as obvious as that of crocodile liver peptides prepared from experimental group 1.

[0056] Animal experiments verified:

[0057] Experimental animals and grouping: Sixty male ICR mice were randomly divided into 6 groups of 10 mice each: normal control group, model control group, positive drug control group, experimental group 1 low-dose crocodile liver peptide group (20 mg / kg), experimental group 1 medium-dose crocodile liver peptide group (50 mg / kg), and experimental group 1 high-dose crocodile liver peptide group (100 mg / kg).

[0058] Modeling and Drug Administration: Except for the normal control group, mice were administered alcohol (50% concentration) via gavage. The alcohol dosage was calculated based on the mouse's body weight, generally 15 mL / kg body weight of alcohol diluted with physiological saline to an appropriate concentration, administered by gavage at regular intervals daily for 4 weeks to establish an acute alcoholic liver injury model. After successful modeling, the normal control group and the model control group were administered the same volume of physiological saline by gavage. Experimental group 1, the low, medium, and high dose groups of crocodile liver peptide, were administered 20 mg / kg, 50 mg / kg, and 100 mg / kg body weight of crocodile liver peptide solution by gavage, twice daily for 14 consecutive days.

[0059] Detection indicators and methods: 24 hours after the last administration, blood was collected from the orbital cavity of mice, serum was separated, and the levels of ALT and AST in the serum were detected using a fully automated biochemical analyzer. After cervical dislocation and sacrifice, the liver was quickly removed, rinsed with physiological saline, dried with filter paper, and a portion of the liver tissue was taken to prepare a homogenate. The activities of SOD and GSH-Px and the content of MDA in the liver tissue were detected using a kit.

[0060] Experimental Results: As shown in Table 1, compared with the model control group, the serum ALT and AST levels of mice in each dose group of crocodile liver peptide were significantly decreased (P<0.05), while the SOD and GSH-Px activities in liver tissue were significantly increased (P<0.05), and the MDA content was significantly decreased (P<0.05). Furthermore, the improvement effect of the medium and high dose groups of crocodile liver peptide was better than that of the low dose group, showing a certain dose-dependent effect. This indicates that the crocodile liver peptide prepared in this invention can effectively improve alcohol-induced acute liver injury in mice, enhance the liver's antioxidant capacity, and protect hepatocytes.

[0061] Table 1. Serum ALT and AST levels (IU / L) in mice

[0062]

[0063] The technical solutions described in the embodiments of this application have at least the following technical effects or advantages:

[0064] Advantages of the preparation method: This invention uses a compound enzyme preparation for enzymatic hydrolysis. By optimizing the type, ratio, and dosage of the enzyme, as well as the hydrolysis conditions (pH, temperature, and time), the extraction rate and activity of peptides are improved. Compared with single enzymatic hydrolysis, compound enzymatic hydrolysis can more comprehensively break down protein peptide chains, releasing more active peptides related to liver detoxification function. It also increases the extraction yield of glutamate-containing peptides and is rich in glutamate and proline, which are beneficial for alcohol metabolism. The entire preparation process is green and environmentally friendly, using no toxic or harmful chemical reagents, and has no impact on the environment or product safety. Furthermore, the process is simple to operate, easy to scale up for industrial production, and reduces production costs.

[0065] Effects on Liver Function Improvement: Experimental studies have shown that the crocodile liver peptide extracted in this invention is rich in glutamic acid and polypeptides related to liver detoxification. Glutamic acid, as an important amino acid in liver metabolism, can participate in the urea cycle, promote ammonia metabolism, and reduce the liver's detoxification burden. Related polypeptides can enhance the liver's antioxidant capacity and reduce free radical damage to hepatocytes by regulating the activity of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px); they can also regulate the metabolic pathways of hepatocytes, promoting the repair and regeneration of damaged hepatocytes, thereby effectively improving liver function. The short peptides are high in glutamic acid and proline, and their structure is beneficial to alcohol metabolism; therefore, the crocodile liver peptides can intervene to improve alcohol-induced liver damage.

[0066] In mice, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum are sensitive indicators of hepatocyte damage and its degree. Both ALT and AST are present in hepatocytes, and their activity increases in the serum when the hepatocyte membrane is damaged. By measuring the activity of these enzymes in serum and plasma, the extent of hepatocyte damage can be reflected.

[0067] In animal experiments, after administering the crocodile liver peptide of this invention to liver injury model animals, the serum ALT and AST levels were significantly reduced, indicating that the degree of hepatocyte damage was alleviated; the SOD and GSH-Px activities in liver tissue were increased, and the malondialdehyde (MDA) content was reduced, indicating that the liver's antioxidant capacity was enhanced and the degree of lipid peroxidation was reduced.

[0068] Example 2: Example 1 above successfully extracted bioactive peptides rich in glutamic acid and specific short peptides using a compound enzymatic hydrolysis technique. Experiments and animal studies showed that the crocodile liver peptides extracted by this method significantly improved liver function, reduced hepatocyte damage, enhanced liver antioxidant capacity, and had an interventional and ameliorative effect on alcoholic liver injury. To further enhance the overall performance of the extracted bioactive peptides, further improvements were made based on Example 1.

[0069] 1. Pretreatment of crocodile liver;

[0070] 1.1 Select fresh crocodile liver and rinse it three times with physiological saline (0.9% w / v) to thoroughly remove the fascia and adipose tissue;

[0071] 1.2 Cut the liver into 1-2 cm pieces 3 Add small pieces of liver to a homogenizing container in a mass ratio of liver to deionized water of 1:2 (w / v);

[0072] 1.3 Add zirconium phosphate molecular sieve (ZrP-MS);

[0073] Addition amount: 0.1-0.3% (w / w) of the total mass of the crocodile liver homogenate;

[0074] Particle size: 100-200 nm (pre-activated with 0.1 mol / L NaOH and dispersed in 0.05 mol / L phosphate buffer);

[0075] Addition method: Add to homogenizer simultaneously with liver blocks and deionized water;

[0076] 1.4 Homogenization parameters: Homogenize at 8000-10000 rpm for 5 minutes to obtain a uniform ZrP-MS-liver homogenate.

[0077] 2. Stepwise enzymatic hydrolysis;

[0078] 2.1 First stage: Proteinase K digestion;

[0079] Adjust the pH of the ZrP-MS liver homogenate to 8.0-9.0 (using 1 mol / L NaHCO3 or HCl).

[0080] Add proteinase K: 0.8%-1.0% (w / w) (based on the mass of liver homogenate);

[0081] Reaction conditions: 56-60℃, 200 rpm, 4-5 hours;

[0082] 2.2 Second stage: Synergistic enzymatic hydrolysis by complex enzymes and Bacillus subtilis protease;

[0083] Cool to 52-55℃ and adjust pH to 8.5±0.2;

[0084] Add premixed enzyme preparation:

[0085] Enzyme ratio: Alkaline protease: Trypsin: Bacillus subtilis protease (BsP) = 2:1:0.3 (mass ratio);

[0086] Total enzyme addition: 0.7%-2.0% (w / w) of liver homogenate mass (actual percentage of Bacillus subtilis protease: 0.2%-0.5%).

[0087] Reaction parameters: 52-55℃, 200-250 rpm, enzymatic hydrolysis to a degree of hydrolysis (DH) of 25%-30% (approximately 3-4 hours).

[0088] 3. Enzyme inactivation and ZrP-MS separation and regeneration;

[0089] 3.1 Heat to 95-100℃ and maintain for 10-15 minutes to inactivate the enzyme;

[0090] 3.2 Cool to 25℃ and centrifuge at 8000 rpm for 20 minutes;

[0091] Supernatant: Transfer to the next purification step (containing the target peptide);

[0092] Precipitation: Recovery of ZrP-MS-metal complex;

[0093] 3.3 ZrP-MS regeneration;

[0094] The precipitate was washed with 0.5 mol / L HCl by shaking for 30 minutes, then washed with deionized water until neutral (pH 7.0), and then dried at 60°C.

[0095] Recycling: After regeneration, ZrP-MS can be reused ≤5 times (metal desorption rate ≥85%).

[0096] 4. Ultrafiltration purification;

[0097] 4.1 The supernatant was treated with a 2000 Da ultrafiltration membrane (operating pressure 0.1 MPa), and the permeate with a molecular weight <2000 Da was collected.

[0098] 5. Phosphorylation modification;

[0099] 5.1 Concentrate the permeate to a peptide concentration of 8%-10% (w / v) and dissolve it in 0.02 mol / L Tris-HCl buffer (pH 7.0).

[0100] 5.2 Add the compound phosphate solution;

[0101] Composition: Sodium hexametaphosphate: Sodium tripolyphosphate = 2:1 (mass ratio);

[0102] Concentration: 0.15-0.20 mol / L;

[0103] Addition ratio: peptide solution: phosphate solution = 1:1 (v / v);

[0104] 5.3 Adjust the pH of the mixture to 4.0-4.2 (using 0.1 mol / L HCl) and react at 25℃ for 30-60 minutes;

[0105] 5.4 Spray drying: Inlet air temperature 180℃, outlet air temperature 80℃, to obtain phosphorylated crocodile liver peptide powder.

[0106] The technical solution of this embodiment is based on the experiment of experimental group 1; the experimental groups and operating parameters of this embodiment are shown in Table 2 below;

[0107] Table 2

[0108]

[0109] The proportion of Bacillus subtilis protease refers to its mass fraction in the complex enzyme (the total enzyme content is fixed at 1.2%).

[0110] Crocodile liver homogenate: liver:water = 1:2 (w / v), homogenize at 10000 rpm × 5 min;

[0111] Enzymatic hydrolysis: Proteinase K 0.9% (56℃×4.5h), compound enzyme (54℃×3h, pH 8.5);

[0112] Phosphorylation: 0.18 mol / L complex phosphate, pH 4.1 × 40 min;

[0113] Detection indicators and methods:

[0114] 1. Small peptide yield (<1 kDa): Ultrafiltration permeate solids mass / initial liver homogenate solids mass × 100%;

[0115] 2. Glutathione (GSH) content: HPLC method (C18 column, mobile phase 0.1% TFA-acetonitrile, detection wavelength 210 nm);

[0116] 3. Antioxidant Activity: ABTS⁺ Scavenging Rate: Measure 25 mL of ABTS solution (7.4 mmol / L) and 25 mL of K₂S₂O₈ solution (2.6 mmol / L), mix well, and react at room temperature in the dark for 12 h. Take 1 mL of the reaction solution, dilute 40 times with anhydrous ethanol, and let stand at room temperature in the dark for 30 min to obtain the ABTS working solution. Take 20 μL of the sample solution (20 mg / mL) and 280 μL of the ABTS working solution, mix well, and measure the absorbance (A1) at 734 nm in a 96-well microplate. The absorbance of deionized water instead of the sample solution is A2. ABTS + • Clearance rate (%) = (A2 - A1) / A2 × 100;

[0117] DPPH scavenging rate: 3 mL of DPPH solution (0.2 mmol / L, 95% methanol as solvent) was mixed with 1 mL of the test sample (20 mg / mL) and reacted at room temperature for 20 min. The mixture was centrifuged at 10000 g for 10 min (4℃), and the supernatant was collected. The absorbance value A1 was measured at 517 nm. The absorbance value A2 was obtained by replacing the sample solution with 95% anhydrous ethanol. DPPH scavenging rate (%) = (A2 - A1) / A2 × 100. The experimental results are shown in Table 3 below.

[0118] Table 3

[0119]

[0120] 4. Improvement effect on liver damage:

[0121] This experiment used the same alcoholic liver injury model (50% alcohol by gavage, 15 mL / kg / d × 4 weeks) to verify the effect of the improved crocodile liver peptide (containing subtilisin + ZrP-MS). The experimental groups were the same as in Example 1, except that the original crocodile liver peptide was replaced with the peptide product prepared in experimental group c of this example. The experimental results are shown in Table 4 below;

[0122] Table 4

[0123]

[0124] The technical solutions described in the embodiments of this application have at least the following technical effects or advantages:

[0125] Introducing Bacillus subtilis protease and zirconium phosphate molecular sieve (ZrP-MS) into the preparation scheme of crocodile liver peptide significantly improved the antioxidant properties and liver damage repair effect of the product through synergistic effect.

[0126] Subtilisin is a serine protease with a unique substrate-binding pocket structure at its active site. It can specifically recognize proline-rich sequences (such as GPPG) in crocodile hepatin and release the short peptide EPE by hydrolyzing the Pro-X bond (where X is a hydrophobic amino acid). This tripeptide structure is a key signaling molecule for activating the liver's antioxidant pathway.

[0127] The introduction of Bacillus subtilis protease can improve the yield of active peptides. By targeting and cleaving cryptic peptides, the yield of small molecule peptides is increased, with a significant increase in glutathione content. In addition, it can also enhance liver damage repair. The released EPE tripeptide can directly bind to Nrf2 protein in hepatocytes and promote the expression of superoxide dismutase and glutathione peroxidase.

[0128] Zirconium phosphate molecular sieves (ZrP-MS) have a layered porous structure, and their surface phosphate groups capture heavy metal ions (such as Fe) in the homogenate through an ion exchange mechanism.3+ Zn 2+ These metal ions are key catalysts for the burst of oxygen free radicals in alcohol metabolism. Meanwhile, the nanoscale pores of ZrP-MS can adsorb hydrophobic impurities and reduce pigments and lipid peroxides.

[0129] The introduction of zirconium phosphate molecular sieves can block the oxidation chain reaction, adsorb metal ions, reduce the free radical generation rate, and improve the DPPH scavenging rate of the final product. In addition, it can protect enzyme activity, prevent metal ions from inhibiting the activity of Bacillus subtilis protease, and maintain its activity in complex systems.

[0130] The synergistic effect of these two mechanisms can activate spatial conformation, allowing the phosphate groups on the ZrP-MS surface to interact with the Ser of subtilisin. 195 Hydrogen bonds are formed at the active site, which broadens the enzyme substrate binding channel and enhances its affinity for the proline-rich region. At the same time, metal-enzyme decoupling is achieved. ZrP-MS pre-chelates metal ions, eliminating their competitive occupation of the enzyme catalytic site, thus significantly improving the hydrolysis efficiency of Bacillus subtilis protease.

[0131] The two work synergistically to achieve breakthroughs in yield and activity, increasing the yield of small molecule peptides and ABTS. + • Improved clearance rate; enhanced liver damage repair, with AST values ​​in the high-dose group decreasing to near normal levels, and mitochondrial damage reduced due to synergistic effects (a core target of alcohol metabolism); furthermore, ZrP-MS is recyclable, and cost increases are controllable.

[0132] This embodiment achieves a triple synergistic effect of enzymatic hydrolysis, adsorption, and phosphorylation. At the process level, the metal pre-capture by ZrP-MS during the homogenization stage creates an efficient environment for the action of Bacillus subtilis protease, and phosphorylation modification after enzymatic hydrolysis further enhances antioxidant activity. At the product level, high-purity phosphorylated crocodile liver peptide with high GSH content is obtained, exhibiting high DPPH scavenging rate and containing specific liver repair peptides (EPE). At the functional level, in animal models, a significant reduction in AST was achieved even with a medium dose, and ZrP-MS reduced Fe... 3+ It directly inhibits the acetaldehyde-free radical damage pathway, while the EPE tripeptide released by Bacillus subtilis protease activates the antioxidant enzyme system, forming a dual-pathway liver protection, and achieving synergistic enhancement of hepatocyte membrane repair and mitochondrial function protection.

[0133] Example 3: Example 2 introduced subtilisin and zirconium phosphate molecular sieve. Subtilisin releases liver repair active substances by targeting key peptide segments, while ZrP-MS blocks the oxidation chain reaction and protects enzyme activity through atomic-level metal capture. The integration of the two in the homogenization stage forms a synergistic mechanism of enzyme conformation activation and metal decoupling, thereby improving the antioxidant properties, liver damage repair efficiency, and product purity of crocodile liver peptides. Further improvements were made based on Example 2 to further enhance the comprehensive performance of the extracted active peptides.

[0134] The zirconium phosphate molecular sieve comprises small particles with a diameter of 50±5 nm and large particles with a diameter of 300±20 nm, with a mass ratio of 1:2.

[0135] Based on experimental group c in Example 2, the technical solution of this embodiment was tested as experiment d. The difference between experiment d and experiment group c is that the zirconium phosphate molecular sieve includes small particles with a diameter of 50±5 nm and large particles with a diameter of 300±20 nm, and the mass ratio of the two is 1:2. The experimental results are shown in Table 5 below.

[0136] Table 5

[0137]

[0138] The liver injury improvement effect was observed in the same experimental groups as in Example 1, except that the original crocodile liver peptide was replaced with the peptide product prepared in experimental group d of this example. The experimental results are shown in Table 6 below;

[0139] Table 6

[0140]

[0141] The technical solutions described in the embodiments of this application have at least the following technical effects or advantages:

[0142] By introducing a dual-size zirconium phosphate molecular sieve (ZrP-MS) (50±5 nm small particle size and 300±20 nm large particle size, mass ratio 1:2), further synergy with Bacillus subtilis protease was achieved.

[0143] Small particle size (50 nm) enables atomic-level trapping. 50 nm particles have a high specific surface area, and their densely packed phosphate groups preferentially adsorb transient free metal ions from the homogenate through ion exchange. These metal ions are key mediators of the catalytic burst of oxygen free radicals in alcohol metabolism; the introduction of small particle size can enhance the adsorption rate, increase the proportion of metal ions cleared in the early stages of homogenization, block the lipid peroxidation chain reaction, and improve the DPPH scavenging rate of the final product.

[0144] Large particle size (300 nm) enables the creation of microreactors. The layered channels of the 300 nm particles form a physically confined space that can accommodate alligator liver protein and subtilisin, prolonging the contact time between the substrate and the enzyme. The interlayer electrostatic field can also stabilize the protease conformation and prevent denaturation and inactivation. The introduction of large particle size improves enzymatic hydrolysis efficiency, promotes targeted cleavage of proline-rich regions (such as GPPG), increases the yield of small peptides, and increases the release of EPE tripeptide (Glu-Pro-Glu).

[0145] Both react synergistically with subtilisin, and in terms of time, small-particle ZrP-MS rapidly removes metal ions in the early stage of homogenization (0-10 minutes), eliminating their inhibition of the active site of subtilisin (Ser). 195 The large-particle-size ZrP-MS provides a sustained reaction microenvironment during the mid-stage of enzymatic hydrolysis (30-120 minutes), enhancing the cleavage efficiency of subtilisin on hydrophobic peptide chains (such as Pro-Leu). Conformally, the phosphate groups of the small-particle-size ZrP-MS react with the Arg groups of subtilisin. 170 The side chain forms hydrogen bonds, which widens the substrate binding pocket and enhances the recognition ability of proline. The negative electric field between the layers of the large-particle ZrP-MS repels hydrophobic impurities, reduces competitive binding of substrates, and improves enzyme specificity.

[0146] The synergistic effect produces the following results: breakthroughs in the yield and purity of active peptides, increased yield of small molecule peptides, extended enzyme action time due to larger particle size, release of more <1 kDa short peptides, and blockage of metal interference by small particle size, protecting the enzyme's sustained activity; increased GSH content due to the optimized release efficiency of glutathione precursor (glutamate / cysteine) by the dual-particle size system; enhanced liver damage repair efficacy, with small particle size clearing metal ions and reducing mitochondrial oxidative damage, and large particle size promoting the activation of the Nrf2 pathway by EPE tripeptide, upregulating SOD expression; and improved antioxidant activity, with metal adsorption reducing free radical generation and enhanced electron transport capacity of small molecule peptides after phosphorylation modification.

[0147] Example 4: Example 3 above introduces dual-particle-size ZrP-MS to achieve a complementary scale effect. The small-particle-size (50 nm) ZrP-MS focuses on atomic-level metal removal, blocking the oxidation chain reaction; the large-particle-size (300 nm) ZrP-MS constructs an enzymatic microreactor to enhance the release of targeted peptides. The two ZrP-MS and Bacillus subtilis protease form a synergistic effect of removal, protection and activation, ultimately achieving a further improvement in the yield, purity and function of active peptides. To further improve the overall performance of the extracted active peptides, further improvements are made based on Example 3.

[0148] The large-particle-size zirconium phosphate molecular sieve serves as the core, with a mesoporous silica shell (mSiO2) covering the surface, and Bacillus subtilis protease is immobilized in the pores to form a core-shell structure.

[0149] The preparation of the core-shell structure specifically involves:

[0150] ZrP-MS was dispersed in an ethanol / ammonia mixture, and tetraethyl orthosilicate (TEOS) was added (TEOS, ZrP-MS:TEOS=1:2 w / w). The mixture was reacted at 50°C for 6 h to form a 20 nm thick mSiO2 shell with a pore size of 8 nm.

[0151] Then, it was immersed in a subtilisin solution (8 mg / mL, pH 7.0), and adsorbed by shaking at 25°C for 12 h. The subtilisin accounted for 40% of the total amount of subtilisin in the mSiO2 shell pores, and was frozen to form a core-shell structure.

[0152] Based on Experiment d in Example 3, Experiment e was conducted. The difference between Experiment e and Experiment d is that the large-particle-size zirconium phosphate molecular sieve serves as the core, coated with a mesoporous silica shell (mSiO2), and immobilized with subtilisin within the pores, forming a core-shell structure. The immobilized subtilisin within the mSiO2 shell accounts for 40% of the total content. The experimental results are shown in Table 7 below.

[0153] Table 7

[0154]

[0155] The liver injury improvement effect was observed in the same experimental groups as in Example 1, except that the original crocodile liver peptide was replaced with the peptide product prepared in experimental group e of this example. The experimental results are shown in Table 8 below;

[0156] Table 8

[0157]

[0158] The technical solutions described in the embodiments of this application have at least the following technical effects or advantages:

[0159] By constructing a core-shell structure, spatial synergy and functional division of adsorption and enzymatic hydrolysis are achieved. A large-particle-size zirconium phosphate molecular sieve (ZrP-MS, 300±20 nm) serves as the core, whose layered phosphate groups efficiently capture heavy metal ions in the homogenate through ion exchange, blocking the free radical chain reaction caused by alcohol metabolism at its source and reducing oxidative damage to hepatocyte mitochondria. The surface-coated mesoporous silica shell forms a physically confined space, immobilizing subtilisin within the pores. On the one hand, it locks the enzyme conformation through electrostatic interaction, targeting and cleaving proline-enriched sequences (such as GPPG) in hepatin, specifically releasing liver-repairing active substances such as EPE tripeptide. On the other hand, it shields hydrophobic denaturing factors in the homogenate, thereby improving the enzyme activity retention rate. The core-shell linkage allows adsorbed metal ions to be transferred to the enzyme active center through the mSiO2 pores, activating the peroxidase-like activity of subtilisin, while the hydrolysis products diffuse rapidly through the pores, eliminating the product inhibition effect of traditional enzymatic hydrolysis.

[0160] Retaining free Bacillus subtilis protease and small-particle ZrP-MS (50±5 nm), a spatiotemporal dual-dimensional synergistic network is formed. The free enzyme acts by freely diffusing to all parts of the homogenate, rapidly breaking down large hepatic proteins (such as collagen fibers), exposing more GPPG sequences for precise cleavage by the immobilized enzyme, forming a "pre-depolymerization-targeted cleavage" cascade reaction, shortening the enzymatic digestion time. The small-particle size allows for transient adsorption of free metals (such as Cu) in the early stages of homogenization due to its high specific surface area. 2+ This process removes obstacles to the operation of the core-shell structure, enhances the system's shear resistance due to its mechanical stability, ensures the integrity of the core-shell structure, fills the adsorption blind zone of the core-shell structure, reduces the oxidative denaturation of liver proteins, and improves the dissolution efficiency of GSH precursors (glutamate / cysteine).

[0161] The two work synergistically: small particle size and free enzyme together ensure the efficient operation of the core-shell structure. Small particle size eliminates transient interference, free enzyme provides the initial action site, and the core-shell structure completes deep enzymatic hydrolysis and enrichment of active peptides.

[0162] This approach yields significant results. In terms of process, the core-shell structure enables rapid adsorption and enzymatic hydrolysis, shortening the hydrolysis time and allowing for stable recycling of the immobilized enzyme. At the biological level, it significantly enhances liver damage repair efficacy, primarily through the simultaneous blocking of the Nrf2 pathway activated by the EPE tripeptide and mitochondrial oxidative damage. In the antioxidant system, metal adsorption and enzymatic hydrolysis synergistically scavenge free radicals, while glutathione released from the core-shell structure reduces vitamin E / C to form a continuous antioxidant cycle network, achieving a balance between efficacy and cost.

[0163] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing an alligator liver peptide for improving liver damage, characterized by, The method comprises the following steps: (1) alligator liver pretreatment: fresh alligator liver is washed, fascia and adipose tissue are removed, and then the liver is cut into pieces and mixed with deionized water at a mass ratio of 1:2 (w / v) to obtain a homogenate, wherein zirconium phosphate molecular sieves are added during the homogenization process, and the addition amount is 0.1%-0.3% (w / w) of the mass of the homogenate, and the particle size of the zirconium phosphate molecular sieves is 100-200 nm; (2) enzymatic reaction: first stage: the pH of the homogenate is adjusted to 8.0-9.0, and proteinase K is added, and the addition amount is 0.8%-1.0% (w / w) of the mass of the homogenate, and the enzymolysis is carried out at 56-60 ℃ and 200 rpm for 4-5 hours; second stage: the pH is adjusted to 8.5±0.2, and a complex enzyme preparation is added, wherein the complex enzyme preparation is composed of alkaline protease, trypsin and subtilisin at a mass ratio of 2:1:0.3, and the addition amount is 0.7%-2.0% (w / w) of the mass of the homogenate, and the enzymolysis is carried out at 52-55 ℃ and 200-250 rpm until the degree of hydrolysis is 25%-30%; (3) enzyme inactivation treatment: the temperature is raised to 95-100 ℃ and maintained for 10-15 minutes, and then cooled to 25 ℃, and then centrifuged at 8000 rpm for 20 minutes to obtain a supernatant; (4) ultrafiltration separation: the supernatant is filtered through a 2000 Da ultrafiltration membrane, and the small molecular peptide permeate with a molecular weight of less than 2000 Da is collected; (5) purification and refinement: the permeate is concentrated to a peptide concentration of 8%-10% (w / v), and then dissolved in a Tris-HCl buffer solution with a concentration of 0.02 mol / L and a pH of 7.0 to form a peptide solution; then a complex phosphate solution is added, wherein the complex phosphate solution is composed of sodium hexametaphosphate and sodium tripolyphosphate, and the mass ratio of the two is 2:1, and the concentration is 0.15-0.20 mol / L; the volume ratio of the peptide solution to the complex phosphate solution is 1:1; 0.1 mol / L HCl is used to adjust the pH to 4.0-4.2, and the reaction is carried out at 25 ℃ for 30-60 minutes; after the reaction is completed, spray drying is carried out, the inlet air temperature is 180 ℃, and the outlet air temperature is 80 ℃, to obtain phosphated alligator liver peptide powder.

2. A method of preparing an alligator liver peptide for improving liver damage, characterized by, The method comprises the following steps: (1) alligator liver pretreatment: fresh alligator liver is washed, fascia and adipose tissue are removed, and then the liver is cut into pieces and mixed with deionized water at a mass ratio of 1:2 (w / v) to obtain a homogenate, wherein zirconium phosphate molecular sieves are added during the homogenization process, and the addition amount is 0.1%-0.3% (w / w) of the mass of the homogenate, and the particle size of the zirconium phosphate molecular sieves is 100-200 nm; (2) enzymatic reaction: first stage: the pH of the homogenate is adjusted to 8.0-9.0, and proteinase K is added, and the addition amount is 0.8%-1.0% (w / w) of the mass of the homogenate, and the enzymolysis is carried out at 56-60 ℃ and 200 rpm for 4-5 hours; The second stage: adjusting pH to 8.5±0.2, adding a complex enzyme preparation, the complex enzyme preparation is composed of alkaline protease, trypsin and subtilisin in a mass ratio of 2:1:0.3, the addition amount is 0.7%-2.0% (w / w) of the homogenate liquid, and enzymolysis is carried out at 52-55℃ and 200-250 rpm until the degree of hydrolysis is 25%-30%; (3) enzyme inactivation treatment: heating to 95-100℃ for 10-15 minutes, cooling to 25℃, and then centrifuging at 8000 rpm for 20 minutes to take the supernatant; (4) ultrafiltration separation: the supernatant is filtered through a 2000 Da ultrafiltration membrane, and the small molecular peptide permeate with a molecular weight of <2000 Da is collected; (5) purification and refinement: the permeate is concentrated to a peptide concentration of 8%-10% (w / v), dissolved in a Tris-HCl buffer with a concentration of 0.02 mol / L and a pH of 7.0 to form a peptide solution; Then a complex phosphate solution is added, wherein the complex phosphate solution is composed of sodium hexametaphosphate and sodium tripolyphosphate in a mass ratio of 2:1, and the concentration is 0.15-0.20 mol / L; The volume ratio of the peptide solution to the complex phosphate solution is 1:1; 0.1 mol / L HCl is used to adjust the pH to 4.0-4.2, and the reaction is carried out at 25℃ for 30-60 minutes; After the reaction is completed, spray drying is carried out, the inlet air temperature is 180℃, and the outlet air temperature is 80℃, and zirconium phosphate crocodile liver peptide powder is obtained.

3. The method for preparing crocodile liver peptides for improving liver injury as described in claim 2, characterized in that, The large-particle-size zirconium phosphate molecular sieve has a core-shell structure: The inner core is a 300±20 nm zirconium phosphate molecular sieve; The outer shell is 20 nm thick mesoporous silica with a pore size of 8 nm; The enzyme fixed in the shell channel accounts for 40% of the total amount of subtilisin.

4. The method for preparing crocodile liver peptides for improving liver injury as described in claim 3, characterized in that, The preparation of the core-shell structure includes: Dispersing the zirconium phosphate molecular sieve in an ethanol / ammonia water mixture, adding tetraethyl orthosilicate, and reacting at 50℃ for 6 hours to form a mesoporous silica shell; Immersing in a subtilisin solution with a concentration of 8 mg / mL and a pH of 7.0, and oscillating adsorption at 25℃ for 12 hours, and freezing fixation.

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