Preparation method of codfish skin collagen peptide with high ACE (Angiotensin Converting Enzyme) inhibitory activity

Abstract

The invention discloses a preparation method of cod skin collagen peptide with high ACE (angiotensin converting enzyme) inhibitory activity, which comprises the following steps: firstly, scaling cod skin, cleaning, cutting into small pieces, pretreating with NaOH and n-butyl alcohol to remove impure protein and certain lipid substances, preparing cod skin gelatin by a hot water extraction method, and then adding the cod skin gelatin into the cod skin collagen peptide to obtain cod skin collagen peptide; the cod skin collagen peptide with high ACE inhibitory activity is prepared by using cod skin collagen as a raw material, performing enzymolysis on the cod skin collagen peptide sequentially by using a double-enzyme distribution enzymolysis method, performing enzyme deactivation and centrifugal treatment after enzymolysis, and performing membrane separation on enzymolysis supernate. The whole enzymolysis process is more thorough by adopting a double-enzyme distribution enzymolysis method, more small molecule peptides can be obtained, cod skin collagen peptide with high ACE inhibitory activity can be further enriched by combining a membrane separation method, the utilization degree of cod skin resources is effectively improved, and the cod skin collagen peptide has a relatively wide application prospect.

Description

Technical Field

[0001] The invention relates to the field of food biotechnology, and in particular to a method for preparing cod skin collagen peptide with high ACE inhibitory activity. Background Art

[0002] Hypertension is a common chronic disease and one of the important risk factors for cardiovascular disease. Increased blood pressure is closely related to the renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS). Angiotensin converting enzyme (ACE) can convert angiotensin I into angiotensin II in the RAS system, promote the synthesis and release of aldosterone, produce reactive oxygen species, and lead to increased blood pressure. ACE inhibitors can block the conversion of angiotensin I to angiotensin II, thereby lowering blood pressure. Conventional hypertension drugs include synthetic drugs such as captopril and lisinopril. Long-term use can cause side effects such as dizziness, nausea, and hyperkalemia. It is particularly important to find natural inhibitors with few side effects and significant ACE inhibitory effects.

[0003] Cod is one of the most fished fish in the world, with important edible and economic value. my country processes more than 500,000 tons of cod each year, of which cod skin accounts for about 7-8% of processing waste. Except for a small amount used to produce aquaculture feed, most of these wastes are discarded, with low economic benefits. Studies have shown that collagen peptides, a hydrolyzate of cod skin, have potential ACE inhibitory activity and can be used as a natural antihypertensive substance. How to extract and prepare collagen peptides with high ACE inhibitory activity from cod skin has become a goal and direction of people's attention.

[0004] At present, the preparation methods of cod skin collagen peptides include water extraction, acid-base extraction, microbial fermentation, enzymatic method, etc. Among them, the water extraction method has low extraction efficiency and low product purity, and it is difficult to obtain highly active components. Although the acid-base extraction method can quickly extract protein, it is easy to destroy the protein structure and affect its biological activity; although the microbial fermentation method can improve the activity of the product to a certain extent, the fermentation cycle is long and the process is complicated, which is not conducive to industrial production, so the enzymatic method has attracted more and more attention. Guo Hongxing et al. established a marine biological source collagen peptide and its extraction method. First, the cod skin was treated with alkali and acid, and then the collagen peptide obtained after enzymatic hydrolysis with a composite protease had inhibitory activity against ACE (patent application number: CN202111579484.6). Liao Daiping et al. established a cod peptide and its enzymatic extraction method. First, defatted cod skin fragments were prepared, and then enzymatic hydrolysis with papain was performed to obtain marine biological collagen peptides (patent application number 201810644088.9); Du Ming et al. established a cod peptide preparation method with immunomodulatory activity by grinding, enzymatic hydrolysis, spray drying, alcohol extraction, dealcoholization, desalting and other methods (patent application number 202210025039.3); Zhuo Xiaoyan et al. established a method for preparing sturgeon skin protein peptides by enzyme combined with membrane separation, but this method only uses one protease preparation, and the maximum inhibition rate of the obtained collagen peptide on ACE is only 23% (patent application number: 202310169089.3). Collagen peptides can be obtained by one-step enzymatic hydrolysis combined with membrane separation, but the quality and function of the obtained product can be further improved. How to construct a new method for enriching and preparing cod skin collagen peptides with high ACE inhibitory activity is particularly important.

[0005] This patent constructs a method for preparing cod skin ACE inhibitory peptides with high ACE inhibitory activity by dual-enzyme step-by-step enzymatic hydrolysis combined with membrane separation. Through multiple steps such as pretreatment, extraction, dual-enzyme distributed enzymatic hydrolysis, membrane separation, etc., cod skin collagen peptides with high ACE inhibitory activity are enriched, effectively solving many deficiencies in the existing technology. Summary of the invention

[0006] In order to obtain cod skin collagen peptides with high ACE inhibitory activity, the present invention provides a more advanced technical solution: a method for preparing high ACE inhibitory peptide cod skin collagen peptides by dual enzyme step-by-step enzymatic hydrolysis combined with membrane separation. The dual enzyme step-by-step extraction method adopted is simple to operate and utilizes the specificity of different enzymes to produce more abundant and smaller molecular weight polypeptide species, which significantly improves the inhibitory activity of the enzymatic hydrolysis product on ACE. Combined with the membrane separation method, the components with strong ACE inhibitory activity are separated and purified from the mixture, and the obtained product has good ACE inhibitory activity. The present invention adopts the following technical solution, including the following steps:

[0007] (1) Cod skin pretreatment

[0008] Descale the cod skin, clean it and cut it into small pieces with a side length of 1 to 2 cm, add 0.05 to 0.20 mol / L NaOH according to a material-liquid ratio of 1:6 to 1:20, soak it at 4 to 10°C for 12 to 24 hours, then wash it with distilled water until it is neutral, then add 5 to 10% n-butanol according to a material-liquid ratio of 1:10 to 1:20, soak it at 4 to 10°C for 12 to 24 hours, then rinse it with distilled water and set it aside;

[0009] (2) Cod skin gelatin extraction

[0010] The pretreated cod skin is added with distilled water at a material-liquid ratio of 1:10-1:20, and then extracted at 50-100°C hot water for 6-12 hours. After the extraction is completed, centrifuge at 4000-10000 r / min for 10-30 minutes, and then the supernatant is concentrated and dried under reduced pressure under certain conditions to obtain cod skin gelatin;

[0011] (3) First enzymatic hydrolysis

[0012] The obtained cod skin gelatin is added with distilled water with a pH of 7 to 11 at a material-liquid ratio of 1:10 to 1:40, and then a certain amount of protease preparation is added, and the enzymatic hydrolysis temperature is set to 40 to 60° C. and the enzymatic hydrolysis time is set to 2 to 10 hours. After the enzymatic hydrolysis is completed, the enzyme is inactivated at 90 to 100° C. for 10 to 30 minutes, and then cooled to room temperature, and centrifuged at 4000 to 10000 r / min for 10 to 30 minutes, and then the supernatant is taken for subsequent enzymatic hydrolysis;

[0013] (4) Second enzymatic hydrolysis

[0014] After adjusting the pH of the supernatant after the first enzymatic hydrolysis to 5-9, a certain amount of protease preparation is added, the enzymatic hydrolysis temperature is set to 35-55°C, the enzymatic hydrolysis time is set to 2-10h, after the enzymatic hydrolysis is completed, the enzyme is inactivated at 90-100°C for 10-30min, then cooled to room temperature, centrifuged at 4000-10000r / min for 10-30min, and the supernatant is separated;

[0015] (5) Membrane separation

[0016] The supernatant obtained after double-enzyme step-by-step enzymatic hydrolysis and centrifugation is passed through an ultrafiltration membrane of a certain specification to obtain a permeate which is then concentrated and dried under reduced pressure under certain conditions to obtain cod skin collagen peptide with high ACE inhibitory activity.

[0017] As a further improvement of the present invention: after rinsing with distilled water in step 1, continue to soak in citric acid for a period of time, clean until neutral and dry for later use. Preferably, the concentration of citric acid is 1% and the soaking time is 8 hours.

[0018] As a further improvement of the preparation method of the present invention: the protease preparation in step 3 can be one of pepsin, trypsin, alkaline protease, collagenase, neutral protease, composite protease, and flavor protease, preferably one of alkaline protease, collagenase, neutral protease, and composite protease;

[0019] As a further improvement of the preparation method of the present invention: the amount of the protease preparation added in step 3 is 0.5% to 5%, preferably 2% to 4%, and more preferably 4%;

[0020] As a further improvement of the preparation method of the present invention: the enzymatic hydrolysis temperature in step 3 is 45-55°C, preferably 50°C;

[0021] As a further improvement of the preparation method of the present invention: the enzymatic hydrolysis pH in step 3 is 8-10, preferably 9;

[0022] As a further improvement of the preparation method of the present invention: the enzymatic hydrolysis time in step 3 is 2-6 hours, preferably 4 hours;

[0023] As a further improvement of the preparation method of the present invention: the material-liquid ratio in step 3 is 1:10-1:30, preferably 1:20;

[0024] As a further improvement of the preparation method of the present invention: the protease preparation in step 4 can be one of pepsin, trypsin, alkaline protease, collagenase, neutral protease, composite protease, and flavor protease, preferably flavor protease, trypsin, and pepsin;

[0025] As a further improvement of the preparation method of the present invention: the amount of the protease preparation added in step 4 is 0.5% to 5%, preferably 2% to 4%, and more preferably 3%;

[0026] As a further improvement of the preparation method of the present invention: the enzymatic hydrolysis temperature in step 4 is 35-45°C, preferably 40°C;

[0027] As a further improvement of the preparation method of the present invention: the enzymatic hydrolysis pH in step 4 is 5-7, preferably 6;

[0028] As a further improvement of the preparation method of the present invention: the enzymatic hydrolysis time in step 4 is 3-5h, preferably 4h;

[0029] As a further improvement of the preparation method of the present invention: the type of ultrafiltration membrane in step 5 can be one or more combinations of 1000, 3000, 5000 or 10000 Da, preferably a combination of 10000, 3000 and 1000;

[0030] As a further improvement of the preparation method of the present invention: the reduced pressure concentration temperature in step 5 is 50-60° C., and the drying method can be freeze drying, spray drying, or a combination of the two, preferably spray drying.

[0031] The present invention further includes the use of the collagen peptide obtained by the preparation method of the cod skin collagen peptide in the preparation of blood pressure lowering drugs.

[0032] Compared with the prior art, the present invention provides a method for preparing cod skin collagen inhibitory peptides with high ACE inhibitory activity. First, the cod skin is pretreated, and after the pretreatment, gelatin is prepared by a water extraction method, and then a double-enzyme step-by-step enzymatic hydrolysis combined with a membrane separation method is used to prepare cod skin collagen peptides with high ACE inhibitory activity.

[0033] (1) Pretreatment of cod skin can effectively remove fish scales, impurities, proteins, fat, etc., avoiding the impact on subsequent extraction processes;

[0034] (2) Gelatin can be prepared first by water extraction, which can increase the dissolution rate of cod skin collagen and facilitate the subsequent enzymatic hydrolysis process;

[0035] (3) The most suitable protease types for preparing cod skin collagen peptides with high ACE inhibitory activity by enzymatic hydrolysis of cod skin are flavor protease and collagenase, and the temperature, pH, time, enzyme addition amount, and material-liquid ratio are screened, and double enzyme step-by-step enzymatic hydrolysis is used to improve the contact efficiency between the enzyme and the substrate, making the enzymatic hydrolysis more thorough, and more types of cod skin collagen peptides with smaller molecules can be obtained by enzymatic hydrolysis. The yield of ACE inhibitory peptides is significantly improved, which helps to improve its ACE inhibitory activity;

[0036] (4) The membrane separation method was used to obtain freeze-dried parts of four components: >10, 3-10, 1-3, and <1 kDa. The ACE inhibitory activity of the <1 kDa part was as high as 87.65%, which further improved the ACE inhibition rate of the prepared cod skin collagen peptide and significantly improved the accuracy of the final product. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a flow chart of the method of the present invention;

[0038] Figure 2 The effect of treating cod skin with collagenase at different temperatures on ACE inhibition rate and hydrolysis degree;

[0039] Figure 3 The effect of collagenase treatment on ACE inhibition rate and hydrolysis degree at different pH values ​​on cod skin;

[0040] Figure 4 The effect of treating cod skin with collagenase at different times on ACE inhibition rate and hydrolysis degree;

[0041] Figure 5 The effect of different enzyme dosages on ACE inhibition rate and hydrolysis degree of cod skin treated with collagenase;

[0042] Figure 6 The effect of collagenase treatment on ACE inhibition rate and hydrolysis degree of cod skin at different material-liquid ratios;

[0043] Figure 7 The effect of flavor protease treatment on cod skin on ACE inhibition rate and hydrolysis degree at different temperatures;

[0044] Figure 8 The effect of flavor protease treatment on ACE inhibition rate and hydrolysis degree of cod skin at different pH;

[0045] Fig. 9 The effect of flavor protease treatment on cod skin on ACE inhibition rate and hydrolysis degree at different time periods;

[0046] Fig.10 The effect of flavor protease treatment on ACE inhibition rate and hydrolysis degree of cod skin at different enzyme addition amounts;

[0047] Fig.11 The effect of flavor protease treatment on ACE inhibition rate and hydrolysis degree of cod skin at different material-liquid ratios;

[0048] Fig.12 The effect of double-enzyme step-by-step enzymatic hydrolysis and single-enzyme enzymatic hydrolysis on ACE inhibition rate and hydrolysis degree;

[0049] Fig.13 The ACE inhibition rate and hydrolysis degree are affected by membranes of different molecular weights after the protein peptides are separated. DETAILED DESCRIPTION

[0050] In order to make the purpose of the invention more clear, the technical solution of the present invention is described below in conjunction with embodiments and drawings, but the protection scope is not limited thereto.

[0051] Example 1 Preparation of ACE inhibitory peptide from cod skin by enzymatic method

[0052] The prepared cod skin gelatin was dissolved in distilled water at a solid-liquid ratio of 1:20, and 5 different types of enzymes were added respectively at an enzyme amount of 2%. Enzymatic hydrolysis was performed according to the optimum temperature and pH in the instructions (see Table 1). After 4 hours of enzymatic hydrolysis, the enzyme was inactivated by heating in boiling water for 20 minutes, and then centrifuged at 9000 r / min for 20 minutes. This was repeated three times, and the supernatant was taken to determine its hydrolysis degree and its ACE inhibition rate.

[0053] Table 1 Optimal reaction conditions for 5 proteases

[0054]

[0055]

[0056] Selecting the appropriate protease can optimize the enzymatic conditions, improve the hydrolysis efficiency and ACE inhibition rate. Therefore, this study first screened the types of enzymes, and the results are shown in Table 2. As can be seen from the table, flavor protease and collagenase have high ACE inhibition activity, with inhibition rates of 45.98±0.59% and 40.70±0.77%, respectively. Protease can improve the hydrolysis efficiency, and flavor protease can reduce the generation of bitter peptides, thereby improving the sensory quality of ACE inhibitory peptide products and making them more suitable as functional food ingredients. Therefore, flavor protease and collagenase can be used as preferred enzymes for subsequent research.

[0057] Table 2 Effects of five proteases on ACE inhibition rate and hydrolysis degree

[0058]

[0059] Example 2: Preparation of ACE inhibitory peptides from cod skin by collagen hydrolase method

[0060] S1, fish skin pretreatment: fresh frozen cod skin was taken, thawed at 4°C, the cod skin was descaled, residual fat was scraped off the inner skin, washed three times and then dried, and cut into 2×2 cm pieces after drying;

[0061] S2, protein removal and defatting: the pretreated cod skin was added with 0.05 mol / L (0.2%) NaOH at a material-liquid ratio of 1:6 and soaked at 4°C for 24 hours, and washed with distilled water until neutral. The fish skin was added with 10% n-butanol at a material-liquid ratio of 1:10 and soaked for 24 hours, rinsed until neutral, and then soaked with 1% citric acid for 8 hours, washed until neutral, and dried.

[0062] S3. Preparation of cod skin gelatin: fish skin and distilled water were extracted at 50°C hot water with a solid-liquid ratio of 1:10 for 12 h, centrifuged at 9000 r / min for 20 min, and the supernatant was taken. The supernatant was lyophilized by rotary evaporation to obtain gelatin.

[0063] S4. Preparation of collagen peptides: collagen hydrolase, enzyme addition amount: 2%, solid-liquid ratio 1:20, pH 9, enzymatic hydrolysis temperature 50°C, enzymatic hydrolysis time 4.0 h.

[0064] S5. Enzyme inactivation treatment: heat to 100°C and maintain for 20 min, then centrifuge at 9000 r / min for 20 min to obtain the supernatant.

[0065] S6, finished product: rotary evaporation concentration and vacuum freeze drying to obtain cod skin ACE inhibitory peptide.

[0066] Comparative Example 1

[0067] Effects of different temperatures on ACE inhibition rate and hydrolysis degree. The present invention investigates the effects of different temperatures on enzymatic hydrolysis products, takes 1g of cod skin gelatin, sets the enzymatic hydrolysis temperature to 40, 45, 50, 55, 60°C, adds 2% enzyme, enzymatic hydrolysis for 4.0h, pH 9, and a solid-liquid ratio of 1:20. The optimal temperature is determined by measuring the ACE inhibition rate and the hydrolysis degree.

[0068] The effects of different temperatures on ACE inhibition rate and hydrolysis degree are shown in Figure 2 . ACE inhibitory activity decreases as the hydrolysis temperature increases to 50°C. ACE inhibitory activity reaches a maximum of 40.72% at 50°C and the degree of hydrolysis reaches a maximum of 30.40%. However, a decrease in process efficiency was observed with increasing enzymatic hydrolysis temperature; the deleterious effects of high temperature on the molecular stability of the enzyme and the ability of its binding site to attach to the substrate may be the root cause of the problem, resulting in a decrease in the degree of hydrolysis. Therefore, 50°C was determined to be the optimal temperature for proteolytic enzymes.

[0069] Comparative Example 2

[0070] The temperature remains unchanged, and the effects on ACE inhibition rate and hydrolysis degree at different pH values ​​are also different. The present invention investigates the effects on the enzymatic hydrolysis products at different pH values, takes 1.0 g of cod skin gelatin, sets the enzymatic hydrolysis temperature to 50°C, adds 2% enzyme, enzymatic hydrolysis for 4.0 h, sets the pH to 7, 8, 9, 10, 11, and the material-liquid ratio to 1:20. The optimal pH is determined by measuring the ACE inhibition rate and hydrolysis degree.

[0071] The effects of different pH on ACE inhibition rate and hydrolysis degree are shown in Figure 3 . In the range of pH 7.0 to 11.0, the degree of hydrolysis increased from pH 7 to pH 9. The optimal value of hydrolysis was 30.85% at pH 9. The degree of hydrolysis began to decrease as the pH continued to rise. This was because the excessively high pH value prevented the enzyme from binding to the substrate, thereby reducing the degree of hydrolysis. The ACE inhibitory activity initially showed an upward trend. At pH 9, the cod skin ACE inhibitory peptide had the maximum activity of 40.33%. When the pH continued to rise, the ACE inhibitory activity began to decrease. The main reason for the analysis was that the hydrolytic protease was an alkaline protease that could act on the site to the maximum extent under alkaline conditions. Therefore, in order to protect the efficacy of the peptide from the harmful effects of excessive pH, and also for the optimal degree of hydrolysis and to increase the activity of the peptide, it is crucial to maintain a moderate pH value throughout the enzymatic process. Finally, the optimal pH of collagen hydrolase was determined to be 9.

[0072] Comparative Example 3

[0073] The pH remains unchanged, and the effects on ACE inhibition rate and degree of hydrolysis are different at different times. The present invention investigates the effects on the enzymatic hydrolyzate at different times, takes 1.0g of cod skin gelatin, sets the enzymatic hydrolysis temperature to 50°C, adds 2% enzyme, sets the enzymatic hydrolysis time to 2, 4, 6, 8, 10h, pH 9, and a solid-liquid ratio of 1:20. The optimal time is determined by measuring the ACE inhibition rate and degree of hydrolysis.

[0074] The effects of different time on ACE inhibition rate and hydrolysis degree are shown in Figure 4 . When the hydrolysis time is between 2.0 and 4.0 h, the degree of hydrolysis and ACE inhibitory activity also increase steadily with the extension of the hydrolysis time; the progress is rapid in the first four hours of the reaction because the enzyme quickly binds to the substrate at the beginning of the reaction and acts on the site to decompose the peptide. After 4.0 h, the degree of hydrolysis increases slightly and the ACE inhibitory activity begins to decrease. The ACE inhibitory activity of the product produced after 4.0 h of hydrolysis is the strongest, with an ACE inhibitory activity of 40.86%; if the reaction continues, although the degree of hydrolysis increases slightly, the stability of the ACE inhibitory peptide structure is destroyed due to excessive hydrolysis. 4.0 h was selected as the enzymatic hydrolysis time of collagen hydrolase.

[0075] Comparative Example 4

[0076] The time is unchanged, and the effects on ACE inhibition rate and hydrolysis degree are different under different enzyme addition amounts. The present invention investigates the effects on enzymolysis products under different enzyme addition amounts, takes 1.0g of cod skin gelatin, sets the enzymolysis temperature to 50°C, sets the enzyme addition amount to 1%, 2%, 3%, 4%, 5%, sets the enzymolysis time to 4.0h, pH 9, and the material-liquid ratio to 1:20. The optimal time is determined by measuring the ACE inhibition rate and hydrolysis degree.

[0077] The effects of different enzyme addition amounts on ACE inhibition rate and hydrolysis degree are shown in Figure 5 . When the enzyme dosage is in the range of 1.0% to 4.0%, the larger the enzyme dosage, the more sufficient the enzyme and substrate are combined, so the rate of the enzymatic reaction also increases. When the enzyme dosage is 4%, the hydrolysis degree is 31.04%, and the ACE inhibitory activity is 41.48%; the enzyme and substrate reaction has approached saturation, and the reaction rate has been flat. The subsequent increase in the enzyme dosage only slightly increases the hydrolysis degree to 31.47%, while the ACE inhibitory activity of the hydrolyzate is significantly reduced to 33.68%. Therefore, 4% is the optimal enzyme addition amount.

[0078] Comparative Example 5

[0079] The amount of enzyme added remains unchanged, and the effects of different material-liquid ratios on ACE inhibition rate and hydrolysis degree are also different. The present invention investigates the effects of different material-liquid ratios on enzymatic hydrolyzates, takes 1.0g of cod skin gelatin, sets the enzymatic hydrolysis temperature to 50°C, adds 4% enzyme, sets the enzymatic hydrolysis time to 4.0h, pH 9, and material-liquid ratios of 1:10, 1:20, 1:30, 1:40, and 1:50. The optimal time is determined by measuring the ACE inhibition rate and hydrolysis degree.

[0080] The effects of different material-liquid ratios on ACE inhibition rate and hydrolysis degree are shown in Figure 6 . When the material-liquid ratio is 1:20, the ACE inhibitory activity of the hydrolyzate is optimal at 42.89%, and the degree of hydrolysis also reaches a maximum value of 31.06%; as the material-liquid ratio gradually decreases, the contact area between the cod skin and the enzymatic hydrolyzate increases, achieving full exposure, and the mass transfer drive of the solvation is enhanced, which is beneficial to the dissolution of the ACE inhibitory active substances in the cod skin. When the material-liquid ratio is greater than 1:20g / mL, increasing the proportion of the solvent will lead to a decrease in the degree of hydrolysis and ACE inhibitory activity. This may be because the dilution of the solvent will reduce the full reaction of the enzyme with the substrate, thereby resulting in insufficient decomposition of the structure within the fish skin. The decomposition effect becomes less obvious, thereby reducing the ACE inhibitory activity. Therefore, the present invention selects 1:20g / mL as the optimal material-liquid ratio.

[0081] Example 3: Preparation of ACE inhibitory peptides from cod skin using the flavor protease method

[0082] S1, fish skin pretreatment: fresh frozen cod skin was taken, thawed at 4°C, the cod skin was descaled, residual fat was scraped off the inner skin, washed three times and then dried, and cut into 2×2 cm pieces after drying;

[0083] S2, impurity protein removal and defatting: the pretreated cod skin was added with 0.05 mol / L (0.2%) NaOH at a material-liquid ratio of 1:6 and soaked at 4°C for 24.0 hours, and washed with distilled water until neutral. The fish skin was added with 10% n-butanol at a material-liquid ratio of 1:10 and soaked for 24.0 hours, rinsed and rinsed until neutral, and then soaked with 1% citric acid for 8.0 hours, washed until neutral, and dried.

[0084] S3. Preparation of cod skin gelatin: fish skin and distilled water were extracted at 50°C hot water with a solid-liquid ratio of 1:10 for 12.0 h, centrifuged at 9000 r / min for 20 min, and the supernatant was taken. The supernatant was lyophilized by rotary evaporation to obtain gelatin.

[0085] S4. Preparation of collagen peptide: flavor protease, enzyme addition amount: 3%, solid-liquid ratio 1:20, pH 6, enzymatic hydrolysis temperature 40°C, enzymatic hydrolysis time 4.0h.

[0086] S5. Enzyme inactivation treatment: heat to 100°C and maintain for 20 min, then centrifuge at 9000 r / min for 20 min to obtain the supernatant.

[0087] S6, finished product: rotary evaporation concentration and vacuum freeze drying to obtain cod skin ACE inhibitory peptide.

[0088] Comparative Example 1

[0089] Effects of different temperatures on ACE inhibition rate and hydrolysis degree. The present invention investigates the effects of different temperatures on enzymatic hydrolysis products, takes 1.0 g of cod skin gelatin, sets the enzymatic hydrolysis temperature to 35, 40, 45, 50, 55°C, adds 2% enzyme, enzymatic hydrolysis for 4 hours, pH 7, and a material-liquid ratio of 1:20. The optimal temperature is determined by measuring the ACE inhibition rate and hydrolysis degree.

[0090] The effects of different temperatures on ACE inhibition rate and hydrolysis degree are shown in Figure 7 . In the temperature range of 35-55°C, as the hydrolysis temperature increases, the ACE inhibition rate and protein hydrolysis degree first increase and then decrease. When the hydrolysis temperature is 40°C, the ACE inhibition activity reaches a peak of 46.31%, and the hydrolysis degree at this time is 13.45%. The hydrolysis degree reaches a peak of 14.02% at 50°C, which is only slightly increased without obvious changes. As the temperature continues to rise, the enzyme structure stability decreases and the ACE inhibition rate decreases. Therefore, 40°C is determined to be the optimal temperature for proteolytic enzymes.

[0091] Comparative Example 2

[0092] The temperature remains unchanged, and the effects on ACE inhibition rate and hydrolysis degree at different pH values ​​are also different. The present invention investigates the effects on the enzymatic hydrolysis products at different pH values, takes 1.0 g of cod skin gelatin, sets the enzymatic hydrolysis temperature to 40°C, adds 2% enzyme, enzymatic hydrolysis for 4 hours, sets the pH to 5, 6, 7, 8, 9, and the solid-liquid ratio to 1:20. The optimal pH is determined by measuring the ACE inhibition rate and hydrolysis degree.

[0093] The effects of different pH on ACE inhibition rate and hydrolysis degree are shown in Figure 8 . The ACE inhibition rate and the degree of protein hydrolysis both increased first and then decreased with the increase of pH value. When the pH value was 6, the ACE inhibition activity reached a peak of 46.32%, and the protein hydrolysis degree was 13.56%. Although the hydrolysis degree increased slightly at pH = 7, it did not exceed 1%. The reason may be that the optimal pH of flavor protease is 6.0-7.0. If the pH is further increased, the spatial structure and active center of the flavor enzyme will be destroyed, and the catalytic hydrolysis activity will be reduced. Finally, the optimal pH of collagenase was determined to be 6.

[0094] Comparative Example 3

[0095] The pH remains unchanged, and the effects on ACE inhibition rate and hydrolysis degree at different times are also different. The present invention investigates the effects on the enzymatic hydrolysis product at different times, takes 1.0g of cod skin gelatin, sets the enzymatic hydrolysis temperature to 40°C, adds 2% enzyme, sets the enzymatic hydrolysis time to 2, 4, 6, 8, 10h, pH 6, and the solid-liquid ratio to 1:20. The optimal time is determined by measuring the ACE inhibition rate and hydrolysis degree.

[0096] The effects of different time on ACE inhibition rate and hydrolysis degree are shown in Fig. 9 . As time went on, both the degree of protein hydrolysis and the ACE inhibitory activity increased steadily until the duration of enzymatic hydrolysis was 4.0 h. The ACE inhibition rate of the hydrolysate began to decrease above 4.0 h, but the degree of protein hydrolysis continued to increase and then stabilized. The enzymatic hydrolysis reaction proceeded rapidly within 4.0 h, and the cod skin protein was continuously hydrolyzed, resulting in enhanced accumulation of ACE inhibitory peptides. Prolonged reaction time (>4.0 h) will lead to excessive degradation of cod skin protein, thereby reducing the content of ACE inhibitory peptides. After comprehensive consideration, a 4.0 h enzymatic hydrolysis time was selected.

[0097] Comparative Example 4

[0098] The time remains unchanged, and the effects on ACE inhibition rate and hydrolysis degree under different enzyme addition amounts are also different. The present invention investigates the effects on the enzymatic hydrolysis products under different enzyme addition amounts, takes 1.0g of cod skin gelatin, sets the enzymatic hydrolysis temperature to 40°C, sets the enzyme addition amount to 1%, 2%, 3%, 4%, 5%, sets the enzymatic hydrolysis time to 4h, pH 6, and the material-liquid ratio to 1:20. The optimal time is determined by measuring the ACE inhibition rate and hydrolysis degree.

[0099] The effects of different enzyme addition amounts on ACE inhibition rate and hydrolysis degree are shown in Fig.10 . The enzyme dosage has a significant effect on the ACE inhibitory activity of the protein hydrolysate. The ACE inhibitory activity of the protein hydrolysate prepared at a dosage of 3% reaches a peak of 58.75%, which is significantly stronger than the hydrolysates prepared with other enzyme additions. In addition, when the dosage is higher than 3%, the ACE inhibitory activity of the protein hydrolysate slowly decreases, and the degree of hydrolysis only increases slightly. The reason may be that when the enzyme addition amount is 1%-3%, the binding of cod skin protein and protein substrate in the reaction system does not reach a certain saturation, thereby increasing the reaction rate of enzymatic hydrolysis. However, when the enzyme dosage continues to increase, the binding of the enzyme and the substrate is closer to saturation. When the enzyme dosage continues to increase by more than 3%, the substrate is limited, which makes the protease unable to hydrolyze, and it will also cause varying degrees of resource waste. Therefore, 3% is the optimal enzyme addition amount.

[0100] Comparative Example 5

[0101] The amount of enzyme added remains unchanged, and the effects of different material-liquid ratios on ACE inhibition rate and hydrolysis degree are also different. The present invention investigates the effects of different material-liquid ratios on enzymatic hydrolyzates, takes 1.0g of cod skin gelatin, sets the enzymatic hydrolysis temperature to 40°C, adds 3% enzyme, sets the enzymatic hydrolysis time to 4.0h, pH 6, and the material-liquid ratios are 1:10, 1:20, 1:30, 1:40, and 1:50. The optimal time is determined by measuring the ACE inhibition rate and hydrolysis degree.

[0102] The effects of different material-liquid ratios on ACE inhibition rate and hydrolysis degree are shown in Fig.11 . Increasing the ratio of liquid to substrate to 1:10-1:20 can increase the chance of collision between substrate and enzyme in the reaction system, thereby increasing the rate of enzymatic hydrolysis reaction. When the ratio of liquid to substrate is 1:20, the ACE inhibitory activity reaches a peak value of 57.22% and the degree of hydrolysis is 14.75%. Increasing the solid-liquid ratio will cause the solvent volume to increase, thereby resulting in insufficient enzyme-catalyzed substrate reaction. On the other hand, the increase in solvent volume will affect the free diffusion and enzymolysis efficiency of the protein, thereby affecting the ACE inhibitory activity. Therefore, the present invention selects 1:20g / mL as the optimal solid-liquid ratio.

[0103] Example 4: Preparation of cod skin ACE inhibitory peptides by step-by-step enzymatic hydrolysis

[0104] S1, fish skin pretreatment: fresh frozen cod skin was taken, thawed at 4°C, the cod skin was descaled, residual fat was removed from the inner skin, washed three times and then dried, and cut into 2×2 cm pieces after drying;

[0105] S2, impurity protein removal and defatting: the pretreated cod skin was added with 0.05 mol / L (0.2%) NaOH at a material-liquid ratio of 1:6 and soaked at 4°C for 24 hours, and washed with distilled water until neutral. The fish skin was added with 10% n-butanol at a material-liquid ratio of 1:10 and soaked for 24 hours, rinsed until neutral, and then soaked with 1% citric acid for 8 hours, washed until neutral, and dried.

[0106] S3. Preparation of cod skin gelatin: fish skin and distilled water were extracted at 50°C hot water with a solid-liquid ratio of 1:10 for 12 h, centrifuged at 9000 r / min for 20 min, and the supernatant was taken. The supernatant was lyophilized by rotary evaporation to obtain gelatin.

[0107] S4. Preparation of collagen peptides: First step enzymatic hydrolysis: enzyme type: collagen hydrolase, enzyme amount: 4%, material-liquid ratio 1:20, pH 9, enzymatic hydrolysis temperature 50°C, enzymatic hydrolysis time: 4.5h; Second step enzymatic hydrolysis: enzyme type: flavor protease, enzyme amount: 3%, material-liquid ratio 1:20, pH 6, enzymatic hydrolysis temperature 40°C, enzymatic hydrolysis time 4.0h.

[0108] S5. Enzyme inactivation treatment: heat to 100°C and maintain for 20 min, then centrifuge at 9000 r / min for 20 min to obtain the supernatant.

[0109] S6. Membrane separation: Use 10, 3, and 1 kDa ultrafiltration membranes to separate the enzymatic hydrolysate in turn to obtain four components of >10, 3-10, 1-3, and <1 kDa.

[0110] S7, finished product: rotary evaporation concentration and vacuum freeze drying are performed in sequence to obtain cod skin ACE inhibitory peptide.

[0111] Comparative Example 1

[0112] According to Example 2 and Example 3, the order of the double enzyme combination was discussed, and the order of addition was shown in Table 3. After the double enzymes completed enzymolysis, the enzymes were inactivated at 100°C for 20 minutes, cooled to room temperature, and centrifuged at 9000 r / min for 20 minutes. This was repeated three times, and the supernatant was taken to determine its hydrolysis degree and its ACE inhibition rate.

[0113] Table 3 Step-by-step enzymatic hydrolysis sequence

[0114]

[0115] During the hydrolysis process, a single enzyme can often only act specifically on one or several types of peptide bonds, resulting in limited diversity and functionality of the enzymatic hydrolysis products. However, the dual-enzyme step-by-step enzymatic hydrolysis can utilize the specificity of different enzymes to act on different types of peptide bonds in turn, producing a richer variety of peptides, thereby improving ACE inhibitory activity. The results are shown in Table 4. It can be seen from the table that the ACE inhibition rate of collagenase followed by flavor protease is significantly improved compared with that of flavor protease followed by collagenase. The ACE inhibitory activity is mostly positively correlated with the degree of hydrolysis. The highest value of collagenase followed by flavor protease is 32.07±0.62%, and its ACE inhibition rate is also the maximum value of 65.44±0.53%.

[0116] Table 4 Step-by-step enzymatic hydrolysis results

[0117]

[0118] Studies have shown that the synergistic effect of different enzymes can increase the yield of ACE active peptides. Through double enzyme step-by-step enzymatic hydrolysis, peptides with ACE inhibitory activity can be generated more effectively. The results of single enzyme and double enzyme hydrolysis are as follows: Fig.12 As shown in the figure, it can be seen that the hydrolysis degree and ACE inhibition rate of cod skin collagen ACE inhibitory peptide 10 mg / mL were 14.70±0.49% and 45.43±0.66%, respectively, with IC 50 The value was 3.78 mg / mL. After process optimization, the hydrolysis degree and ACE inhibition rate of double enzyme step-by-step hydrolysis were significantly improved to 32.07%±0.62% and 65.44%±0.53%, respectively. 50The value is 1.46mg / mL.

[0119] Comparative Example 2

[0120] The above-mentioned optimal enzymatic hydrolysis conditions and enzymatic hydrolysis sequence were determined, and the cod skin ACE inhibitory peptides were separated by membranes of different molecular weights using step-by-step enzymatic hydrolysis. The enzymatic hydrolysate was separated by ultrafiltration membranes of 10, 3, and 1 kDa to obtain freeze-dried powders of four components of >10, 3-10, 1-3, and <1 kDa, and their ACE inhibition rates were determined. The results are as follows Fig.13 shown.

[0121] Membrane separation is to study the distribution of ACE inhibitory activity of cod skin ACE inhibitory peptides in different molecular weight ranges. Previous studies have shown that small molecule peptides often have higher biological activity and better absorption characteristics. Therefore, ultrafiltration separation can not only optimize the activity of the product, but also further reveal the relationship between the molecular weight and activity of cod skin ACE inhibitory peptides. This study used membrane separation to divide the enzymatic hydrolysate into 4 components: F1 (>10kDa), F2 (10kDa~3kDa), F3 (1kDa~3kDa) and F4 (<1kDa), and measured the ACE inhibitory activity of each component at 10mg / mL. Fig.13 It can be seen that the inhibitory activities of F1, F2 and F3 are 73.75%, 76.62%, 80.47% and 87.65% respectively. The ACE inhibitory activity of F4 is higher with IC 50 The value is 0.86mg / mL.

[0122] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is determined by the appended claims and their equivalents.

Claims

1. A method for preparing cod skin collagen peptide with high ACE inhibitory activity, characterized in that The following steps are involved: (1) Cod skin pretreatment Descale and clean the cod skin, soak it in NaOH, wash it with distilled water until it is neutral, soak it in n-butanol, rinse it with distilled water and set it aside; (2) Gelatin extraction The pre-treated cod skin is added with distilled water and hot water for extraction, and after the extraction is completed, the supernatant is centrifuged and concentrated under reduced pressure and dried under certain conditions to obtain cod skin gelatin; (3) First enzymatic hydrolysis of cod skin gelatin The obtained cod skin gelatin is added to distilled water with a pH of 7 to 11, and then a certain amount of protease preparation is added, and the enzymatic hydrolysis temperature is set to 40 to 60° C. and the enzymatic hydrolysis time is set to 2 to 10 hours. After the enzymatic hydrolysis is completed, the enzyme is inactivated at 90 to 100° C. for 10 to 30 minutes, and then cooled to room temperature, and centrifuged at 4000 to 10000 r / min for 10 to 30 minutes, and then the supernatant is taken for subsequent enzymatic hydrolysis; (4) Second enzymatic hydrolysis After adjusting the pH of the first enzymatic hydrolysis supernatant to 5-9, a certain amount of protease preparation is added, the enzymatic hydrolysis temperature is set to 35-55°C, the enzymatic hydrolysis time is set to 2-10h, after the enzymatic hydrolysis is completed, the enzyme is inactivated at 90-100°C for 10-30min, then cooled to room temperature, centrifuged at 4000-10000r / min for 10-30min, and the supernatant is separated; (5) Membrane separation The supernatant obtained after double-enzyme step-by-step enzymatic hydrolysis and centrifugation is passed through an ultrafiltration membrane of a certain specification to obtain a permeate which is then concentrated and dried under reduced pressure under certain conditions to obtain cod skin collagen peptide with high ACE inhibitory activity.

2. The preparation method according to claim 1, characterized in that: In step 2, the pretreated cod skin is added with distilled water at a material-liquid ratio of 1:10-1:20, and then extracted with hot water at 50-100° C. for 6-12 hours. The reduced pressure concentration temperature is 50-60° C., and the drying method can be freeze drying, spray drying, or a combination of the two.

3. The preparation method according to claim 2, characterized in that: In step 3, the amount of the protease preparation added is 0.5% to 5%, and the protease preparation can be one of pepsin, trypsin, alkaline protease, collagenase, neutral protease, composite protease, and flavor protease.

4. The preparation method according to claim 3, characterized in that: In the step 3, the amount of the protease preparation added is 2% to 5%, and the protease preparation is collagen hydrolase.

5. The preparation method according to claim 4, characterized in that: In step 4, the amount of the protease preparation added is 0.5% to 5%, and the protease type can be one of pepsin, trypsin, alkaline protease, collagenase, neutral protease, composite protease, and flavor protease.

6. The preparation method according to claim 5, characterized in that: In step 4, the amount of the protease preparation added is 2% to 5%, and the protease preparation is flavor protease.

7. The preparation method according to claim 6, characterized in that: The type of ultrafiltration membrane in step 5 may be one or a combination of 1000, 3000, 5000 or 10000 Da.

8. The preparation method according to claim 7, characterized in that: The reduced pressure concentration temperature in step 5 is 50-60° C., and the drying method can be freeze drying, spray drying, or a combination of the two.

9. The preparation method according to claim 8, characterized in that: The maximum inhibition rate of the cod skin collagen peptide obtained in step 5 on ACE is ≥80%.

10. Use of the collagen peptide obtained by the method for preparing the cod skin collagen peptide according to any one of claims 1 to 9 in the preparation of antihypertensive drugs.

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