Method for improving cholesterol lowering activity effect of crassostrea hongkongensis polypeptide
By optimizing the compound enzymatic hydrolysis process, flavor protease and animal protease were used to enzymatically hydrolyze Hong Kong oysters, which solved the problem of low cholesterol-lowering activity of peptides in Hong Kong oysters and achieved a significant increase in peptide content and activity.
Patent Information
- Application Number
- CN202511398172.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-28
- Publication Date
- 2026-02-06
AI Technical Summary
The cholesterol-lowering activity of Hong Kong oyster peptides in existing technologies is relatively low and difficult to further improve.
A compound enzymatic hydrolysis process was adopted, using a specific ratio of flavor protease and animal protease to hydrolyze Hong Kong oysters, and the hydrolysis conditions were optimized to increase peptide content and cholesterol-lowering activity.
It significantly improved the cholesterol micelle inhibition rate and bile salt binding rate of Hong Kong oyster peptides, and also improved the peptide content and yield, providing better cholesterol-lowering activity.
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Figure CN121472354A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biological medicine, in particular to a method for improving the cholesterol-lowering activity of oyster polypeptide. BACKGROUND
[0002] Oysters, as a traditional tonic marine product, have shown a variety of biological activities of their enzymatic polypeptide products, and have become a hot research field of marine active peptides. Oyster polypeptide is a small molecule active substance extracted from oysters by enzymatic or fermentation technology, which has high biological activity and easy absorption, and is widely used in health care, food and pharmaceutical fields. However, due to the inherent differences in the amino acid composition, sequence and structure of proteins in different oyster species, the cleavage sites and efficiency of different enzymes during enzymolysis are different, and the amino acid sequences of the active peptides produced are also different. Since the specific biological activity of oyster peptides (such as cholesterol-lowering, antioxidant activity, etc.) is highly dependent on specific amino acid sequences, the final results of enzymolysis using different oysters must be different.
[0003] Hong Kong oyster (Crassostrea hongkongensis) Crassostrea hongkongensis ), also known as white oyster, contains rich protein and trace elements, and has various biological activities such as anti-depression, anti-thrombosis, anti-bacterial, anti-tumor, anti-coagulation, anti-fatigue, etc. At present, there are few studies on the cholesterol-lowering activity of Hong Kong oyster polypeptide at home and abroad. The previous research of the present application shows that the polypeptide content prepared by animal protease enzymolysis of Hong Kong oyster is as high as 642.09 μg / mL, but its cholesterol inhibition rate is relatively low, with a maximum of 46.65%, and it is difficult to further improve the cholesterol-lowering activity of the polypeptide. Therefore, it is necessary to provide more methods to maintain the high polypeptide content of Hong Kong oyster polypeptide while improving its cholesterol-lowering activity.
[0004] Enzymatic process is the most core and key link in the preparation of oyster polypeptide, which directly determines the yield, composition, biological activity of polypeptide and application value of the final product. Oyster protein has large molecular weight and complex structure, and usually exists in a folded or aggregated state. Different proteases are used for enzymolysis, and different peptide sequences are produced due to different cleavage sites and hydrolysis degrees, which affects the yield of polypeptide and its biological activity. Even if the content of polypeptide prepared by enzymolysis is high, if the hydrolysis is not complete or excessive, free amino acids will be generated, which will also reduce the activity of oyster polypeptide. Therefore, the enzymolysis process directly determines the activity (cholesterol-lowering, antioxidant, ACE inhibition, etc.) and strength of the final product. For example, polypeptide prepared by using alkaline protease has good antioxidant activity, polypeptide prepared by using trypsin has good antibacterial activity, polypeptide prepared by using papain has good effect on enhancing endurance and reducing blood lactic acid level, and polypeptide prepared by using flavor protease can effectively reduce bitterness and improve flavor. At present, there are few studies on oyster polypeptide with cholesterol-lowering activity, and how to improve the cholesterol-lowering activity of Hong Kong oyster polypeptide is unknown, based on which the present application is proposed. SUMMARY
[0005] The technical problem to be solved by the present application is to overcome the problem that the cholesterol-lowering activity of the existing Hong Kong oyster polypeptide needs to be further improved. The present application provides a method for improving the cholesterol-lowering activity of Hong Kong oyster polypeptide.
[0006] The first object of the present application is to provide a method for improving the cholesterol-lowering activity of Hong Kong oyster polypeptide.
[0007] The second object of the present application is to provide a Hong Kong oyster polypeptide.
[0008] The third object of the present application is to provide the use of the above-mentioned method.
[0009] The fourth object of the present application is to provide the use of Hong Kong oyster polypeptide.
[0010] The above objects of the present application are achieved by the following technical solutions: The present application provides a method for preparing polypeptide with high cholesterol-lowering activity, comprising the following steps: S1. Preparation of Hong Kong oyster peptide: wash the Hong Kong oyster meat, remove impurities, uniformly beat the slurry to obtain oyster homogenate, mix with ultrapure water, and adjust the pH to 6.8-7.5; S2. Complex enzymatic hydrolysis: Adjust the enzymatic hydrolysis temperature to 45~55℃, add 3~4.5% of a complex enzyme of flavor protease and animal protease, and enzymatically hydrolyze for 3~4 h. Then, inactivate the enzyme at 100~120℃ for 8~10 min, centrifuge and take the supernatant to obtain Hong Kong oyster polypeptide; the complex enzyme is a neutral protease and an animal protein hydrolase, or a flavor protease and an animal protease.
[0011] To address the problem of low cholesterol-lowering activity of Hong Kong oyster peptides prepared by existing single-enzyme hydrolysis methods, this invention provides a method that significantly enhances the cholesterol-lowering activity of Hong Kong oyster peptides by using specific enzymatic hydrolysis process conditions while maintaining a high peptide content. The method provided by this invention employs a compound enzyme hydrolysis process under specific ratio conditions to prepare the crude extract of Hong Kong oyster polypeptides, resulting in significantly improved polypeptide content and yield. Compared to the optimal single-enzyme hydrolysis process in previous studies, which yielded oyster polypeptides with a content of 642.09 μg / mL, a cholesterol micelle inhibition rate of 46.65%, and a polypeptide yield of 63.25%, with binding rates of 63.20%, 51.21%, and 51.62% for sodium taurocholate, sodium glycocholate, and sodium cholate, respectively, this invention, using a specific ratio of compound enzymes—flavor protease and animal protease—results in a high polypeptide content of 784.28 μg / mL, a cholesterol micelle inhibition rate as high as 65.6%, a polypeptide yield of 73.99%, and binding rates of 80.79%, 79.91%, and 83.37% for sodium cholate, sodium glycocholate, and sodium taurocholate, respectively. The method significantly improved the cholesterol micelle inhibition rate and the binding rate of sodium cholate, sodium glycocholate, and sodium taurocholate of Hong Kong oyster polypeptides. Compared with existing single-enzyme prepared polypeptides with cholesterol-lowering activity, the Hong Kong oyster polypeptides prepared by this invention have better cholesterol-lowering activity and further improved polypeptide content and yield, providing an effective method for preparing more and better active oyster polypeptides.
[0012] Preferably, the ratio of oyster homogenate to ultrapure water in S1 is 1:1~4 g / mL.
[0013] More preferably, the ratio of oyster homogenate to ultrapure water is 1:3.5 g / mL.
[0014] Preferably, in S1, a 0.1-1 mol / L sodium hydroxide solution is used to adjust the pH to 6.8-7.5.
[0015] More preferably, the pH is adjusted to 7.0.
[0016] Preferably, after adjusting the pH in S1, stirring is performed at 500~800 r / min.
[0017] Preferably, the enzymolysis temperature in S2 is 45-53 DEG C, and the enzymolysis time is 3.25-3.75 h.
[0018] Further preferably, the enzymolysis temperature is 50-52 DEG C, and the enzymolysis time is 3.5-3.6 h.
[0019] Preferably, the mass ratio of the compound enzyme in S2 is (1-5):(1-4).
[0020] Preferably, the enzyme addition amount in S2 is 3.25-3.75%.
[0021] More preferably, the enzyme addition amount is 3.5%.
[0022] More preferably, the compound enzyme in S2 is a combination of flavor protease and animal protease, and the mass ratio is (2-5):1.
[0023] Further preferably, the mass ratio of the flavor protease and the animal protease is 3:1.
[0024] The application provides a Hong Kong oyster polypeptide prepared by the method.
[0025] The application provides application of the Hong Kong oyster polypeptide in preparation of a cholesterol-lowering product.
[0026] The application provides application of the method in improving the polypeptide content and yield of the Hong Kong oyster polypeptide.
[0027] The application provides application of the method in improving the cholesterol-lowering activity effect of the Hong Kong oyster polypeptide.
[0028] The application also provides a product containing the Hong Kong oyster polypeptide prepared by the specific method, wherein the cholesterol micelle inhibition rate is not less than 50%, the polypeptide yield is not less than 70%, and the sodium cholate, sodium glycocholate and sodium taurocholate combination rates are not less than 80%, 79% and 83% respectively.
[0029] The application has the following beneficial effects: The application provides a method for improving the cholesterol-lowering activity effect of a Hong Kong oyster polypeptide, which is prepared by using a compound enzyme enzymolysis process under specific conditions, so that the cholesterol-lowering activity of the Hong Kong oyster polypeptide crude extract is obviously improved; after the compound enzyme neutral protease and animal protease or the compound enzyme flavor protease and animal protease are used in a specific ratio for enzymolysis, the cholesterol-lowering activity effect of the Hong Kong oyster polypeptide is obviously improved while the polypeptide content and yield are maintained, compared with the polypeptide with cholesterol-lowering activity prepared by using the existing enzymolysis method, the method can obviously improve the cholesterol-lowering activity effect of the Hong Kong oyster polypeptide, and more effective methods and products of the oyster polypeptide with specific activity effect are provided. Attached Figure Description
[0030] Figure 1 The graph shows the results of peptide content and cholesterol micelle inhibition rate of enzymatic hydrolysis products of different complex enzyme groups.
[0031] Figure 2 The graph shows the results of bile salt binding rates of enzymatic hydrolysis products from different complex enzyme groups.
[0032] Figure 3 The effect of temperature on cholesterol micelle inhibition rate (different lowercase letters in the figure indicate significant differences). p <0.05).
[0033] Figure 4 The effect of time on cholesterol micelle inhibition rate (different lowercase letters in the figure indicate significant differences). p <0.05).
[0034] Figure 5 The effect of pH on cholesterol micelle inhibition rate (different lowercase letters in the figure indicate significant differences). p <0.05).
[0035] Figure 6 The effect of different enzyme dosages on cholesterol micelle inhibition rate (different lowercase letters in the figure indicate significant differences). p <0.05).
[0036] Figure 7 The effect of different feed-to-liquid ratios on cholesterol micelle inhibition rate (different lowercase letters in the figure indicate significant differences). p <0.05).
[0037] Figure 8 The graph shows the effect of different factors on the inhibition rate of cholesterol micelles, along with contour plots.
[0038] Figure 9 The graphs and contour plots show the effects of different factors on peptide concentration. Detailed Implementation
[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.
[0040] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.
[0041] The experiments in the examples were repeated in triplicate, and the results were expressed as mean ± standard deviation. SPSS 27.0 was used for data analysis, Origin 2021 software was used for plotting, and Design Expert 13.0 was used for response surface experimental design.
[0042] Example 1 Complex enzymatic hydrolysis 1. Compound scheme In order to improve the existing Hong Kong oyster polypeptide's cholesterol-lowering activity, the oyster peptide's enzymatic hydrolysis process was optimized in this embodiment. The Hong Kong oyster meat was washed and impurities were removed, then it was uniformly pulped. A certain mass of oyster homogenate was weighed, and ultrapure water was added according to a certain solid-liquid ratio (g / mL) ratio, mixed uniformly, and 0.1 mol / L sodium hydroxide solution was used to adjust the pH of enzymatic hydrolysis. It was placed in a constant temperature water bath with stirring at 700 r / min, and when the temperature was raised to the enzymatic hydrolysis temperature, a certain mass of enzyme was added, which was the mass of the substrate. After enzymatic hydrolysis for a certain time, the enzyme was inactivated at 100 ℃ for 10 min, and centrifuged at 12000 r / min for 10 min. The supernatant was taken and stored at -18 ℃ to -24 ℃.
[0043] Using complex enzymatic hydrolysis process, neutral protease + animal protein hydrolyzing enzyme, flavor protease + animal protein hydrolyzing enzyme, and flavor protease + neutral protease were set up respectively, and the mass ratio (g / g) of each compound enzyme was set as 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, and 4:1. The specific group settings are shown in Table 1. 10.0 g of oyster homogenate was weighed for each group, and the compound enzyme was used for enzymatic hydrolysis under the conditions of enzymatic hydrolysis temperature 50℃, enzymatic hydrolysis time 3.0h, enzymatic hydrolysis pH 7.0, enzyme addition amount 3.0%, and solid-liquid ratio 1:3 (g / mL). The polypeptide content, cholesterol micelle inhibition rate, and three kinds of cholic acid salt binding rate were determined as the determination indexes to determine the best compound enzyme and compound ratio.
[0044] Table 1 Different compound sample treatment groups
[0045] In the table: "neutral" is neutral protease; "animal" is animal protein hydrolyzing enzyme; "flavor" is flavor protease.
[0046] 2. Determination method (1) Polypeptide content determination: Folin-phenol method was used to determine the polypeptide content, and the polypeptide content standard curve (equation y1=0.00111x1+0.00027, R 2=0.9981. In the formula: x1 is the sample polypeptide content, pg / mL, y1 is the absorbance). Take 1.00 mL of sample, add 1.00 mL of 10% TCA, mix and stand after centrifugation for 5 min, take 1.00 mL of supernatant, and dilute with 5% TCA. According to the kit method, sample solution is taken and Folin-phenol reagent is added, after treatment, it is determined at 500 nm, and the absorbance is brought into the standard curve to obtain the corresponding polypeptide content of the sample.
[0047] (2) Cholesterol micelle inhibition rate determination: according to the method of Zhang Huijuan, ultrasonic is used to dissolve oleic acid, cholesterol, lecithin in a certain amount of methanol. Add sodium chloride, sodium taurocholate and 0.1 mol / L phosphate buffer solution with pH=7.4, ultrasonic treatment for 15 min, constant temperature culture in water bath for 12.0 h (the final cholesterol micelle solution contains 5 mmol / L cholesterol and oleic acid, 10 mmol / L lecithin and sodium taurocholate, 132 mmol / L sodium chloride and 15 mmol / L phosphate buffer solution). Take 1.0 mL of micelle solution and 1.0 mL of sample, constant temperature culture at 37 ℃ for 2.0 h, then centrifuge at 12000 r / min for 10 min. Take the blank group without sample as control, and use cholesterol kit to determine the content of cholesterol micelles. The cholesterol micelle inhibition rate is calculated according to the following formula.
[0048] ; ; In the formula: V1, V2 are the sample and standard sample volume, mL, and D is the dilution multiple.
[0049] (3) Simulated intestinal environment bile salt binding experiment: according to the method of Zhong Siyan, the concentration standard curve of sodium cholate, sodium glycocholate and sodium taurocholate is prepared (the equations are y4=3.12193x4+0.01411, R 2 =0.9980; y5=1.83472x5+0.00096, R 2 =0.9984; y6=2.69988x6+0.01824, R 2 =0.9981. In the formula: x4, x5, x6 represent the concentration of three kinds of bile salts, mmol / L, and y4, y5, y6 are the measured absorbance). Take 1.0 mL of sample, add 1.0 mL of 0.01 mol / L hydrochloric acid, constant temperature oscillation at 37 ℃ for 2.0 h, adjust the pH to 7.6, add 4.0 mL of bile salt solution and continue constant temperature oscillation for 2.0 h, then centrifuge at 12000 r / min for 10 min, and measure the absorbance of the supernatant at 387 nm. At the same time, a group of substrate blank and a group of bile salt blank are prepared. Calculate according to the following formula.
[0050] ; C1, C2 respectively represent the concentration of bile salt in blank and sample, mmol / L.
[0051] The results of enzymolysis using the complex enzymes are shown in Table 2. The results in the table show that the cholesterol micelle inhibition rate and the polypeptide content of the products after enzymolysis using different complex enzyme combinations and ratios have significant differences. The cholesterol micelle inhibition rate of some groups is high, but the polypeptide content is relatively low (E4, E8, E19, E21). The polypeptide content of some groups is high, but the cholesterol micelle inhibition rate is relatively low (E17, E18). It is shown that the polypeptide content and its cholesterol-lowering activity are not positively correlated. The polypeptide content of the group is not necessarily good in terms of its cholesterol-lowering activity. In the table, under the same cholesterol micelle inhibition rate results, the polypeptide content difference is very significant (such as E6 and E8, or E12 and E19, E14 and E16). This is because different complex enzyme combinations and their ratios are used, the enzyme cutting sites are different, and the activity effect of the prepared polypeptides has significant differences.
[0052] Table 2 Polypeptide content and cholesterol micelle inhibition rate results of different complex enzyme groups
[0053] The polypeptide content, cholesterol micelle inhibition rate and bile salt binding rate results measured by using 21 groups of complex enzyme combinations are shown in Table 2. Figure 1 and Figure 2 It is shown that the polypeptide content, cholesterol micelle inhibition rate are higher when the complex enzymes use neutral protease and animal protein hydrolysis enzyme, and flavor protease and animal protease. Among them, the comprehensive complex enzyme hydrolysis ability of flavor protease: animal protease = 3:1 (E13 group) is the best. The polypeptide content of the enzyme hydrolysis is 784.28 μg / mL, the cholesterol micelle inhibition rate is 58.29 %, the binding rates of sodium cholate, sodium glycocholate and sodium taurocholate are 80.79 %, 79.91 % and 83.37 % respectively. The polypeptide content, cholesterol micelle inhibition rate and bile salt binding rate are the highest in the 21 groups of enzyme hydrolysis experiments. Therefore, the flavor protease: animal protease mass ratio (g / g) of 3:1 is selected for subsequent enzyme hydrolysis process optimization experiments.
[0054] Example 2 Condition optimization of complex enzyme enzyme hydrolysis method 1. Condition optimization The flavor protease screened according to Example 1: animal protein hydrolyzing enzyme = 3:1 complex enzyme (E13) group was subjected to single factor experiment of condition optimization, and the oyster homogenate was freeze-dried to obtain oyster dry powder. The oyster dry powder: water = 1:8 g / mL was prepared into oyster solution, and 2.0 g of the oyster solution was subjected to single factor enzyme hydrolysis experiment, and the effects of temperature, time, pH, enzyme amount and solid-liquid ratio on the enzyme hydrolysis effect were investigated. The F test and significance analysis of the cholesterol-lowering activity index of the screened enzyme (detection method see Example 1) were carried out, the cholesterol micelle inhibition rate was selected as the single factor index, and the activity index analysis results are shown in Table 3. The F test and significance analysis were carried out by using SPSS 27.0, since the significant differences between the index groups were all extremely significant differences, the cholesterol micelle inhibition rate was selected as the determination index of condition optimization; the polypeptide concentration and the cholesterol micelle inhibition rate were selected as the response surface determination index.
[0055] Table 3 Data analysis of cholesterol-lowering activity index
[0056] Note: * in the table indicates that the comparison between each index has significant difference p <0.05), ** indicates that the comparison between each index has extremely significant difference p <0.01).
[0057] (1) Enzymolysis temperature: the enzyme hydrolysis temperature (35, 40, 45, 50, 55, 60, 65 ℃) was used for enzyme hydrolysis, with the enzyme hydrolysis time being fixed at 3.0 h, pH = 7.0, enzyme amount being 3.0 %, and solid-liquid ratio being 1:3 (g / mL).
[0058] (2) Enzymolysis time: the enzyme hydrolysis time (1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 h) was used for enzyme hydrolysis, with the enzyme hydrolysis temperature being fixed at 50 ℃, pH = 7.0, enzyme amount being 3.0 %, and solid-liquid ratio being 1:3 (g / mL).
[0059] (3) pH: the enzyme hydrolysis pH (5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5) was used for enzyme hydrolysis, with the enzyme hydrolysis temperature being fixed at 50 ℃, enzyme hydrolysis time being 3.5 h, enzyme amount being 3.0 %, and solid-liquid ratio being 1:3 (g / mL).
[0060] (4) Enzyme amount: the enzyme amount (2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 %) was used for enzyme hydrolysis, with the enzyme hydrolysis temperature being fixed at 50 ℃, enzyme hydrolysis time being 3.5 h, pH = 7.0, and solid-liquid ratio being 1:3 (g / mL).
[0061] (5) Material-liquid ratio: The enzymatic hydrolysis temperature was fixed at 50 °C, the enzymatic hydrolysis time was 3.5 h, the pH was 7.0, and the enzyme dosage was 3.5%. Enzymatic hydrolysis was carried out at material-liquid ratios of (1:1.0, 1:1.5, 1:2.0, 1:2.5, 1:3.0, 1:3.5, 1:4.0 g / mL).
[0062] The effect of different enzymatic hydrolysis temperatures on cholesterol micelle inhibition rate is as follows: Figure 3 As shown, the cholesterol micelle inhibition rate reaches its maximum value of 65.41% when the enzymatic hydrolysis temperature reaches 50℃. Furthermore, when the enzymatic hydrolysis temperature reaches 35℃ to 50℃, the cholesterol micelle inhibition rate increases with increasing reaction temperature; while when the enzymatic hydrolysis temperature reaches 50℃ to 65℃, the cholesterol micelle inhibition rate decreases with increasing reaction temperature.
[0063] The effect of different enzymatic hydrolysis times on cholesterol micelle inhibition rate is as follows: Figure 4 As shown, the cholesterol micelle inhibition rate reached its maximum value of 65.60% when the enzymatic hydrolysis time reached 3.5 h. Furthermore, when the enzymatic hydrolysis time was 1.5 h–2.0 h and 2.5 h–3.5 h, the cholesterol micelle inhibition rate increased with increasing reaction time; however, when the enzymatic hydrolysis time was 2.0 h–2.5 h and 3.5 h–4.5 h, the cholesterol micelle inhibition rate decreased with increasing reaction time.
[0064] The effect of different pH values on cholesterol micelle inhibition rate is as follows: Figure 5 As shown, the cholesterol micelle inhibition rate reached its maximum of 46.82% at pH 7.0. The results of enzymatic hydrolysis at pH 7.0 showed a significant difference compared to other pH experiments. p <0.05 indicates that the result is reliable. When the pH is in the range of 5.5 to 7.0, and in the range of 7.5 to 8.0, the cholesterol micelle inhibition rate increases with increasing pH, but the cholesterol micelle inhibition rate never exceeds 46.82%, and when the pH is in the range of 8.0-8.5, the cholesterol micelle inhibition rate shows a decreasing trend.
[0065] The effect of different enzyme dosages on cholesterol micelle inhibition rate is as follows: Figure 6 As shown, when the enzyme dosage was 3.5% of the substrate mass, the cholesterol micelle inhibition rate reached a peak of 57.5%, and this result was significantly different from the results of other enzyme dosage experiments. p <0.05), the result is reliable.
[0066] The effect of different feed-to-liquid ratios on cholesterol micelle inhibition rate is as follows: Figure 7 As shown, the highest cholesterol micelle inhibition rate of 40.74% was achieved when the material-to-liquid ratio was 1:3.5 g / mL, and this result was significantly different from the results of experiments with other material-to-liquid ratios.p <0.05).
[0067] 2. Optimization of response surface methodology Based on the optimization of the single-factor experiments described above, F-tests and significance analyses were performed, as shown in Table 4. Enzymatic hydrolysis temperature (A), hydrolysis time (B), and enzyme dosage (C) were selected as independent response variables. Response surface methodology was conducted using Design Expert software. Following the Box-Behnken central composite design, with a fixed pH of 7.0 and a solid-liquid ratio of 1:3.5 g / mL, a three-factor, three-level experiment was designed, resulting in 17 experimental groups. The enzymatic hydrolysis process conditions were optimized using peptide content and cholesterol micelle inhibition rate as response values. The influencing factors and levels are shown in Table 5.
[0068] Table 4. Single-factor data analysis
[0069] Table 5. Experimental Factors and Levels in the Box-Behnken Method
[0070] The results of response surface methodology optimization are shown in Table 6. The results show that the condition parameters significantly affect the cholesterol micelle inhibition rate. Under the condition of high peptide content, the cholesterol micelle inhibition rate is still low. Under different conditions, the cholesterol micelle inhibition rate and peptide concentration of oyster enzymatic hydrolysate show significant differences.
[0071] Table 6 Response Surface Design for Enzymatic Hydrolysis Process Optimization
[0072] 3. Model Variance Analysis After fitting the data in Table 6 using Design-Expert 13 software, the bivariate polynomial regression equation of the model was obtained. Using the cholesterol micelle inhibition rate as the response value, the ternary quadratic regression equation relating the cholesterol micelle inhibition rate to temperature (A), time (B), and enzyme dosage (C) is as follows: Cholesterol micelle inhibition rate = 53.528 + 2.1669A + 7.0199B + 5.0590C + 1.2100AB - 0.8582AC - 4.0935BC - 9.8468A 2 -16.1890B 2 -16.7290C 2 .
[0073] The results of the analysis of variance for the regression model of cholesterol micelle inhibition rate are shown in Table 7. It can be seen that the regression model of cholesterol micelle inhibition rate... P <0.01, the model is significant; lack of fit term PThe value is 0.0672 > 0.05, which is not significant, indicating that the equation is not lacking in fit and the experimental results are less affected by unknown factors; the coefficient of determination R0 2 =0.9023, the equation is relatively reliable. In summary, the regression model fits well and can be used to predict and analyze changes in cholesterol micelle inhibition rate. Among the factors, A 2 B has a significant impact on the model. P <0.05), quadratic term B 2 C 2 The impact on the model is extremely significant. P <0.01). According to the F-test, the influence of the three factors on the cholesterol micelle inhibition rate, from largest to smallest, is: enzymatic hydrolysis time, enzyme dosage, and enzymatic hydrolysis temperature.
[0074] Table 7. Analysis of variance of the regression model for cholesterol micelle inhibition rate
[0075] Note: * indicates a significant difference (P<0.05), and ** indicates an extremely significant difference (P<0.01).
[0076] Using peptide concentration as the response value, the ternary quadratic regression equation fitting the relationship between cholesterol micelle inhibition rate and temperature (A), time (B), and enzyme dosage (C) is as follows: Peptide concentration = 565.01 + 12.554A - 4.0218B - 32.964C + 44.353AB - 17.140AC - 0.37587BC - 53.532A 2 -64.432B 2 -74.656C 2 .
[0077] The results of the analysis of variance for the regression model of peptide content are shown in Table 8. It can be seen that the peptide concentration regression model... P <0.01, model is significant; lack of fit term P The value is 0.0513 > 0.05, which is not significant, indicating that the equation is not lacking in fit and the experimental results are less affected by unknown factors; the coefficient of determination R0 2 =0.9251, the equation is relatively reliable, and in summary, the regression model fits well and can be used to predict and analyze changes in peptide concentration. Among the factors, the linear terms C and AB have a significant impact on the model. P <0.05), quadratic term A 2 B 2 C 2 The impact on the model is extremely significant. P <0.01). According to the F-test, the influence of the three factors on the cholesterol micelle inhibition rate, from largest to smallest, is: enzyme dosage, enzymatic hydrolysis temperature, and enzymatic hydrolysis time.
[0078] Table 8 Analysis of variance of regression model of polypeptide content
[0079] Note: * indicates significant difference (P < 0.05), ** indicates extremely significant difference (P < 0.01).
[0080] 4. Response surface analysis of multi-factor interaction The 3D response surface graph can be drawn according to the regression model by using Design Expert 13 software, which can clearly and intuitively reflect the influence of each factor and its interaction on the response value, so as to explore and predict the optimal enzymolysis process parameter range. The sensitivity of the response surface to the change of the independent variable can be reflected by the change of the slope of the response surface. The more significant the influence of a single factor on the response value, the more steep the curve surface will be. The response surface graphs of the cholesteryl ester micelle inhibition rate and the polypeptide concentration are shown in Figure 8 and Figure 9 When the enzymolysis temperature is fixed, the cholesteryl ester micelle inhibition rate and the polypeptide concentration increase first and then decrease with the increase of the enzymolysis time. When the enzymolysis temperature is fixed, the cholesteryl ester micelle inhibition rate and the polypeptide concentration increase first and then decrease with the increase of the enzyme amount.
[0081] The regression equation is solved by using Design Expert 13 software, and the cholesteryl ester micelle inhibition rate and the polypeptide concentration are set as Maximize. After slightly correcting and adjusting the optimal process, the optimal enzymolysis process parameter conditions of the cholesteryl ester micelle inhibition rate and the polypeptide concentration are obtained as follows: the enzymolysis temperature is 51℃, the enzymolysis time is 3.56 h, and the enzyme amount is 3.5%. The cholesteryl ester micelle inhibition rate is 54.121%, and the polypeptide concentration is 565.91 μg / mL.
[0082] Example 3 A method for improving the cholesterol-lowering activity of oyster polypeptide S1. Preparation of Hong Kong oyster peptide The Hong Kong oyster meat was washed and cleaned, and then uniformly pulped. A certain mass of oyster homogenate was mixed with ultrapure water at a ratio of 1:3.5 (g / mL), and the pH of the enzymolysis was adjusted to 7.0 using 0.1 mol / L sodium hydroxide solution, and then placed in a constant temperature water bath with stirring at 700 r / min.
[0083] S2. Compound enzymolysis When the enzymolysis temperature was adjusted to 51℃, 3.5% flavor protease: animal protease = 3:1 compound enzyme was added, and the enzymolysis was carried out for 3.56 h. Then the enzyme was inactivated at 100℃ for 10 min, and centrifuged at 12000 r / min for 10 min. The supernatant was oyster enzymolysis product (crude polypeptide), which could be further purified.
[0084] Under the optimal enzymolysis process parameter conditions, the reliability of the model was verified through three groups of parallel tests, the average inhibition rate of cholesterol micelles was 54.891%, the average concentration of polypeptides was 587.220 μg / mL, the relative error was less than 5%, which was consistent with the model prediction results, and the polypeptide yield was calculated to be 73.99%.
[0085] Similarly, after enzymolysis by the flavor protease: animal protease = 5: 1 complex enzyme in the same way as the above method, the cholesterol micelle inhibition rate was 56.77%, compared with the prior art single enzyme method (Comparative Example 1-2), the cholesterol-lowering activity of oyster polypeptides was obviously improved, on the basis of maintaining a high polypeptide content, the yield of polypeptides was improved and had better activity effect, and could be used to prepare more efficient cholesterol-lowering drugs.
[0086] Comparative Example 1 Animal protease enzymolysis Animal protease was used for enzymolysis, the enzymolysis method was the same as in Example 3, and the polypeptide content of the product after enzymolysis was 642.09 μg / mL under the optimal conditions of the enzyme, and the cholesterol micelle inhibition rate was 46.65%.
[0087] Comparative Example 2 Flavor protease enzymolysis Flavor protease was used for enzymolysis, the enzymolysis method was the same as in Example 3, and the polypeptide content of the product after enzymolysis was 525.12 μg / mL under the optimal conditions of the enzyme, and the cholesterol micelle inhibition rate was 45.47%.
[0088] Comparative Example 3 Complex enzymolysis Flavor protease: animal protease = 1:5 complex enzyme combination was used for enzymolysis, the enzymolysis method was the same as in Example 3 under the optimal conditions, and the polypeptide content of the product after enzymolysis was 546.78 μg / mL, and the cholesterol micelle inhibition rate was 46.57%.
[0089] Comparative Example 4 Complex enzymolysis Flavor protease: animal protease = 6:1 complex enzyme combination was used for enzymolysis, the enzymolysis method was the same as in Example 3 under the optimal conditions, and the polypeptide content of the product after enzymolysis was 556.47 μg / mL, and the cholesterol micelle inhibition rate was 41.48%.
[0090] Comparative Example 5 Complex enzymolysis Flavor protease, animal protease and neutral protease were compounded in a ratio of 1:1:1, and the enzymolysis was carried out under the optimal conditions of Example 3, and the polypeptide content of the product after enzymolysis was 501.26 μg / mL, and the cholesterol micelle inhibition rate was 38.14%.
[0091] The above comparative example results show that there is no significant difference in the effect of using only flavour protease or animal protease for enzymolysis, although the polypeptide content is higher, the cholesterol-lowering activity is lower (Comparative Examples 1-2); after using the complex enzymolysis process, the polypeptide content is also higher after flavour protease and animal protease are used for enzymolysis at a ratio of 6:1 or 1:5, but the cholesterol-lowering activity is still lower (Comparative Examples 3-4), which shows that the complex enzymolysis process can only improve the cholesterol-lowering activity of oyster polypeptide under specific conditions and ratios. After using three enzymes for complex enzymolysis, the cholesterol micelle inhibition rate is even lower (Comparative Example 5), which shows that the more enzymes used for enzymolysis, the better the effect. Therefore, it can be seen that different enzymolysis process conditions have a greater effect on the cholesterol-lowering activity of oyster polypeptide, and the oyster polypeptide with high polypeptide content does not necessarily have good cholesterol-lowering activity effect, only under specific complex enzymolysis conditions can the cholesterol-lowering activity of oyster polypeptide be improved. The method provided by the present application can significantly improve the cholesterol-lowering activity and maintain a high polypeptide content, and has a better cholesterol-lowering effect than the existing oyster polypeptide enzymolysis.
[0092] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.
Claims
1. A method for enhancing the cholesterol-lowering activity of peptides in Hong Kong oysters, characterized in that, Includes the following steps: S1. Preparation of Hong Kong oyster peptides: Take Hong Kong oyster meat, wash it, remove impurities, and then beat it evenly to obtain oyster homogenate. Add ultrapure water and mix well, and adjust the pH to 6.8~7.
5. S2. Compound enzymatic hydrolysis: Adjust the enzymatic hydrolysis temperature to 45~55℃, add 3~4.5% compound enzyme, enzymatically hydrolyze for 3~4 h, then inactivate the enzyme at 100~120℃ for 8~10 min, centrifuge and take the supernatant to obtain Hong Kong oyster polypeptide; the compound enzyme is a neutral protease and an animal protein hydrolase, or a flavor protease and an animal protease.
2. The method according to claim 1, characterized in that, In S1, the ratio of oyster homogenate to ultrapure water is 1:1~4 g / mL.
3. The method according to claim 1, characterized in that, In S1, the pH is adjusted to 6.8-7.5 using a 0.1-1 mol / L sodium hydroxide solution, and then stirred at 500-800 r / min.
4. The method according to claim 1, characterized in that, The mass ratio of the complex enzyme in S2 is (1~5):(1~4).
5. The method according to claim 4, characterized in that, The amount of enzyme added in S2 is 3.25~3.75%.
6. The method according to claim 5, characterized in that, S2 uses a combination of flavor protease and animal protease in a mass ratio of (2~5):
1.
7. The method according to claim 6, characterized in that, The enzymatic hydrolysis temperature in S2 is 50~55℃, and the enzymatic hydrolysis time is 3.5~4 h.
8. The Hong Kong oyster polypeptide prepared by the method according to any one of claims 1 to 7.
9. The use of the Hong Kong oyster polypeptide according to claim 8 in the preparation of cholesterol-lowering products.
10. The application of the method according to any one of claims 1 to 7 in improving the cholesterol-lowering activity, peptide content, and yield of Hong Kong oyster peptides.