Preparation method of artemia polypeptide

Abstract

The invention provides a preparation method of artemia polypeptide, and belongs to the technical field of polypeptide preparation. The preparation method of the artemia polypeptide comprises the following steps: performing enzymolysis on artemia slurry under the action of protease to obtain the artemia polypeptide, the protease comprises alkaline protease and / or papain. According to the method, specific parameters such as the addition amount of protease, the pH value of enzymolysis and the enzymolysis temperature are further optimized, so that the artemia hydrolysis degree reaches 68.28%. According to the preparation method provided by the invention, a large amount of artemia polypeptides are obtained, and a new technical means is provided for reasonable development and high-valued resource utilization of artemia.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide preparation, and particularly relates to a method for preparing an Artemia polypeptide. Background Art

[0002] Artemia (Artemia) belongs to the phylum Arthropoda, subphylum Crustacea, class Branchiopoda, order Acropora, family Artemia, genus Artemia. It is a small crustacean widely distributed in high-salinity environments such as salt lakes and salt pans. As a typical organism tolerant of extreme environments, Artemia has a strong salinity tolerance and can survive in salinity ranges of 30 to 200 g / L. It possesses a robust osmotic pressure regulation system, which facilitates the synthesis of a variety of active substances with specialized functions. Furthermore, Artemia contains a high protein content of 45% to 55% (dry weight), is rich in nutrients such as highly unsaturated fatty acids, and has a balanced amino acid profile, making it a high-quality source for the development of bioactive peptides. Existing research has confirmed that Artemia protein extracts possess certain biological activities and show significant application potential and prospects in functional foods, cosmetics, and aquaculture. However, the current industrial application of Artemia adult resources is limited to supplementary feed for aquatic animals, with a relatively limited market application and low conversion rate to high added value. The development of Artemia peptide products through enzymatic hydrolysis is a promising approach to further develop Artemia adult resources. At present, there is almost no research on the preparation process and structural identification of Artemia-derived polypeptides, which seriously hinders the application of Artemia polypeptides. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide a method for preparing Artemia polypeptides, by optimizing the protein types and improving the Artemia hydrolysis degree, thereby increasing the Artemia polypeptide content.

[0004] The present invention provides a method for preparing an Artemia polypeptide, comprising the following steps:

[0005] Artemia slurry is enzymatically hydrolyzed under the action of protease to obtain Artemia polypeptide;

[0006] The protease includes alkaline protease and / or papain.

[0007] Preferably, the added amount of the protease is 500 to 2500 U / g Artemia dry weight.

[0008] Preferably, the added amount of the protease is 600 to 2100 U / g Artemia dry weight.

[0009] Preferably, the enzymatic hydrolysis temperature of the protease is 45-65°C.

[0010] Preferably, the enzymatic hydrolysis temperature of the protease is 50-60°C.

[0011] Preferably, the enzymatic hydrolysis temperature of the protease is 55-55.6°C.

[0012] Preferably, the enzymatic hydrolysis pH value of the protease is 8-10.

[0013] Preferably, the enzymatic hydrolysis pH value of the protease is 8.5-9.5.

[0014] Preferably, the enzymatic hydrolysis pH value of the protease is 9.

[0015] Preferably, the enzymatic hydrolysis time is 1.5 to 2.5 hours;

[0016] After the enzymatic hydrolysis, the method further includes inactivating the enzymes in the obtained enzymatic hydrolysis products and separating the Artemia polypeptides.

[0017] The present invention provides a method for preparing an Artemia polypeptide, comprising the following steps: enzymatically hydrolyzing an Artemia slurry under the action of a protease to obtain the Artemia polypeptide; the protease includes alkaline protease and / or papain. The present invention uses Artemia slurry as raw material and optimizes the type of protease used for enzymatic hydrolysis. When enzymatically hydrolyzed with alkaline protease or papain alone, the degree of hydrolysis of Artemia can be greatly increased, thereby increasing the Artemia polypeptide content. This provides a new technical means for the rational development and high-value resource utilization of Artemia.

[0018] The method for preparing Artemia polypeptides provided herein further defines specific parameters such as the amount of protease added, the pH value of the enzymatic hydrolysis, and the enzymatic hydrolysis temperature. Through single-factor optimization, steepest-hill climb experiments based on the single-factor results, and Box-Behnken response surface optimization, the present invention achieved a degree of hydrolysis of Artemia greater than 50%, with an optimal value of 68.28%. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The results show the effects of different proteases on the hydrolysis degree of Artemia adult protein;

[0020] Figure 2 The results show the effect of enzyme addition on the degree of protein hydrolysis of Artemia adult.

[0021] Figure 3 The results show the effect of enzymatic hydrolysis temperature on the degree of protein hydrolysis of Artemia adult;

[0022] Figure 4 The results show the effect of enzymatic hydrolysis pH on the degree of protein hydrolysis of Artemia adult.

[0023] Figure 5 The results show the effect of enzymatic hydrolysis time on the hydrolysis degree of Artemia adult protein. DETAILED DESCRIPTION

[0024] The present invention provides a method for preparing an Artemia polypeptide, comprising the following steps:

[0025] Artemia slurry is enzymatically hydrolyzed under the action of protease to obtain Artemia polypeptide; the protease includes alkaline protease and / or papain.

[0026] In the present invention, the Artemia slurry is a slurry obtained by crushing adult Artemia slurry. The adult Artemia slurry includes fresh Artemia slurry and / or frozen Artemia slurry. The present invention does not particularly limit the method of crushing, and crushing methods well known in the art can be used, such as homogenization, grinding, etc. In an embodiment of the present invention, the homogenization is performed using a homogenizer for 3 minutes.

[0027] In the present invention, the protease is preferably alkaline protease. In an embodiment of the present invention, in order to improve the hydrolysis degree of Artemia, an experiment was carried out to optimize the type of protease. Alkaline protease, acidic protease, pepsin and papain were used as protease for enzymolysis, and the degree of hydrolysis was measured. The results showed that alkaline protease and papain were more effective than acidic protease and pepsin in terms of the degree of hydrolysis, and alkaline protease had the advantage of hydrolyzing Artemia more than papain. The enzymatic activity of the papain was preferably 2,000,000 U / g. The enzymatic activity of the alkaline protease was 200,000 U / g.

[0028] In the present invention, the amount of the protease added is preferably 500-2500U / g Artemia dry weight, can be 600-2100U / g Artemia dry weight, further 900-1600U / g Artemia dry weight, can also be 1000-1800U / g Artemia dry weight, can also be 1100-1500U / g Artemia dry weight. The enzymatic hydrolysis temperature of the protease is preferably 45-65°C, can be 50-60°C, further 55-55.6°C. The enzymatic hydrolysis pH value of the protease is preferably 8-10, can also be 8.5-9.5, can also be 9. The enzymatic hydrolysis time is preferably 1.5-2.5h, can also be 2h. In one embodiment of the present invention, the degree of hydrolysis of different enzymatic hydrolysis products was compared with the enzyme addition amount, enzymatic hydrolysis pH, and enzymatic hydrolysis temperature as variable factors. The results show that the degree of influence on the hydrolysis degree of Artemia is ranked as follows: amount of protease added > enzymatic hydrolysis temperature > enzymatic hydrolysis pH. After the establishment of response surface regression model and variance analysis, the optimal process for preparing peptides from Artemia adult enzymatic hydrolysis was as follows: enzyme dosage 2500U / g, enzymatic hydrolysis temperature 55.6℃, and enzymatic hydrolysis pH 9.0. The results showed that the hydrolysis degree of Artemia adult protein was 68.28%.

[0029] In the present invention, after the enzymatic hydrolysis, the enzymatic hydrolysis product is further inactivated and the Artemia polypeptide is separated. The enzyme inactivation method preferably includes inactivating the enzyme in a high-temperature water bath. The temperature of the high-temperature water bath inactivation is preferably 90-100°C, and can be 95°C. The enzyme inactivation time is 5-20 minutes, and can be 10 minutes. The method for isolating the Artemia polypeptide is preferably centrifugation, and the supernatant is collected.

[0030] The preparation method provided by the present invention can obtain Artemia polypeptides with a high degree of hydrolysis, providing a new technical means for the resource utilization of Artemia and providing a raw material basis for the functional utilization of the active ingredients of Artemia.

[0031] The following is a detailed description of the method for preparing a Artemia polypeptide provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1

[0033] A method for preparing Artemia polypeptide

[0034] 1 Materials and Methods

[0035] 1.1 Test materials

[0036] Artemia adult frozen blocks were obtained from Tangshan Caofeidian Huitong Aquatic Technology Co., Ltd.

[0037] 1.2 Reagents and Instruments

[0038] Reagents: Alkaline protease (200,000 U / g) was from Tianjin Noo Enzyme Productivity Promotion Co., Ltd.; papain (2 million U / g) and acid protease (700,000 U / g) were from Macklin; pepsin (3 million U / g) was from Bid Pharmaceuticals; DL-dithiothreitol (DTT), o-phthalaldehyde, sodium dodecylbenzenesulfonate, and sodium tetraborate were from Aladdin; L-serine was from Solebo Company.

[0039] Instruments: electronic balance; pH meter; constant temperature oscillator; disperser; high-speed refrigerated centrifuge; freeze dryer; UV-visible spectrophotometer.

[0040] 1.3 Experimental methods

[0041] 1.3.1 Extraction process of Artemia adult polypeptide

[0042] Peptide extraction steps

[0043] Weigh 100 g of Artemia adult frozen blocks and thaw them in a beaker. Use a homogenizer to homogenize for 3 minutes to obtain Artemia adult slurry, adjust to the optimal pH value, add protease, and enzymatically hydrolyze at the optimal temperature for 2 hours. After the enzymatic hydrolysis is completed, quickly transfer to a 100°C water bath to inactivate the enzyme for 15 minutes. After cooling to room temperature, centrifuge at 8000 r / min for 12 minutes, collect the supernatant, filter, and freeze-dry the supernatant for later use.

[0044] 1.3.2 Optimization of Artemia adult peptide extraction process

[0045] (1) Determination of the degree of protein hydrolysis of Artemia adult

[0046] The degree of Artemia protein hydrolysis was determined using the OPA method. OPA reagent: First, prepare Solution A (dissolve 3.810g of sodium tetraborate and 100mg of SDS in 75mL of distilled water), then prepare Solution B (dissolve 80mg of 97% OPA in 2mL of ethanol). Mix the two solutions, add 88mg of 99% DTT, and dilute to 100mL with distilled water. Because this reagent is light-sensitive, protect from light during preparation and use.

[0047] To create a serine standard curve: Weigh 100 mg of serine and dilute to 1000 mL with deionized water. Add 0, 100 μL, 200 μL, 300 μL, and 400 μL of the serine standard solution to a 5 mL centrifuge tube. Fill the volume to 400 μL with deionized water. Add 3 mL of LOP A reagent, mix thoroughly, and incubate in the dark for 2 minutes. Measure the absorbance at 340 nm. Use the absorbance versus serine concentration to create a standard curve.

[0048] Determination of hydrolysis degree of hydrolyzate: Take 400 μL of appropriately diluted Artemia adult hydrolyzate and add 3 mL OPA reagent. Incubate in the dark for 2 minutes and measure the absorbance of the sample at 340 nm. The corresponding serine equivalent value C is obtained by the standard curve. Serine-NH2 (mmol·L -1 ), and calculate the degree of hydrolysis according to Formula I to Formula III.

[0049]

[0050] Where: W serine-NH2 (mmol·g -1 ): the amount of serine-NH2 per gram of protein;

[0051] X (g): sample mass; P (%): protein mass fraction in the sample;

[0052] V(L): volume of sample hydrolyzate; N: dilution multiple of hydrolyzate;

[0053] h(mmol·g -1 ): the number of peptide bonds broken per gram of sample protein during the hydrolysis process; h tot (mmol·g -1 ): total number of peptide bonds per gram of sample protein;

[0054] α, β: represented by constants 1.00 and 0.40 respectively.

[0055] (2) Selection of protease

[0056] Artemia adult worms were used as raw material. Four enzymes, alkaline protease, acidic protease, pepsin and papain, were selected and enzymolyzed for 2 h at the optimal enzymolysis temperature and pH conditions at an enzyme dosage of 100 U / g. After the enzymolysis, the enzymes were inactivated in a boiling water bath and the supernatant was collected by centrifugation. The hydrolysis degree of Artemia adult worms was used as an indicator to determine the optimal enzyme for enzymolysis.

[0057] (3) Single-factor experiment

[0058] 100 g of Artemia adult frozen blocks were weighed, the enzyme addition amount was 100 U / g dry matter, the enzymatic hydrolysis pH was 9, the enzymatic hydrolysis temperature was 50℃ and the enzymatic hydrolysis time was 2 h as the benchmark conditions, other factors and corresponding level conditions were fixed, and the protein hydrolysis degree was used as an indicator to explore the effects of enzyme addition amount (100 U / g, 600 U / g, 1100 U / g, 1600 U / g, 2100 U / g), enzymatic hydrolysis temperature (35℃, 40℃, 45℃, 50℃, 55℃, 60℃), enzymatic hydrolysis pH (6, 7, 8, 9, 10) and enzymatic hydrolysis time (0.5h, 1h, 2h, 3h, 4h, 5h, 6h) on the enzymatic hydrolysis effect of Artemia adults, so as to obtain the optimal single factor conditions.

[0059] (4) Steepest climbing test

[0060] Based on the results of the single-factor test, the steepest climbing test was designed. The enzyme addition, hydrolysis temperature and hydrolysis pH were increased according to a certain gradient, and the hydrolysis degree was measured to determine the optimal conditions for each single factor.

[0061] (5) Box-Behnken response surface optimization design

[0062] Based on the ramp test, three factors, enzyme addition amount (A), enzymatic hydrolysis temperature (B), and enzymatic hydrolysis pH, were selected as independent variables, and the hydrolysis degree was used as the response value. The response surface Box-Behnken Design experiment was designed using Design-Expert 13 software. The factor level table is shown in Table 1.

[0063] Table 1 Response surface analysis factors and levels

[0064]

[0065] 1.4 Data Processing

[0066] Each group of experiments was conducted in parallel with three groups. SPSS22.0 software was used for data processing and analysis, and Design-Expert13 was used for response surface design.

[0067] 2. Results and Analysis

[0068] 2.1 Effect of different proteases on hydrolysis degree

[0069] Effects of different proteases on the hydrolysis degree of Artemia hydrolysates Figure 1 At the same time, the degree of enzymatic hydrolysis of Artemia by the four proteases is alkaline protease > papain > acidic protease > pepsin, so alkaline protease is selected to enzymatically hydrolyze Artemia adults.

[0070] 2.2 Single-factor experimental results

[0071] 2.2.1 Effect of enzyme addition on the hydrolysis degree of Artemia adult polypeptides

[0072] Depend on Figure 2 As can be seen, as the amount of protease added increases, the degree of hydrolysis first increases and then stabilizes, reaching its maximum at an enzyme dosage of 1100 U / g. When the enzyme dosage is too high, the interaction between the substrate and the enzyme reaches a saturation level and the degree of hydrolysis no longer increases. However, when the enzyme dosage is too low, the Artemia adult raw material cannot be fully enzymatically hydrolyzed, resulting in a waste of resources. Therefore, 1100 U / g was selected as the protease dosage for subsequent experiments.

[0073] 2.2.2 Effect of enzymatic hydrolysis temperature on the degree of hydrolysis of Artemia adult polypeptides

[0074] As shown in Figure 3, the degree of hydrolysis reaches its maximum at 55°C. This is because a moderate increase in temperature increases the frequency of collisions between the enzyme and substrate, promoting the reaction. However, excessively high temperatures can inactivate the enzyme. Therefore, a hydrolysis temperature of 55°C is considered optimal.

[0075] 2.2.3 Effect of enzymatic hydrolysis pH on the degree of hydrolysis of Artemia adult polypeptides

[0076] Depend on Figure 4 As can be seen, the degree of hydrolysis first increases and then decreases with increasing pH. Each enzyme has an optimal pH range; excessively high or low pH values ​​directly affect enzyme activity. When the pH is greater than 9, the enzyme's biological activity decreases, and its enzymatic ability also decreases. Therefore, 9 was selected as the pH level for subsequent experiments.

[0077] 2.2.4 Effect of enzymatic hydrolysis time on the degree of hydrolysis of Artemia adult polypeptides

[0078] Depend on Figure 5 It can be seen that the degree of protein hydrolysis of Artemia adult worms increases with the extension of enzymatic hydrolysis time. Considering the time cost, 2 h was selected as the enzymatic hydrolysis time in all experiments.

[0079] 2.3 Enzymatic hydrolysis process optimization

[0080] 2.3.1 Hill climbing test

[0081] The results of the hill climbing test are shown in Table 2.

[0082] Table 2 Climbing test design and results

[0083]

[0084]

[0085] As shown in Table 2, when the enzyme addition amount was 1500 U / g, the enzymatic hydrolysis temperature was 55°C, and the enzymatic hydrolysis pH was 9, the degree of hydrolysis of the polypeptide reached the maximum, so it was selected as the center point of the response surface experiment.

[0086] 2.3.2 Response surface optimization test results

[0087] According to the design in Table 2, a three-factor, three-level response surface optimization experiment for Artemia protein hydrolysis was conducted on the three experimental variables, namely, enzyme addition amount (A), hydrolysis temperature (B), and hydrolysis pH (C). The degree of hydrolysis of Artemia adult protein was used as the evaluation index. The experimental results are shown in Table 3.

[0088] Table 3 Response surface experimental design and results

[0089] Test No. A amount of enzyme added / (U / g) B temperature / ℃ CpH value Degree of hydrolysis / % 1 500 45 9 53.70 2 2500 45 9 63.18 3 500 65 9 55.64 4 2500 65 9 63.70 5 500 55 8 57.20 6 2500 55 8 64.16 7 500 55 10 57.33 8 2500 55 10 65.08 9 1500 45 8 57.73 10 1500 65 8 53.67 11 1500 45 10 54.91 12 1500 65 10 55.89 13 1500 55 9 66.27 14 1500 55 9 67.19 15 1500 55 9 67.53 16 1500 55 9 66.91 17 1500 55 9 65.78

[0090] 2.3.3 Establishment of response surface regression model and variance analysis

[0091] The multivariate regression fitting of the protein hydrolysis degree of Artemia adult was performed using Design-Expert 13, and the quadratic polynomial regression model equation was obtained as Y=66.74+4.03A-0.0770B+0.0562C-0.3559AB+0.1959AC+1.26BC-1.15A 2 -6.54B 2 -4.65C 2 According to the quadratic polynomial regression model equation, the regression model equation obtained by the response surface experiment was subjected to variance analysis, and the significance test of each coefficient of the model was performed. The obtained variance analysis results of the response surface experiment are shown in Table 4.

[0092] Table 4 Analysis of variance of regression model

[0093]

[0094] Note: * indicates significant difference, P < 0.05; ** indicates extremely significant difference, P < 0.01.

[0095] As shown in Table 4, the model value is 47.15, P < 0.0001, indicating that the response surface fitting model is extremely significant and can well reflect the influence of the experimental factors on the response value. The lack of fit term P = 0.1326, which is not significant (P > 0.05), indicates that the regression model has a good degree of fit and the non-experimental factors have little influence on the test results. The first-order term A of the model is extremely significant, and the second-order term B is 2 、C 2Very significant, the interaction term BC is significant. Corrected correlation coefficient R 2 The corrected model determination coefficient R 2 adj =0.9629, indicating that the regression equation has a good fit and the model can explain 96.26% of the variation in the response values. This model can be used to analyze and predict the effects of various factors on the degree of hydrolysis and to determine the optimal experimental process for extracting peptides from Artemia adult artemia. The order of influence of each factor is: A > B > C, i.e., enzyme addition (A) > hydrolysis temperature (B) > hydrolysis pH (C). The factor with the greatest impact on the degree of hydrolysis of Artemia adult artemia hydrolysis products is enzyme addition. Using Design-Expert 13, the model was analyzed and predicted, and the optimal process for preparing peptides from Artemia adult artemia enzymatic hydrolysis was determined to be an enzyme addition of 2500 U / g, a hydrolysis temperature of 55.6°C, and a hydrolysis pH of 9.0, with a maximum response value of 70.15%.

[0096] 2.3.4 Response surface optimal condition verification test

[0097] Parallel experiments were conducted three times according to the predicted conditions, and the arithmetic mean of the measured results was taken. The protein hydrolysis degree of Artemia adult was obtained to be 68.28%, which was close to the predicted value of 70.15%, proving that the model parameters were reliable and of reference value.

[0098] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing Artemia polypeptide, characterized in that: The following steps are involved: Artemia slurry is enzymatically hydrolyzed under the action of protease to obtain Artemia polypeptide; The protease includes alkaline protease and / or papain.

2. The preparation method according to claim 1, characterized in that The added amount of the protease is 500-2500 U / g Artemia dry weight.

3. The preparation method according to claim 2, characterized in that: The added amount of the protease is 600-2100 U / g Artemia dry weight.

4. The preparation method according to claim 1, characterized in that The enzymatic hydrolysis temperature of the protease is 45-65°C.

5. The preparation method according to claim 1, characterized in that: The enzymatic hydrolysis temperature of the protease is 50-60°C.

6. The preparation method according to claim 1, characterized in that: The enzymatic hydrolysis temperature of the protease is 55-55.6°C.

7. The preparation method according to claim 1, characterized in that: The enzymatic hydrolysis pH value of the protease is 8-10.

8. The preparation method according to claim 1, characterized in that: The enzymatic hydrolysis pH value of the protease is 8.5-9.

5.

9. The preparation method according to claim 1, characterized in that: The enzymatic hydrolysis pH value of the protease is 9.

10. The preparation method according to any one of claims 1 to 9, characterized in that: The enzymatic hydrolysis time is 1.5 to 2.5 hours; After the enzymatic hydrolysis, the method further includes inactivating the enzymes in the obtained enzymatic hydrolysis products and separating the Artemia polypeptides.

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