Preparation method of litopenaeus vannamei shell antioxidant peptide based on response surface optimization
Through the optimization of the response surface enzymatic solution method, the problems of shrimp shell treatment and antioxidant peptide extraction were solved, and the efficient extraction of Vannabinoid shrimp shell antioxidant peptide was achieved, reducing environmental pollution and providing technical support.
Patent Information
- Application Number
- CN202411787315.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-06
- Publication Date
- 2025-04-08
AI Technical Summary
The treatment of shrimp shells and their by-products is difficult to effectively carry out, resulting in environmental pollution, and the prior art is difficult to efficiently extract antioxidant peptides in shrimp shells.
The enzymatic solution was adopted based on response surface optimization, and the enzymatic solution conditions were optimized to efficiently extract the antioxidant peptide of vannabinoid shrimp shell through single-factor screening and five-factor three-level response surface experiments.
It has achieved efficient digestion of shrimp shells, fully released and prepared antioxidant peptides, improved extraction rate and antioxidant activity, reduced environmental pollution, and provided important technical support for the development and utilization of shrimp shells.
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Figure CN119785932A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biochemistry and relates to a preparation method of Litopenaeus vannamei shell antioxidant peptide based on response surface optimization. Background Art
[0002] China is one of the important countries in shrimp farming in the world. In 2022, the total production of marine shrimp in China reached about 1.66 million tons, of which Litopenaeus vannamei contributed about 1.34 million tons, accounting for about 81% of the total. Litopenaeus vannamei is an important aquatic species not only in my country but also in the international aquaculture industry. It not only has important socioeconomic value, but also plays an indispensable role in ensuring global food supply. However, while enjoying the economic benefits brought by shrimp, it also faces an environmental challenge: the disposal of shrimp shells and their by-products such as shrimp heads. These are usually regarded as food processing waste and are often classified as kitchen waste or swill and discarded. Because shrimp shells are strong and rich in calcium, they are difficult to effectively treat by conventional means, and the natural degradation process is slow. In addition, as the shrimp shells decay, a variety of harmful gases and substances such as ammonia and sulfur compounds are released, which leads to more serious environmental pollution problems.
[0003] Shrimp shells are rich in protein, which can be converted into short peptides and amino acids by hydrolyzing them with proteases. Modern scientific research has revealed that the human body does not completely absorb the ingested protein in the form of individual amino acids during digestion. In fact, more of it is absorbed by the body in the form of small molecule peptides, which have multiple physiological functions and are called "bioactive peptides." Studies have shown that certain peptides in shrimp shells have good antioxidant properties, protect cell membranes and DNA from oxidative damage, and have the effect of preventing cardiovascular disease, cancer, neurodegenerative diseases and other chronic diseases. Therefore, the research on extracting such small molecule active peptides from shrimp shells not only helps to improve resource utilization and reduce environmental pollution, but also has important significance for the development of new functional foods and health products.
[0004] Enzymatic extraction of protein is an emerging technology. Enzymatic hydrolysis breaks high molecular weight proteins into low molecular weight polypeptides and releases them from the raw materials into the solution, thereby achieving the purpose of extracting protein. Enzymatic preparation of bioactive peptides has the advantages of mild reaction conditions, no pollution, maximum preservation of flavor, high product activity, and minimal impact on the nutritional value of protein, and has been widely used in current research and production. Therefore, the present invention finally established an efficient enzymatic process for extracting and preparing antioxidant peptides from Penaeus vannamei shells through single factor screening and combined response surface optimization reaction. The method uses a green, efficient and safe enzymatic method to digest Penaeus vannamei shells, which is conducive to obtaining low molecular weight, easily absorbable, and strong antioxidant activity Penaeus vannamei shell peptides, and provides important technical support for subsequent research and development and application of the antioxidant activity of Penaeus vannamei shells. Summary of the invention
[0005] The purpose of the present invention is to utilize abundant shrimp shell waste resources and invent an efficient preparation method of Penaeus vannamei shell antioxidant peptides based on response surface optimization, which can efficiently digest Penaeus vannamei shells to achieve the purpose of fully releasing and efficiently preparing Penaeus vannamei shell antioxidant peptides.
[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present invention is as follows: an efficient preparation method of antioxidant peptides from Penaeus vannamei shells based on response surface optimization, comprising the following steps: screening enzymes for enzymatic hydrolysis, enzymatic hydrolysis pH value, enzymatic hydrolysis temperature, enzyme addition amount, material-liquid ratio and enzymatic hydrolysis time by using a single factor experiment, taking the optimal point of the single factor experiment as the center, taking a level value above and below the optimal point as the level of the response surface, designing a five-factor three-level experiment by using the Box-Behnken response surface design method, taking the extraction rate of antioxidant peptides from Penaeus vannamei shells as the response value, and determining the extraction conditions of antioxidant peptides from Penaeus vannamei shells in combination with a three-dimensional response surface diagram.
[0007] Preferably, the protein hydrolysis degree is analyzed and fitted to obtain a regression equation of the protein hydrolysis degree, and the extraction conditions of the antioxidant peptides from the shell of Litopenaeus vannamei are determined with the maximum protein hydrolysis degree as the goal.
[0008] Preferably, the enzymolysis enzyme is alkaline protease.
[0009] Preferably, the enzymatic hydrolysis pH is 8.0-10.0.
[0010] Preferably, the enzymatic hydrolysis temperature is 50-60°C.
[0011] Preferably, the amount of enzyme added is 2500-3500 U / g.
[0012] Preferably, the solid-liquid ratio is 1:8-1:10.
[0013] Preferably, the enzymatic hydrolysis time is 4-6 h.
[0014] Compared with the prior art, the present invention has the following advantages: 1. Based on the single factor experiment, the present invention combines the five-factor three-level response surface experiment to establish the optimal enzymatic hydrolysis preparation process of antioxidant peptides from Penaeus vannamei shells, providing important technical support for the development and utilization of Penaeus vannamei shells; 2. The present invention optimizes and screens out alkaline protease to digest the shell of Penaeus vannamei. The reaction process is environmentally friendly and provides technical support for the functional research and development and application of antioxidant peptides from Penaeus vannamei shells. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 This is a flow chart of a method for preparing antioxidant peptides from Litopenaeus vannamei shell based on response surface optimization in an embodiment of the present invention; Figure 2 This is a graph showing the effects of different proteases on the extraction rate of antioxidant peptides from Litopenaeus vannamei shells in Example 1 of the present invention; Figure 3 This is a graph showing the influence of different factors on the extraction rate of shrimp shell antioxidant peptides prepared by alkaline protease in Example 2 of the present invention; wherein A. pH value; B. temperature; C. enzyme addition amount; D. material-liquid ratio; E. enzymolysis time; Figure 4 The response surface diagram of the interaction of various factors on the extraction rate of antioxidant peptides from Litopenaeus vannamei shells in Example 3 of the present invention; Figure 5 The analysis of the DPPH free radical scavenging rate of the antioxidant peptide from the shell of Litopenaeus vannamei prepared according to the optimal extraction conditions in Example 4 of the present invention; Figure 6 The reducing power analysis of the antioxidant peptides from the shell of Penaeus vannamei prepared according to the optimal extraction conditions in Example 4 of the present invention; Figure 7 This is an analysis of the hydroxyl radical scavenging rate of the antioxidant peptides from the shell of Penaeus vannamei prepared according to the optimal extraction conditions in Example 4 of the present invention. DETAILED DESCRIPTION
[0016] In order to facilitate the understanding of the present invention, the present invention is described in more detail below in conjunction with the accompanying drawings and specific embodiments. Preferred embodiments of the present invention are provided in the accompanying drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.
[0017] Example 1 This example provides a method for screening enzymes for enzymatic hydrolysis of Litopenaeus vannamei shells, which is as follows:
[0018] Fresh shrimp shells were dried and crushed, and 1.00 g of shrimp shell powder was taken and dispersed in different pH buffers at a material-liquid ratio of 1: 10 (g: mL). Different proteases were added at 2000 U / g (enzyme activity / sample amount), and the reaction was carried out for 2 h according to the optimal temperature and pH of each protease (Table 1). After the reaction, the enzyme was inactivated by heating in a boiling water bath for 10 minutes, and the mixture was centrifuged at 4°C and 10,000 rpm for 10 minutes in a centrifuge. The supernatant was collected and the degree of hydrolysis of shrimp shell protein was measured. The degree of hydrolysis was used as an indicator to screen out the protease with the highest efficiency in enzymatic hydrolysis of shrimp shells. The above experiments were conducted in parallel three times.
[0019] Table 1 Optimum temperature and pH value of different proteases .
[0020] according to Figure 2 The results showed that at the optimal temperature and pH value of different proteases, alkaline protease had the highest efficiency of hydrolyzing Litopenaeus vannamei shells, which was 11.08%.
[0021] Example 2 This example provides a single-factor experimental design for preparing Penaeus vannamei shells. The alkaline protease with the highest enzymatic hydrolysis efficiency in Example 1 is selected to study the effects of five factors, namely, different extraction temperatures (37, 45, 50, 55, 60 ° C), extraction times (2, 3, 4,5, 6, 7 h), extraction pH (7.5, 8.0, 8.5, 9.0, 9.5, 10.0), enzyme addition amounts (1000, 1500, 2000, 2500, 3000 and 3500 U / g), and solid-liquid ratios (1:8, 1:9, 1:10, 1:11, 1:12, 1:13 g / mL), on the extraction capacity of antioxidant peptides from Penaeus vannamei shells.
[0022] according to Figure 3 The results shown in A show that the optimal pH for the enzymatic reaction is 9.0; according to Figure 3 The results shown in B show that the optimum temperature for the enzymatic hydrolysis reaction is 55°C; according to Figure 3 The results shown in C in the middle show that as the amount of enzyme added increases, the hydrolysis degree of protein in the hydrolyzate gradually increases, and the growth rate slows down around 3000 U / g; according to Figure 3 The results shown in D in the figure show that the optimal solid-liquid ratio for the enzymatic hydrolysis reaction is 1:9; according to Figure 3 The results shown in E show that as the enzymatic hydrolysis time increases, the degree of hydrolysis of the protein in the hydrolysate gradually increases, and the growth rate slows down around 5 hours.
[0023] Example 3 This example provides a response surface optimization scheme for enzymatic hydrolysis of Litopenaeus vannamei shells, as follows: The optimal conditions for enzymatic hydrolysis of Litopenaeus vannamei shells were determined by single-factor experiments as a benchmark, and three levels were selected for the five factors of enzymatic pH, enzymatic temperature, enzyme addition, feed-liquid ratio, and enzymatic time. A five-factor three-level response surface experimental table was designed and coded (Table 2) to optimize the extraction conditions. A variance analysis was performed on the experimental data to estimate the significance of the second-order polynomial equation (Table 3).
[0024] Table 2 Factor levels of response surface experiment .
[0025] Table 3 Analysis of variance of second-order polynomial model fitting .
[0026] The protein hydrolysis degree was input into Design Expert 13 software for analysis and fitting, and the regression equation of protein hydrolysis degree was obtained as follows: Y = 10.79 +1.46 A +0.3556 B - 0.0084 C + 0.225 D + 0.6116 E +0.9960 AB +0.3724 AC -0.75 AD + 0.2624 AE -1.13 BC - 0.22 BD - 0.405 BE - 0.5425 CD + 1.08 CE -0.415 DE -1.79 A 2 - 0.5177 B 2 - 3.24 C 2 -1.75 D 2 - 0.8344 E 2 ; Among them, A represents pH; B represents time; C represents temperature; D represents the material-liquid ratio; and E represents the amount of enzyme added.
[0027] The quadratic regression model was significant (P < 0.05), confirming that the model was reliable for determining the degree of protein hydrolysis. In addition, the lack-of-fit term was not significant (P > 0.05), further supporting the validity of the model.2 The adjusted coefficient of determination was 0.8774, indicating that the experimental value was in good agreement with the predicted value. The coefficient of variation (CV) was less than 10%, indicating that the results were accurate and reproducible. These findings validated the adequacy, accuracy, and reliability of the model for optimizing the extraction conditions to increase the content of peptides in shrimp shells.
[0028] With the goal of maximizing the degree of protein hydrolysis, the optimal enzymatic hydrolysis conditions of the response surface methodology were a temperature of 54.69 °C, a hydrolysis time of 5.88 h, and a solid-liquid ratio of 1:8.85 g mL -1 , pH 9.71, enzyme dosage 3112.29 U / g, correspondingly, the predicted protein hydrolysis degree is 11.58%, and the ideal degree is 0.8774. Experimental verification under these conditions showed that the protein hydrolysis degree was 11.56%, which was close to the predicted result. The consistency between the predicted and experimental results confirmed the reliability of the model. The response surface of the interaction of various factors on the extraction rate of antioxidant peptides from shrimp shells is shown in Figure 2. Figure 4 shown.
[0029] Example 4 This example provides the determination of the antioxidant capacity of antioxidant peptides from the shell of Penaeus vannamei, wherein the DPPH free radical scavenging ability is implemented with reference to GB / T 39100-2020, the reducing power is determined by the potassium ferrocyanide reduction method, and the hydroxyl radical scavenging rate is determined by using saffron red T as a color developer and implemented by the Fenton colorimetric method.
[0030] according to Figure 5-7 It can be seen that the antioxidant capacity of antioxidant peptides from Litopenaeus vannamei shell increased in a dose-dependent manner. When the protein concentration of the enzymatic hydrolysate was 0.359 mg / mL, the hydroxyl radical scavenging rate was 34.21%, the reducing power was 0.298, and the hydroxyl radical scavenging rate was 34.21%.
Claims
1. A method for preparing antioxidant peptides from Litopenaeus vannamei shell based on response surface optimization, characterized in that: A single factor experiment was used to screen the enzyme for enzymatic hydrolysis, enzymatic hydrolysis pH value, enzymatic hydrolysis temperature, enzyme addition amount, solid-liquid ratio and enzymatic hydrolysis time. Then, with the optimal point of the single factor experiment as the center, a level value above and below the optimal point was taken as the level of the response surface. The Box-Behnken response surface design method was used to design a five-factor three-level experiment. The extraction rate of antioxidant peptides from vannamei shell was used as the response value, and the extraction conditions of antioxidant peptides from vannamei shell were determined in combination with the three-dimensional response surface diagram.
2. The method for preparing antioxidant peptides from Litopenaeus vannamei shell based on response surface optimization according to claim 1, characterized in that: The protein hydrolysis degree was analyzed and fitted to obtain the regression equation of the protein hydrolysis degree. The extraction conditions of antioxidant peptides from the shell of Litopenaeus vannamei were determined with the maximum protein hydrolysis degree as the goal.
3. The method for preparing antioxidant peptides from Litopenaeus vannamei shell based on response surface optimization according to claim 1 or 2, characterized in that: The enzyme for enzymolysis is alkaline protease.
4. The method for preparing antioxidant peptides from Litopenaeus vannamei shell based on response surface optimization according to claim 1 or 2, characterized in that: The enzymatic hydrolysis pH value is 8.0-10.
0.
5. The method for preparing antioxidant peptides from Litopenaeus vannamei shell based on response surface optimization according to claim 1 or 2, characterized in that: The enzymolysis temperature is 50-60°C.
6. The method for preparing antioxidant peptides from Litopenaeus vannamei shell based on response surface optimization according to claim 1 or 2, characterized in that: The enzyme addition amount is 2500-3500 U / g.
7. The method for preparing antioxidant peptides from Litopenaeus vannamei shell based on response surface optimization according to claim 1 or 2, characterized in that: The solid-liquid ratio is 1:8-1:
10.
8. The method for preparing antioxidant peptides from Litopenaeus vannamei shell based on response surface optimization according to claim 1 or 2, characterized in that: The enzymatic hydrolysis time is 4-6 h.