CN110710592A - A kind of antioxidative activity method of improving walnut cake meal protein - Google Patents

Abstract

The invention discloses a method for improving the antioxidant activity of walnut cake protein, comprising the following steps: S1: testing the effect of enzyme types on the protein yield of walnut cake; S2: testing the effect of enzyme addition on the protein yield of walnut cake Influence; S3: Test the effect of solid-liquid ratio on the protein yield of walnut cake; S4: Test the effect of particle size on the protein yield of walnut cake; S5: Test the effect of extraction time on the protein yield of walnut cake; S6: Determination of the protein content of the extract; S7: Enzyme-assisted extraction of walnut cake protein response surface experiment; S8: The effect of enzyme types on the yield of walnut cake protein; Solve the problem that the existing methods cannot be applied in the field of food and medicine, and in the experimental process There are problems that the single factor cost of temperature control is high, the safety factor is low, the antioxidant activity of the obtained meal protein is extremely low, and the buffer solution cannot obtain a high protein yield.

Description

A kind of antioxidative activity method of improving walnut cake meal protein

technical field

The invention relates to the field of antioxidant activity methods of walnut cake protein, in particular to a method for improving the antioxidant activity of walnut cake protein.

Background technique

Walnut cake is derived from walnut kernels. After walnut kernels are pressed to extract oil, the remaining part is called walnut cakes. The products obtained by extracting oils and fats by different methods have different names. The products obtained by the physical pressing method are called "cakes"; the products obtained by the leaching method are called "meal". The recycling and utilization of contract cakes in my country is still in the preliminary stage, mainly focusing on the composition analysis of walnut cakes, protein extraction, walnut cakes feed and fertilizers. Studies have shown that walnut meal contains about 50% protein.

In recent years, walnut meal protein has been widely used in medical research as a raw material for the production of walnut polypeptides because of its various physiological functions such as antioxidant, anti-cardiovascular disease, anti-obesity, and anti-diabetes. Studies have found that proteins are enzymatically hydrolyzed into active peptides by the digestive tract, which can be absorbed and utilized faster. At present, the bioactive peptides used as food on the market are mainly soy products, and walnuts, which are high protein sources, are rarely used in actual production.

At present, studies have found that the water product of walnut defatted powder with a protein content of 46.14% is enzymatically hydrolyzed by trypsin, and its water product has high antioxidant function, which can remove reactive oxygen species in mouse tumor cells. The walnut polypeptide nutrient solution was prepared by anion and cation mixed bed desalination method and biofilm filtration, which has high antioxidant activity.

Up to now, the research and preparation of the biological activity of walnut cake is not perfect, the system is also in the primary exploration stage, and there are few studies on the molecular structure of the active agent in the physiological period.

SUMMARY OF THE INVENTION

In order to solve the problems existing in the prior art, the present invention provides a method for improving the antioxidant activity of walnut cake protein, which solves the problem that the existing method cannot be applied to the fields of food and medicine, and there is temperature control in the experiment process. The cost is high, the safety factor is low, and the obtained cake protein has very low antioxidant activity, and the buffer cannot obtain a high protein yield.

The technical scheme adopted in the present invention is a method for improving the antioxidant activity of walnut cake protein, comprising the following steps:

S1: Test the effect of enzyme types on the protein yield of walnut cake;

S2: Test the effect of enzyme addition on the protein yield of walnut cake;

S3: Test the effect of solid-liquid ratio on the protein yield of walnut cake;

S4: Test the effect of particle size on the protein yield of walnut cake;

S5: Test the effect of extraction time on the protein yield of walnut cake;

S6: determine the protein content of the extract;

S7: Enzyme-assisted extraction of protein response surface experiment from walnut cake;

S8: Effect of enzyme types on protein yield of walnut cake;

S9: analyze the results of the single factor experiment;

S10: Obtain the response surface analysis result;

S11: Optimize the response surface;

S12: Do the protein antioxidant experiment of walnut cake;

S13: Obtain the test result.

Preferably, S1 includes the following steps:

S1-1: Weigh about 1g of walnut cake powder with a particle size of 60 mesh and add it to a 50ml glass conical flask. According to the ratio of feed liquid 1:30, according to the optimum pH value of different types of enzymes, add buffers of different pH , the extraction time is set to 4h, and at room temperature, it is placed on a magnetic stirrer and stirred at a speed of 500r/min for 4h to make it fully dissolved;

S1-2: After reaching the specified time, transfer it into a 1.5ml centrifuge tube and put it into a centrifuge for centrifugation. Each sample was repeated three times, and the temperature was kept at 4 °C and the rotation speed was 12000 rpm during centrifugation. After 5 minutes, sampling was performed, and 0.2 ml of supernatant was extracted for the next experiment.

Preferably, S2 includes the following steps:

S2-1: Weigh about 1g of walnut cake powder with a particle size of 60 mesh and add it to a 50ml glass conical flask. According to the ratio of material to liquid of 1:30, add 30ml of buffer solution with a pH value of 7.5 respectively. Put it into a water bath for constant temperature heating at 40°C, then add 1%, 2%, 3%, 4% and 5% of α-amylase respectively, and set the extraction time to 1, 2, 4, 6 and 8 h. At room temperature, stir on a magnetic stirrer at 500r/min for 4h to fully dissolve;

S2-2: After reaching the specified time, transfer it into a 1.5ml centrifuge tube and put it into a centrifuge for centrifugation. Each sample was repeated three times, and the temperature was kept at 4 °C and the rotation speed was 12000 rpm during centrifugation. After 5 minutes, sampling was performed, and 0.2 ml of supernatant was extracted for the next experiment.

Preferably, S3 includes the following steps:

S3-1: Weigh about 1g of walnut cake powder with a particle size of 60 mesh and add it to a 50ml glass conical flask. According to the corresponding material-to-liquid ratio, add 30ml of buffer solution with a pH value of 7.5, and put the conical flask into the conical flask. Heating at a constant temperature of 40 °C in a water bath, then adding 3% α-amylase, the extraction time was set to 1, 2, 4, 6 and 8 h, at room temperature, placed on a magnetic stirrer at a speed of 500 r/min. Stir for 4h to fully dissolve;

S3-2: After reaching the specified time, transfer it into a 1.5ml centrifuge tube and put it into a centrifuge for centrifugation. Each sample was repeated three times, and the temperature was kept at 4 °C and the rotation speed was 12000 rpm during centrifugation. After 5 minutes, sampling was performed, and 0.2 ml of supernatant was extracted for the next experiment.

Preferably, S5 includes the following steps:

S5-1: Weigh about 1g of walnut cake powder with a particle size of 60 mesh and add it to a 50ml glass conical flask, add 30ml of buffer solution with a pH value of 7.5 according to the material-to-liquid ratio of 1:30, and put the conical flask into the conical flask. Heating at a constant temperature of 40 °C in a water bath, then adding 3% α-amylase, the extraction time was set to 1, 2, 4, 6 and 8 h, at room temperature, placed on a magnetic stirrer at a speed of 500 r/min. Stir to fully dissolve;

S5-2: After reaching the specified time, transfer it into a 1.5ml centrifuge tube and put it into a centrifuge for centrifugation. Each sample was repeated three times, and the temperature was kept at 4 °C and the rotation speed was 12000 rpm during centrifugation. After 5 minutes, sampling was performed, and 0.2 ml of supernatant was extracted for the next experiment.

Preferably, the protein yield calculation formula of S6 is:

Preferably, the enzyme species includes alkaline proteases, cellulases, alpha-amylases, pectinases and beta-amylases.

Preferably, S12 includes the following steps:

S12-1: Determination of DPPH free radical scavenging ability;

S12-2: Determination of reducing power;

S12-3: Process membrane filtration.

The beneficial effects of the method for improving the antioxidant activity of the walnut meal protein of the present invention are as follows:

1. The phosphate buffer of the present invention, Tris-HCl buffer and NaHCO buffer solution to extract walnut cake protein, the experimental results show that the antioxidant activity of buffer extraction is significantly improved, and the protein yield of walnut cake can be effectively improved by adding enzymes , in this way, while obtaining soluble active protein, the yield of walnut cake protein is also guaranteed. At the same time, the obtained walnut cake protein solution is filtered by using an acetate cellulose membrane to obtain a walnut protein solution with a higher concentration of small molecular protein.

2. The present invention uses cellulose acetate membrane to obtain walnut cake protein with molecular weight below 10kDa by sweep flow filtration and suction filtration. The experiment used the method of extracting protein with alkali and buffer to obtain the protein of walnut cake with larger molecular weight. The same method was used to analyze the difference between the results of single factor and orthogonal test, and the optimal extraction process before and after was compared.

Description of drawings

Fig. 1 is the influence figure of the enzyme species of the antioxidative activity method of improving walnut meal protein of the present invention on walnut meal protein yield

Fig. 2 is the single-factor experiment result of the enzyme-assisted extraction of walnut cake protein of the method for improving the antioxidant activity of walnut cake protein according to the present invention

Fig. 3 is the fitting degree diagram of the model prediction value and the actual experimental value of the method for improving the antioxidant activity of walnut cake protein according to the present invention

Fig. 4 is the response surface diagram and contour diagram of the interaction of the enzyme addition amount (X ₁ ) and the solid-liquid ratio (X ₃ ) of the method for improving the antioxidant activity of walnut meal protein according to the present invention

Fig. 5 is the response surface graph and the contour graph of the interaction of the enzyme addition amount (X ₁ ) particle size (X ₃ ) in the method for improving the antioxidant activity of walnut meal protein of the present invention

Fig. 6 is the response surface diagram and contour diagram of the interaction of enzyme addition amount (X ₁ ) time (X ₃ ) in the method for improving the antioxidant activity of walnut meal protein of the present invention

Fig. 7 is the response surface diagram and contour diagram of the interaction of solid-liquid ratio (X ₁ ) particle size (X ₃ ) in the method of improving the antioxidant activity of walnut cake protein according to the present invention

Fig. 8 is the response surface diagram and the contour diagram of the interaction of solid-liquid ratio (X ₁ ) particle size (X ₃ ) in the method for improving the antioxidant activity of walnut cake protein according to the present invention

Fig. 9 is the response surface diagram and contour diagram of the interaction between the enzyme addition amount (X ₁ ) and the particle size (X ₃ ) of the method for improving the antioxidant activity of walnut cake protein according to the present invention

Figure 10 is the prediction profiler of the response surface of the walnut cake protein yield response surface of the method for improving the antioxidant activity of walnut cake protein according to the present invention

Fig. 11 is the antioxidative ability of walnut cake protein under membrane filtration conditions of the method for improving the antioxidant activity of walnut cake protein according to the present invention

Figure 12 is the antioxidative ability of walnut cake protein under membrane filtration conditions of the method for improving the antioxidant activity of walnut cake protein according to the present invention

Fig. 13 is the general flow chart of the method for improving the antioxidant activity of walnut meal protein according to the present invention

Detailed ways

The specific embodiments of the present invention are described below to facilitate those skilled in the art to understand the present invention, but it should be clear that the present invention is not limited to the scope of the specific embodiments. For those of ordinary skill in the art, as long as various changes Such changes are obvious within the spirit and scope of the present invention as defined and determined by the appended claims, and all inventions and creations utilizing the inventive concept are within the scope of protection.

As shown in Figure 13, a method for improving the antioxidant activity of walnut meal protein, comprising the following steps:

S1: Test the effect of enzyme types on the protein yield of walnut cake;

S2: Test the effect of enzyme addition on the protein yield of walnut cake;

S3: Test the effect of solid-liquid ratio on the protein yield of walnut cake;

S4: Test the effect of particle size on the protein yield of walnut cake;

S5: Test the effect of extraction time on the protein yield of walnut cake;

S6: determine the protein content of the extract;

S7: Enzyme-assisted extraction of protein response surface experiment from walnut cake;

S8: Effect of enzyme types on protein yield of walnut cake;

S9: analyze the results of the single factor experiment;

S10: Obtain the response surface analysis result;

S11: Optimize the response surface;

S12: Do the protein antioxidant experiment of walnut cake;

S13: Obtain the test result.

Enzyme assistance has the characteristics of efficient extraction technology, strong pertinence, and easy to meet experimental conditions, and there are many related researches on enzyme assistance. Wang Peng et al. used blueberries as raw materials to extract blueberry polysaccharides by alkaline leaching and cellulase-assisted methods. The conditions of the alkaline leaching method were the extraction temperature, the amount of alkali added, the number of times of extraction and the extraction time, and the conditions of the cellulase-assisted method were pH, the amount of cellulase added, the extraction time and the extraction temperature, respectively. The yield of polysaccharide obtained by cellulase-assisted method was 26% higher than that obtained by alkaline leaching method. This is because when the biological enzyme is combined with the substrate, the change in the shape of the enzyme molecule will cause the change of the chemical bond of the substrate, which will eventually lead to the breakage of its chemical bond. , dissolved into the extract, so as to achieve full release and efficient extraction of biologically active compounds.

Based on the extraction of walnut cake protein with buffer solution, in order to ensure high protein yield and maintain the activity of walnut cake protein, various enzymes were used to assist in the extraction of walnut cake protein during the experiment. Common enzymes include alkaline protease, neutral protease, acid protease, pectinase, alpha-amylase, beta-amylase, and cellulase. In this experiment, the buffer solution was used to select the enzyme with higher yield to increase the yield of walnut meal protein.

(1) Influence of enzyme types on protein yield of walnut cake

Weigh about 1 g of walnut cake powder with a particle size of 60 mesh and add it to a 50 ml glass conical flask. According to the ratio of feed liquid 1:30, according to the optimal pH value of different types of enzymes, add buffers of different pH, and the extraction time is set. Set as 4h, at room temperature, stir on a magnetic stirrer at 500r/min for 4h to make it fully dissolved. After reaching the predetermined time, it was transferred to a 1.5 ml centrifuge tube, and centrifuged in a centrifuge. Each sample was repeated three times, and the temperature was kept at 4°C and the rotation speed was 12,000 rpm during centrifugation. After 5 minutes, sampling was performed, and 0.2 ml of supernatant was extracted for the next experiment.

(2) Effect of enzyme addition on protein yield of walnut cake

Weigh about 1g of walnut cake powder with a particle size of 60 mesh and add it to a 50ml glass conical flask. According to the ratio of material to liquid of 1:30, add 30ml of buffer solution with pH value of 7.5 respectively, and put the conical flask into a water bath. Heating at a constant temperature of 40 °C in the medium, then adding 1%, 2%, 3%, 4% and 5% of α-amylase respectively, the extraction time was set to 1, 2, 4, 6 and 8 h, at room temperature, let Stir at 500 r/min on a magnetic stirrer for 4 h to make it fully dissolved. After reaching the predetermined time, it was transferred to a 1.5 ml centrifuge tube, and centrifuged in a centrifuge. Each sample was repeated three times, and the temperature was kept at 4 °C and the rotation speed was 12000 rpm during centrifugation. After 5 minutes, sampling was performed, and 0.2 ml of supernatant was extracted for the next experiment.

(3) Effect of solid-liquid ratio on protein yield of walnut cake

Weigh about 1g of walnut cake powder with a particle size of 60 mesh and add it to a 50ml glass conical flask. According to the corresponding material-to-liquid ratio, add 30ml of buffer solution with a pH value of 7.5 respectively, and put the conical flask into a water bath. Heating at a constant temperature of 40 °C, then adding 3% α-amylase respectively, setting the extraction time to 1, 2, 4, 6 and 8 h, and stirring at room temperature on a magnetic stirrer at 500 r/min for 4 h. make it fully dissolved. After reaching the predetermined time, it was transferred to a 1.5 ml centrifuge tube, and centrifuged in a centrifuge. Each sample was repeated three times, and the temperature was kept at 4 °C and the rotation speed was 12000 rpm during centrifugation. After 5 minutes, sampling was performed, and 0.2 ml of supernatant was extracted for the next experiment.

(4) Effect of particle size on protein yield of walnut cake

Weigh about 1g of walnut cake powder with particle sizes of 20, 40, 60, 80 and 100 meshes and add it to a 50ml glass conical flask. According to the ratio of material to liquid of 1:30, add 30ml of buffer solution with pH value of 7.5 respectively. , put the conical flask into a water bath for constant temperature heating at 40°C, then add 3% α-amylase respectively, and set the extraction time to 1, 2, 4, 6 and 8 h, and place them under magnetic stirring at room temperature. Stir at 500r/min for 4h on the device to make it fully dissolved. After reaching the predetermined time, it was transferred to a 1.5 ml centrifuge tube, and centrifuged in a centrifuge. Each sample was repeated three times, and the temperature was kept at 4 °C and the rotation speed was 12000 rpm during centrifugation. After 5 minutes, sampling was performed, and 0.2 ml of supernatant was extracted for the next experiment.

(5) Influence of extraction time on protein yield of walnut cake

Weigh about 1g of walnut cake powder with a particle size of 60 mesh and add it to a 50ml glass conical flask, add 30ml of buffer solution with a pH value of 7.5 according to the material-to-liquid ratio of 1:30, and put the conical flask into a water bath. Heating at a constant temperature of 40 °C, then adding 3% α-amylase respectively, setting the extraction time to 1, 2, 4, 6 and 8 h, and stirring at room temperature on a magnetic stirrer at a speed of 500 r/min, make it fully dissolved. After reaching the predetermined time, it was transferred to a 1.5 ml centrifuge tube, and centrifuged in a centrifuge. Each sample was repeated three times, and the temperature was kept at 4 °C and the rotation speed was 12000 rpm during centrifugation. After 5 minutes, sampling was performed, and 0.2 ml of supernatant was extracted for the next experiment.

(6) Determination of protein content

The protein concentration of the extract was determined by Coomassie brilliant blue G-250 colorimetric method. The formula for calculating protein yield:

Determination of soluble protein content by Coomassie brilliant blue method: add 0.05 ml of extract (set up 3 repeating tubes) and 2.5 ml of Coomassie brilliant blue reagent to the test tube, shake and let stand for 5 minutes, use a spectrophotometer for colorimetry, and set the wavelength to 595nm, record the absorbance value. A standard curve was drawn using bovine serum albumin of known concentration, and the protein concentration was obtained from the standard curve.

(7) Response surface surface experiment of enzyme-assisted extraction of protein from walnut cake

According to the single factor experimental results of enzyme-assisted extraction, the extraction time, solid-liquid ratio, enzyme addition amount and cake particle size were selected as variables, and the protein yield of walnut cake was selected as the index. The Box-Behnken design response surface test of Design-Expert was used. The coding of each factor level is shown in the table below.

Table 4-1 Center combination design factors and level coding table

The protein concentration was determined by the Coomassie brilliant blue method.

(8) Results and Analysis

Effects of Enzyme Types on Protein Yield of Walnut Meal

In order to determine which enzyme to use has a better effect on the yield of walnut protein, the results of the walnut protein extraction experiment using the above-mentioned buffer were selected as alkaline protease, cellulase, α-amylase, pectinase and β-starch. Enzymes were added to the buffer solution, stirring and extraction were carried out under the same parameters. The effect of different enzymes on the protein yield of walnut meal is shown in Figure 1. As can be seen from Figure 1, the yields of alkaline protease, cellulase, pectinase and β-amylase were 13.67%, 8.82%, 7.85% and 5.25%, respectively, indicating that α-amylase had a significant effect on the yield of walnut protein. The effect is significantly higher than that of other enzymes. Using α-amylase extraction, the protein yield of walnut cake reached 17.23%, so α-amylase was selected.

One-factor experiment results

The effect of enzyme addition on the protein yield of walnut cake is shown in Figure 2. As can be seen from Figure 2, Figure 2(a) is the effect of enzyme addition on the protein yield of walnut cake, Figure 2(b) is the effect of solid-liquid ratio on the protein yield of walnut cake, Figure 2(c) For the effect of extraction time on the protein yield of walnut cake, Figure 2(d) shows the effect of particle size on the protein yield of walnut cake. The protein yield of walnut is significantly higher than that of other additions; in the solid-liquid ratio experiment, with the increase of the solid-liquid ratio, the protein yield also increases; similar to the alkali extraction of walnut cake protein, enzyme-assisted extraction is used. The particle size of walnut cake increased to the highest at 80 mesh, and decreased slightly at 100 mesh; when the extraction time reached 1h, the protein yield was the highest, and the yield gradually decreased when the time was prolonged. According to the above experimental results and production practice, it is preliminarily determined that in the enzyme-assisted extraction of walnut protein, the extraction process adopted is: enzyme addition 2-4%, 1:30-1:50, walnut cake particle size 60-100 Mesh, extraction time 0.5-1.5h.

Response Surface Analysis Results

Single factor test showed that the amount of enzyme added, extraction time, solid-liquid ratio and particle size were the main factors affecting the content of soluble protein in the process of protein extraction from walnut cake. Therefore, according to the combined rotation center design method (CCRD for short) in JMP, a four-factor and three-level optimization experiment was carried out on the extraction parameters of walnut cake. The four extraction parameters included buffer pH value, extraction time, solid-liquid ratio and particle size. . The experimental yield and predicted value of walnut meal protein at the design point are shown in Figure 2 and Table 4-2. The study showed that different extraction parameters caused significant differences in protein yields of walnut cakes, ranging from 8.85% to 19.56%. Use CCRD to construct a polynomial regression equation, analyze the interaction between factors and analyze the significant influencing factors in the regression equation. The ANOVA analysis results are shown in Tables 4-3 and 4-4. The calculated probability value (P-value) from the Y-model analysis was less than 0.0001, indicating high significance for the regression model. The value of each item was obtained based on the calculation program of the statistical software JMP, as shown in Table 4-4. The extraction parameter was significant for the Y value when the P value was less than 0.05. Based on the above discussion, we can understand that analyzing variance can well describe the correlation between protein yield (Y) and extraction parameters (X1, X2, X3, X4). In addition, a decision system (R2) will be incorporated to check the agreement between the experimental results and the numerical model. In the present results, the determination system (R2) of the protein yield (Y) target function model is 0.95, which indicates that the model can explain 95% of the target function variance. The adjustment decision system (R2=0.88) was also very high, supporting the high significance of the model. It can be seen from Table 4-4 that the analysis of these extraction parameters with P values shows that the X1, X2, X3, X4, X3X4, X3*X3 and X4*X4 groups have a significant impact on the protein yield of walnut cakes.

Based on single-factor experimental results and literature descriptions, our results show that the currently tested enzyme addition, extraction time, solid-liquid ratio and particle size have significant effects on the protein yield of walnut cakes, and these parameters are also suitable for application in response to In the face analysis, the protein yield was used as the target function value. The results also showed that the effect of X3*X3 and X4*X4 on protein yield was significantly higher than that of X1*X1 and X2*X2. According to the significance test of each variable, it can be concluded that the primary and secondary order of the influence of the four factors on the protein yield of walnut cakes is: buffer particle size, extraction time, α-enzyme addition, and solid-liquid ratio. These findings also contribute to understanding the role and impact of extraction parameters in the protein extraction process.

Comparing the predicted value obtained by the quadratic regression model in the statistical analysis SAS 10.0.0 software with the actual value measured by the experiment, it is concluded that the experimental result is shown in Figure 2. From Figure 3, it can be concluded that the two have better fit.

Table 4-2 Box-Behnken test design scheme and results

X1 Enzyme dosage, Enzyme dosage(%)

Table 4-3 Regression model analysis of variance

Table 4-3 Variance Analysis of Regression Models

Table 4-4 Two-variable multiple equation effect test

Table 4-4 Binary Multiple Equation Effect Test

Table 4-4 is the statistical analysis table of the quadratic regression model. After the regression fitting of the response value and each factor, the regression expression of the protein yield of walnut meal is obtained:

Y=1.0985X ₁ ² -0.0066X ₂ ² -0.01878X ₃ ² +16.27069X ₄ ² -0.03538X ₁ X ₃ +0.1113X ₁ X ₂ -0.0055X ₂ X ₃ +0.809 X ₁ X ₄ +0.41079X ₂ X ₄ +0.7738X ₃ X ₄ -5.89125X ₁ +0.241X ₂ -1.314574X ₃ -98.836X ₄ +23.6924

Response Surface Optimization

According to the regression expression of protein yield, use JMP software to make response surfaces and contour lines, and optimize the walnut meal protein yield with the largest index. The effect of each single factor on the walnut meal protein yield is shown in the figure. 4 to Figure 9.

At the same time, in order to further obtain the optimal parameters of each factor, the regression equation is mathematically processed. Through the analysis of JMP software, the optimal extraction process parameters of walnut cake protein were obtained as follows: enzyme dosage 2.22%, particle size 89.22 mesh, solid-liquid ratio 1:30.280 (g/ml), extraction time 0.98h. Under this parameter, the protein yield of walnut meal reached 27.96%. Considering the actual situation, the parameters were revised as follows: enzyme dosage 2%, particle size 80 mesh, extraction time 1 h, and solid-liquid ratio 1:30. Three confirmatory experiments were carried out under this parameter to verify the effect of the buffer parameters obtained by JMP software and artificial correction on the protein yield of walnut meal.

It can be seen from Figure 10 that within the scope of the investigated factors, the protein yield of walnut cake showed a trend of first decreasing and then increasing with the extension of extraction time; with the increase of solid-liquid ratio, the protein yield remained basically unchanged; The increase in the amount will lead to a more detailed increase in the yield of walnut protein, and the particle size has a greater impact on the yield.

(2) Antioxidative experiment of walnut cake protein

(1) Determination of DPPH free radical scavenging ability

Measure 100 μl of walnut cake protein solution, add 2 ml of 0.1 mM/L DPPH solution and 900 μl of 50 mM/L TRIS-HCL solution. After uniform mixing, the reaction was carried out at room temperature for 30 min in the dark. Use a spectrophotometer to measure the absorbance A _S at 517 nm, use 3 ml of deionized water to replace the sample and TRIS-HCL solution, and measure the absorbance A ₀ , Use 3 ml of the sample and 2 ml of absolute ethanol to remove sample pigment errors, Obtain the absorbance value A _X . Finally, the DPPH free radical scavenging rate of walnut cake protein is obtained by calculation, and the calculation formula is:

Y=A ₀ -(A _S -A _X )/A ₀ ×100%

(2) Determination of reducing power

Weigh 2ml of walnut cake protein liquid sample into a 10ml test tube, add 2ml of phosphate buffer solution with a concentration of 0.2mM/L and 2ml of 1% red dish salt, react in a constant temperature water bath at 50°C for 20min and then rapidly cool down. After the reaction solution was cooled, 2 ml of 10% trichloroacetic acid was added, mixed evenly, and centrifuged for 5-10 min. After centrifugation, 1.5 ml of supernatant was added to 1.5 ml of distilled water and 0.3 ml of 0.1% ferric chloride solution for uniform reaction for 10 min. Use a spectrophotometer to detect the absorbance at 700 nm, and the higher the absorbance, the stronger the reducing power.

(3) Membrane filtration treatment

The extract was first filtered with ordinary filter paper to remove most of the residue, and then the remaining solids were removed by a suction filter device. Finally, 3kDa and 30kDa cellulose acetate membranes were used to filter the swept-flow filter system to obtain small molecular walnut cake protein.

(3) Experimental results

Alkali extraction antioxidant test results

Using the orthogonal experimental parameters of the alkali extraction experiment in the walnut cake protein extraction experiment, the antioxidant capacity was tested, and the results are shown in Table 4-2. The research shows that the DPPH free radical scavenging rate of alkali-extracted walnut cake protein is in the range of 16.61-50.05%. The absorbance value of total reducing power of walnut meal protein ranged from 0.19 to 0.54.

Effect of Membrane Filtration on Antioxidant Activity of Walnut Meal Protein

Using the experimental method of alkali extraction of walnut cake protein, the obtained walnut cake protein solution was tested for antioxidant activity, and its DPPH free radical scavenging ability and reducing power were determined, and then filtered through an acetate fiber membrane to compare the walnut cake protein before and after filtering Differences in antioxidant activity. In the experiment, the optimal extraction process parameters of alkali-extracted walnut cake protein obtained by response surface analysis were used for protein extraction, and the antioxidant activity of the obtained walnut cake protein solution was measured. The experimental results are shown in Figure 11.

Antioxidant test results of buffer extraction

Using the orthogonal experimental parameters of the buffer extraction experiment in the walnut cake protein extraction experiment, the antioxidant capacity test was carried out, and the results are shown in Table 3-2. The research showed that the DPPH free radical scavenging rate of walnut cake protein extracted by buffer solution ranged from 26.26-68.72%. The absorbance value of total reducing power of walnut meal protein ranged from 1.02 to 1.37.

Table 3-2 Box-Behnken test design scheme and results

Table 3-2 Box-Behnken design matrixand responsevalues

X ₁ buffer pH, Buffer pH value; X ₂ walnut oil cake grain size (mesh);

X ₃ solid-liquid ratio, solid-liquid ratio (g/mL); X ₄ extraction time, extraction time/(h)

FICP ferrous ion chelating ability, ferrous ion chelatingpower RP: reducing power, reducing power

Effect of Membrane Filtration on Antioxidant Activity of Extracted Protein

Using the experimental method of alkali extraction of walnut cake protein, the obtained walnut cake protein solution was tested for antioxidant activity, and its DPPH free radical scavenging ability and reducing power were determined, and then filtered through an acetate fiber membrane to compare the walnut cake protein before and after filtering Differences in antioxidant activity. In the experiment, the optimal extraction process parameters of alkali-extracted walnut cake protein obtained by response surface analysis were used for protein extraction, and the antioxidant activity of the obtained walnut cake protein solution was measured. The experimental results are shown in Figure 12.

Antioxidant test results of enzyme-assisted extraction

Using the orthogonal experimental parameters of the enzyme-assisted extraction of walnut cake protein in the walnut cake protein extraction experiment, the DPPH free radical scavenging ability was tested. The results are shown in Table 4-2. The research shows that the enzyme-assisted extraction of walnut cake protein The DPPH free radical scavenging rate ranged from 46.49-60.94%.

Claims (8)

1. a method for improving antioxidant activity of walnut meal protein, is characterized in that, comprises the following steps: S1: Test the effect of enzyme types on the protein yield of walnut cake; S2: Test the effect of enzyme addition on the protein yield of walnut cake; S3: Test the effect of solid-liquid ratio on the protein yield of walnut cake; S4: Test the effect of particle size on the protein yield of walnut cake; S5: Test the effect of extraction time on the protein yield of walnut cake; S6: determine the protein content of the extract; S7: Enzyme-assisted extraction of protein response surface experiment from walnut cake; S8: Effect of enzyme types on protein yield of walnut cake; S9: analyze the results of the single factor experiment; S10: Obtain the response surface analysis result; S11: Optimize the response surface; S12: Do the protein antioxidant experiment of walnut cake; S13: Obtain the test result. 2. the antioxidant activity method of improving walnut meal protein according to claim 1, is characterized in that, described S1 comprises the following steps: S1-1: Weigh about 1g of walnut cake powder with a particle size of 60 mesh and add it to a 50ml glass conical flask. According to the ratio of feed liquid 1:30, according to the optimum pH value of different types of enzymes, add buffers of different pH , the extraction time is set to 4h, and at room temperature, it is placed on a magnetic stirrer and stirred at a speed of 500r/min for 4h to fully dissolve it; S1-2: After reaching the specified time, transfer it into a 1.5ml centrifuge tube and put it into a centrifuge for centrifugation. Each sample was repeated three times, and the temperature was kept at 4 °C and the rotation speed was 12000 rpm during centrifugation. After 5 minutes, sampling was performed, and 0.2 ml of supernatant was extracted for the next experiment. 3. the antioxidant activity method that improves walnut meal protein according to claim 1 is characterized in that, described S2 comprises the following steps: S2-1: Weigh about 1g of walnut cake powder with a particle size of 60 mesh and add it to a 50ml glass conical flask. According to the ratio of material to liquid of 1:30, add 30ml of buffer solution with a pH value of 7.5 respectively. Put it into a water bath for constant temperature heating at 40°C, then add 1%, 2%, 3%, 4% and 5% of α-amylase respectively, and set the extraction time to 1, 2, 4, 6 and 8 h. At room temperature, stir on a magnetic stirrer at 500r/min for 4h to fully dissolve; S2-2: After reaching the specified time, transfer it into a 1.5ml centrifuge tube and put it into a centrifuge for centrifugation. Each sample was repeated three times, and the temperature was kept at 4 °C and the rotation speed was 12000 rpm during centrifugation. After 5 minutes, sampling was performed, and 0.2 ml of supernatant was extracted for the next experiment. 4. the antioxidant activity method of improving walnut meal protein according to claim 1, is characterized in that, described S3 comprises the following steps: S3-1: Weigh about 1g of walnut cake powder with a particle size of 60 mesh and add it to a 50ml glass conical flask. According to the corresponding material-to-liquid ratio, add 30ml of buffer solution with a pH value of 7.5, and put the conical flask into the conical flask. Heating at a constant temperature of 40°C in a water bath, adding 3% α-amylase respectively, setting the extraction time to 1, 2, 4, 6 and 8 h, at room temperature, placing it on a magnetic stirrer at a speed of 500 r/min Stir for 4h to fully dissolve; S3-2: After reaching the specified time, transfer it into a 1.5ml centrifuge tube and put it into a centrifuge for centrifugation. Each sample was repeated three times, and the temperature was kept at 4 °C and the rotation speed was 12000 rpm during centrifugation. After 5 minutes, sampling was performed, and 0.2 ml of supernatant was extracted for the next experiment. 5. the antioxidant activity method that improves walnut meal protein according to claim 1 is characterized in that, described S5 comprises the following steps: S5-1: Weigh about 1g of walnut cake powder with a particle size of 60 mesh and add it to a 50ml glass conical flask, add 30ml of buffer solution with a pH value of 7.5 according to the ratio of material to liquid 1:30, and place the conical flask in Put it into a water bath for heating at a constant temperature of 40 °C, then add 3% α-amylase respectively, set the extraction time to 1, 2, 4, 6 and 8 h, at room temperature, place it on a magnetic stirrer at 500 r/min Stir at the rotating speed to make it fully dissolved; S5-2: After reaching the specified time, transfer it into a 1.5ml centrifuge tube and put it into a centrifuge for centrifugation. Each sample was repeated three times, and the temperature was kept at 4 °C and the rotation speed was 12000 rpm during centrifugation. After 5 minutes, sampling was performed, and 0.2 ml of supernatant was extracted for the next experiment. 6. the antioxidant activity method that improves walnut cake meal protein according to claim 1 is characterized in that, the protein yield calculation formula of described S6 is:

7. The method for improving the antioxidant activity of walnut meal protein according to claim 1, wherein the enzyme species comprises alkaline protease, cellulase, alpha-amylase, pectinase and beta-amylase . 8. the antioxidant activity method that improves walnut meal protein according to claim 1, is characterized in that, described S12 comprises the following steps: S12-1: Determination of DPPH free radical scavenging ability; S12-2: Determination of reducing power; S12-3: Process membrane filtration.

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