CN110710592A - Method for improving antioxidant activity of walnut cake protein - Google Patents
Method for improving antioxidant activity of walnut cake protein Download PDFInfo
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- CN110710592A CN110710592A CN201911122975.0A CN201911122975A CN110710592A CN 110710592 A CN110710592 A CN 110710592A CN 201911122975 A CN201911122975 A CN 201911122975A CN 110710592 A CN110710592 A CN 110710592A
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/001—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from waste materials, e.g. kitchen waste
- A23J1/005—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from waste materials, e.g. kitchen waste from vegetable waste materials
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/14—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds
- A23J1/148—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds by treatment involving enzymes or microorganisms
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Abstract
The invention discloses a method for improving the antioxidant activity of walnut cake protein, which comprises the following steps: s1: testing the influence of the enzyme variety on the walnut cake protein yield; s2: testing the influence of the enzyme addition amount on the walnut cake protein yield; s3: testing the influence of the feed-liquid ratio on the yield of the walnut cake protein; s4: testing the influence of the particle size on the yield of the walnut cake protein; s5: testing the influence of the extraction time on the yield of the walnut cake protein; s6: measuring the protein content of the extracting solution; s7: an enzyme-assisted walnut cake protein extraction response surface experiment; s8: influence of enzyme types on the yield of walnut cake protein; solves the problems that the existing method can not be applied to the field of food and medicine, the cost of the single factor is higher when the temperature is controlled in the experimental process, the safety factor is lower, the obtained cake protein has extremely low antioxidant activity, and the buffer solution can not obtain higher protein yield.
Description
Technical Field
The invention relates to the field of an antioxidant activity method of walnut cake protein, in particular to a method for improving the antioxidant activity of walnut cake protein.
Background
The walnut cake is prepared from walnut kernel, and the rest part of the walnut kernel is called walnut cake after the walnut kernel is squeezed to extract grease. The names of products obtained after the oil is extracted by different methods are different, and the product obtained by using a physical squeezing method is called 'cake'; the product resulting from the leaching process is called "meal". China still has a preliminary stage of recycling contract cakes, and mainly focuses on the aspects of component analysis, protein extraction, walnut cake feed, fertilizer and the like of walnut cakes. Studies have shown that walnut cakes contain about 50% protein.
In recent years, walnut cake protein is widely applied to the field of medical research as a raw material for producing walnut polypeptide because the walnut cake protein has multiple physiological functions of resisting oxidation, cardiovascular diseases, obesity, diabetes and the like. The research shows that the protein is enzymatically hydrolyzed into the active peptide by the digestive tract, so that the protein can be absorbed and utilized more quickly. The bioactive peptide which is taken as food in the market at present mainly takes a bean product as a main part, and is taken as a walnut with a high protein source, but is rarely used for actual production.
At present, researches show that by taking walnut defatted powder with protein content of 46.14% as a raw material and carrying out enzymolysis through trypsin, the water product of the walnut defatted powder has a high antioxidant function and can eliminate active oxygen in mouse tumor cells. The walnut polypeptide nutrient solution is prepared by a mixed anion and cation bed desalting method and biological membrane filtration, and has high antioxidant activity.
At present, the research and preparation aiming at the biological activity of the walnut cake are not complete, the system is also in the primary exploration stage, and the research on the aspects of the molecular structure of the active agent in the physiological period is also little.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a method for improving the antioxidant activity of walnut cake protein, and solves the problems that the existing method cannot be applied to the fields of food and medicines, temperature control is performed in an experimental process, the cost of a single factor is high, the safety coefficient is low, the antioxidant activity of the obtained cake protein is extremely low, and a buffer solution cannot obtain high protein yield.
The technical scheme adopted by the invention is that the method for improving the antioxidant activity of the walnut cake protein comprises the following steps:
s1: testing the influence of the enzyme variety on the walnut cake protein yield;
s2: testing the influence of the enzyme addition amount on the walnut cake protein yield;
s3: testing the influence of the feed-liquid ratio on the yield of the walnut cake protein;
s4: testing the influence of the particle size on the yield of the walnut cake protein;
s5: testing the influence of the extraction time on the yield of the walnut cake protein;
s6: measuring the protein content of the extracting solution;
s7: an enzyme-assisted walnut cake protein extraction response surface experiment;
s8: influence of enzyme types on the yield of walnut cake protein;
s9: analyzing a single-factor experiment result;
s10: obtaining a response surface analysis result;
s11: optimizing a response surface;
s12: performing an anti-oxidation experiment on the walnut cake protein;
s13: and obtaining the test result.
Preferably, S1 includes the steps of:
s1-1: weighing about 1g of walnut cake meal powder with the grain size of 60 meshes, adding the walnut cake meal powder into a 50ml glass conical flask, adding buffer solutions with different pH values according to the optimal pH values of different types of enzymes according to the feed liquid ratio of 1:30, setting the extraction time to be 4h, and stirring the walnut cake meal powder on a magnetic stirrer at the rotating speed of 500r/min for 4h at room temperature to fully dissolve the walnut cake meal powder;
s1-2: after the specified time, the mixture was transferred to a 1.5ml centrifuge tube and centrifuged in a centrifuge. Each sample was repeated 3 times, centrifuged at 4 ℃ and 12000rpm, sampled 5min later, and 0.2ml of supernatant was extracted and prepared for further experiments.
Preferably, S2 includes the steps of:
s2-1: weighing about 1g of walnut cake meal powder with the grain size of 60 meshes, adding the walnut cake meal powder into a 50ml glass conical flask, respectively adding 30ml of buffer solution with the pH value of 7.5 according to the material-liquid ratio of 1:30, placing the conical flask into a water bath kettle for constant temperature heating at 40 ℃, then respectively adding 1%, 2%, 3%, 4% and 5% of alpha-amylase, setting the extraction time to be 1, 2, 4, 6 and 8 hours, and placing the conical flask on a magnetic stirrer for stirring at the rotating speed of 500r/min for 4 hours at room temperature to fully dissolve the alpha-amylase;
s2-2: after the specified time, the mixture was transferred to a 1.5ml centrifuge tube and centrifuged in a centrifuge. Each sample was repeated 3 times, centrifuged at 4 ℃ and 12000rpm, sampled 5min later, and 0.2ml of supernatant was extracted and prepared for further experiments.
Preferably, S3 includes the steps of:
s3-1: weighing about 1g of walnut cake meal powder with the grain size of 60 meshes, adding the walnut cake meal powder into a 50ml glass conical flask, respectively adding 30ml of buffer solution with the pH value of 7.5 according to corresponding material-liquid ratio, putting the conical flask into a water bath kettle, heating at the constant temperature of 40 ℃, then respectively adding 3% of alpha-amylase, setting the extraction time to be 1, 2, 4, 6 and 8 hours, and stirring on a magnetic stirrer at the rotating speed of 500r/min for 4 hours at room temperature to fully dissolve the alpha-amylase;
s3-2: after the specified time, the mixture was transferred to a 1.5ml centrifuge tube and centrifuged in a centrifuge. Each sample was repeated 3 times, centrifuged at 4 ℃ and 12000rpm, sampled 5min later, and 0.2ml of supernatant was extracted and prepared for further experiments.
Preferably, S5 includes the steps of:
s5-1: weighing about 1g of walnut cake meal powder with the grain size of 60 meshes, adding the walnut cake meal powder into a 50ml glass conical flask, respectively adding 30ml of buffer solution with the pH value of 7.5 according to the material-liquid ratio of 1:30, placing the conical flask into a water bath kettle, heating at the constant temperature of 40 ℃, respectively adding 3% of alpha-amylase, setting the extraction time to be 1, 2, 4, 6 and 8 hours, and stirring on a magnetic stirrer at the rotating speed of 500r/min at room temperature to fully dissolve the alpha-amylase;
s5-2: after the specified time, the mixture was transferred to a 1.5ml centrifuge tube and centrifuged in a centrifuge. Each sample was repeated 3 times, centrifuged at 4 ℃ and 12000rpm, sampled 5min later, and 0.2ml of supernatant was extracted and prepared for further experiments.
Preferably, the protein yield of S6 is calculated as:
preferably, the enzyme species include alkaline protease, cellulase, alpha-amylase, pectinase and beta-amylase.
Preferably, S12 includes the steps of:
s12-1: measuring DPPH free radical scavenging ability;
s12-2: measuring the reducing power;
s12-3: and (4) treating membrane filtration.
The method for improving the antioxidant activity of the walnut cake protein has the following beneficial effects:
1. the buffer solutions such as phosphate buffer solution, Tris-HCl buffer solution, NaHCO3 and the like extract the walnut cake protein, and experimental results show that the antioxidant activity of the buffer solution extraction is obviously improved, and the walnut cake protein yield can be effectively improved by adding enzymes, so that the yield of the walnut cake protein is ensured while soluble active protein is obtained. Meanwhile, the obtained walnut cake protein solution is filtered by using an acetate fiber membrane, so that the walnut protein solution with high-concentration micromolecular protein is obtained.
2. The invention uses acetate fiber membrane to obtain walnut cake protein with molecular weight below 10kDa by cross flow filtration and air-suction filtration. The walnut cake protein with larger molecular weight is obtained by a method for extracting the protein by using alkali and buffer solution, and the difference of single factor and orthogonal test results is analyzed by the same method to compare the optimal extraction processes before and after the comparison.
Drawings
FIG. 1 is a graph showing the influence of enzyme species on the walnut cake protein yield in the method for improving the antioxidant activity of walnut cake protein according to the present invention
FIG. 2 is a graph of experimental results of single-factor extraction of walnut cake protein by an enzyme-assisted method of the method for improving the antioxidant activity of walnut cake protein
FIG. 3 is a fitting degree graph of a model predicted value and an actual experimental value of the method for improving the antioxidant activity of walnut cake protein
FIG. 4 shows the enzyme addition amount (X) of the method for improving the antioxidant activity of walnut cake protein according to the invention1) Ratio of material to liquid (X)3) Interactive response surface map and contour map
FIG. 5 shows the enzyme addition amount (X) of the method for improving the antioxidant activity of walnut cake protein according to the invention1) Particle size (X)3) Interactive response surface map and contour map
FIG. 6 shows the enzyme addition amount (X) of the method for improving the antioxidant activity of walnut cake protein according to the present invention1) Time (X)3) Interactive response surface map and contour map
FIG. 7 shows the ratio of material to liquid (X) of the method for improving the antioxidant activity of walnut cake protein according to the present invention1) Particle size (X)3) Interactive response surface map and contour map
FIG. 8 shows the material-to-liquid ratio (X) of the method for improving the antioxidant activity of walnut cake protein according to the invention1) Particle size (X)3) Interactive response surface map and contour map
FIG. 9 shows the enzyme addition amount (X) of the method for improving the antioxidant activity of walnut cake protein according to the invention1) And particle diameter (X)3) Interactive response surface map and contour map
FIG. 10 is a prediction carver of a walnut cake protein yield response curved surface for the method for improving the antioxidant activity of the walnut cake protein
FIG. 11 shows the walnut cake protein antioxidant capacity under the membrane filtration condition of the method for improving the antioxidant activity of walnut cake protein of the invention
FIG. 12 shows the walnut cake protein antioxidant capacity under the membrane filtration condition of the method for improving the antioxidant activity of walnut cake protein of the invention
FIG. 13 is a general flow chart of the method for improving the antioxidant activity of walnut cake protein according to the invention
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.
As shown in fig. 13, a method for improving antioxidant activity of walnut cake protein comprises the following steps:
s1: testing the influence of the enzyme variety on the walnut cake protein yield;
s2: testing the influence of the enzyme addition amount on the walnut cake protein yield;
s3: testing the influence of the feed-liquid ratio on the yield of the walnut cake protein;
s4: testing the influence of the particle size on the yield of the walnut cake protein;
s5: testing the influence of the extraction time on the yield of the walnut cake protein;
s6: measuring the protein content of the extracting solution;
s7: an enzyme-assisted walnut cake protein extraction response surface experiment;
s8: influence of enzyme types on the yield of walnut cake protein;
s9: analyzing a single-factor experiment result;
s10: obtaining a response surface analysis result;
s11: optimizing a response surface;
s12: performing an anti-oxidation experiment on the walnut cake protein;
s13: and obtaining the test result.
The enzyme-assisted extraction method has the characteristics of high efficiency, strong pertinence, easily met experimental conditions and the like, and has a plurality of enzyme-assisted related researches, and Rong and the like extract blueberry polysaccharide by using an alkaline extraction method and a cellulase-assisted method and taking blueberries as raw materials. The alkaline leaching method conditions are respectively extraction temperature, alkali addition amount, extraction times and extraction time, and the cellulase auxiliary method conditions are respectively pH, cellulase addition amount, extraction time and extraction temperature. Compared with the alkaline extraction method, the cellulose-assisted method improves the yield of polysaccharide by 26 percent. This is because when the biological enzyme is combined with the substrate, the change of the molecular shape of the enzyme causes the change of the chemical bond of the substrate to finally cause the breakage of the chemical bond, and the cellulose is broken to break the cell wall, thereby promoting the effective components to flow out of the cytoplasm and dissolve into the extract, and realizing the sufficient release and the high-efficiency extraction of the bioactive compounds.
On the basis of extracting the walnut cake protein by using the buffer solution, various enzymes are used for assisting in extracting the walnut cake protein in the experimental process in order to ensure that higher protein yield is obtained and the activity of the walnut cake protein can be kept. Common enzymes include alkaline proteases, neutral proteases, acid proteases, pectinases, alpha-amylases, beta-amylases, cellulases, and the like. The experiment utilizes the buffer solution experiment condition, selects enzyme with higher yield, and increases the yield of walnut cake protein.
(1) Influence of enzyme variety on walnut cake protein yield
Weighing about 1g of walnut cake meal powder with the grain size of 60 meshes, adding the walnut cake meal powder into a 50ml glass conical flask, adding buffer solutions with different pH values according to the optimal pH values of different types of enzymes according to the feed liquid ratio of 1:30, setting the extraction time to be 4h, and stirring the walnut cake meal powder on a magnetic stirrer at the rotating speed of 500r/min for 4h at room temperature to fully dissolve the walnut cake meal powder. After the specified time, the mixture was transferred to a 1.5ml centrifuge tube and centrifuged in a centrifuge. Each sample was repeated 3 times, centrifuged at 4 ℃ and 12000rpm, sampled 5min later, and 0.2ml of supernatant was extracted and prepared for further experiments.
(2) Influence of enzyme addition amount on walnut cake protein yield
Weighing about 1g of walnut cake meal powder with the grain size of 60 meshes, adding the walnut cake meal powder into a 50ml glass conical flask, respectively adding 30ml of buffer solution with the pH value of 7.5 according to the material-liquid ratio of 1:30, placing the conical flask into a water bath kettle for constant temperature heating at 40 ℃, then respectively adding 1%, 2%, 3%, 4% and 5% of alpha-amylase, setting the extraction time to be 1, 2, 4, 6 and 8 hours, and placing the conical flask on a magnetic stirrer for stirring at the rotating speed of 500r/min for 4 hours at room temperature to fully dissolve the alpha-amylase. After the specified time, the mixture was transferred to a 1.5ml centrifuge tube and centrifuged in a centrifuge. Each sample was repeated 3 times, centrifuged at 4 ℃ and 12000rpm, sampled 5min later, and 0.2ml of supernatant was extracted and prepared for further experiments.
(3) Influence of feed liquid ratio on walnut cake protein yield
Weighing about 1g of walnut cake meal powder with the grain size of 60 meshes, adding the walnut cake meal powder into a 50ml glass conical flask, respectively adding 30ml of buffer solution with the pH value of 7.5 according to corresponding material-liquid ratio, placing the conical flask into a water bath kettle to carry out constant temperature heating at 40 ℃, then respectively adding 3% of alpha-amylase, setting the extraction time to be 1, 2, 4, 6 and 8 hours, and placing the conical flask on a magnetic stirrer to stir for 4 hours at the rotating speed of 500r/min at room temperature to fully dissolve the alpha-amylase. After the specified time, the mixture was transferred to a 1.5ml centrifuge tube and centrifuged in a centrifuge. Each sample was repeated 3 times, centrifuged at 4 ℃ and 12000rpm, sampled 5min later, and 0.2ml of supernatant was extracted and prepared for further experiments.
(4) Influence of particle size on walnut cake protein yield
Weighing about 1g of walnut cake meal powder with the grain sizes of 20 meshes, 40 meshes, 60 meshes, 80 meshes and 100 meshes respectively, adding the walnut cake meal powder into a 50ml glass conical flask, adding 30ml of buffer solution with the pH value of 7.5 respectively according to the material-liquid ratio of 1:30, putting the conical flask into a water bath kettle, heating at the constant temperature of 40 ℃, adding 3% of alpha-amylase respectively, setting the extraction time to be 1, 2, 4, 6 and 8 hours, and stirring on a magnetic stirrer at the rotating speed of 500r/min for 4 hours at room temperature to fully dissolve the alpha-amylase. After the specified time, the mixture was transferred to a 1.5ml centrifuge tube and centrifuged in a centrifuge. Each sample was repeated 3 times, centrifuged at 4 ℃ and 12000rpm, sampled 5min later, and 0.2ml of supernatant was extracted and prepared for further experiments.
(5) Influence of extraction time on walnut cake protein yield
Weighing about 1g of walnut cake meal powder with the grain size of 60 meshes, adding the walnut cake meal powder into a 50ml glass conical flask, respectively adding 30ml of buffer solution with the pH value of 7.5 according to the material-to-liquid ratio of 1:30, placing the conical flask into a water bath kettle, heating at the constant temperature of 40 ℃, respectively adding 3% of alpha-amylase, setting the extraction time to be 1, 2, 4, 6 and 8 hours, and stirring on a magnetic stirrer at the rotating speed of 500r/min at room temperature to fully dissolve the alpha-amylase. After the specified time, the mixture was transferred to a 1.5ml centrifuge tube and centrifuged in a centrifuge. Each sample was repeated 3 times, centrifuged at 4 ℃ and 12000rpm, sampled 5min later, and 0.2ml of supernatant was extracted and prepared for further experiments.
(6) Determination of protein content
The protein concentration of the extract was determined by Coomassie brilliant blue G-250 colorimetry. Calculation formula of protein yield:
soluble protein content was determined by coomassie brilliant blue method: adding 0.05mL of the extractive solution (3 repeat tubes), 2.5mL of Coomassie brilliant blue reagent into the test tube, shaking, standing for 5min, performing color comparison with spectrophotometer at 595nm, and recording the absorbance. A standard curve is drawn by using bovine serum albumin with known concentration, and the protein concentration is found through the standard curve.
(7) Response surface experiment for enzyme-assisted extraction of walnut cake protein
According to the experimental result of enzyme-assisted extraction single factor, selecting extraction time, a material-liquid ratio, an enzyme addition amount and a cake grain size as variables, taking walnut cake protein yield as an index, and adopting Design-Expert Box-Behnken Design response surface test. The factor level codes are shown in the table below.
TABLE 4-1 center combination design factor and horizontal code table
And (3) determining the protein concentration of the protein stock solution by using a Coomassie brilliant blue method, and freeze-drying the protein stock solution by using a freeze dryer and storing for later use.
(8) Results and analysis
Influence of enzyme variety on walnut cake protein yield
In order to determine which enzyme has a better influence on the walnut protein yield, the results of the walnut protein extraction experiment by using the buffer solution are utilized, alkaline protease, cellulase, alpha-amylase, pectinase and beta-amylase are respectively added into the buffer solution, and stirring and extraction are carried out under the same parameters. The effect of different enzymes on the yield of walnut cake protein is shown in figure 1. As can be seen from FIG. 1, the yields of alkaline protease, cellulase, pectinase and beta-amylase are respectively 13.67%, 8.82%, 7.85% and 5.25%, indicating that the effect of alpha-amylase on the yield of walnut protein is significantly higher than that of other enzymes. The alpha-amylase is used for extraction, and the yield of the walnut cake protein reaches 17.23 percent, so the alpha-amylase is selected.
Results of single factor experiments
The effect of enzyme addition on walnut cake protein yield is shown in fig. 2. As can be seen from fig. 2, fig. 2(a) shows the influence of the enzyme addition amount on the walnut cake protein yield, fig. 2(b) shows the influence of the feed liquid ratio on the walnut cake protein yield, fig. 2(c) shows the influence of the extraction time on the walnut cake protein yield, fig. 2(d) shows the influence of the particle size on the walnut cake protein yield, the influence of the alpha-amylase addition amount of 3% on the walnut protein yield is the highest, and the walnut protein yield is significantly higher than that of other addition amounts; in the feed-liquid ratio experiment, the protein yield is increased along with the increase of the feed-liquid ratio; similar to the alkali extraction of walnut cake protein, when the enzyme is used for assisting extraction, the particle size of the walnut cake is increased to the highest when the particle size is 80 meshes, and is slightly reduced when the particle size is 100 meshes; when the extraction time reaches 1h, the protein yield is highest, the time is continuously prolonged, and the yield is gradually reduced. According to the experimental results and the production practice, the extraction process adopted in the enzyme-assisted walnut protein extraction test is preliminarily determined as follows: the enzyme addition amount is 2-4%, the enzyme addition amount is 1:30-1:50, the walnut cake grain size is 60-100 meshes, and the extraction time is 0.5-1.5 h.
Response surface analysis results
Single factor tests show that in the extraction process of the protein of the walnut cake, the enzyme addition amount, the extraction time, the feed-liquid ratio and the particle size are main factors influencing the content of the soluble protein. Therefore, according to a JMP (joint rotation center) combined design method (CCRD for short), a walnut cake extraction parameter optimization test is carried out in four factors and three levels, wherein the four extraction parameters comprise a buffer solution pH value, extraction time, a material-liquid ratio and a particle size. The experimental yield and predicted value of walnut cake protein at the design point are shown in fig. 2 and tables 4-2. Research shows that different extraction parameters cause obvious difference of the protein yield of the walnut cake, and the protein yield ranges from 8.85% to 19.56%. A polynomial regression equation is constructed using CCRD, the interactions between factors are analyzed and significant influencing factors in the regression equation are analyzed. The results of the ANOVA analysis 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 to the regression model. The statistical software JMP based calculation program obtained the values for each entry as shown in tables 4-4. When the P value is less than 0.05, the extraction parameter is significant for the Y value. Based on the above discussion, we can see that analysis of variance can well describe the correlation between protein yield (Y) and extraction parameters (X1, X2, X3, X4). In addition, a decision system (R2) is included to check the agreement of the experimental results with the digital model. In the current results, the decision system (R2) for the protein yield (Y) target functional model is 0.95, which indicates that the model can account for 95% of the target functional variance. The tuning decision system (R2 ═ 0.88) is also very high, supporting the high significance of the model. As can be seen from tables 4-4, analysis of these extracted parameters with P values showed that X1, X2, X3, X4, X3X4, X3X 3 and X4X 4 groups had a significant effect on walnut cake protein yield.
Based on single-factor experimental results and literature description, our results show that the enzyme addition amount, extraction time, feed-liquid ratio and particle size of the current test have significant influence on the walnut cake protein yield, the parameters are also suitable for being applied to response surface analysis, and the protein yield is taken as a target functional value. The results of the study also showed that X3X 3 and X4X 4 had a significantly higher effect on protein yield than X1X 1 and X2X 2. According to the significance test of each variable, the primary and secondary sequence of the influence of four factors on the walnut cake protein yield can be obtained, wherein the primary and secondary sequence comprises buffer solution particle size, extraction time, alpha-enzyme addition amount and feed-liquid ratio. These findings also help to understand the role and influence of extraction parameters in the protein extraction process.
The predicted value obtained by the quadratic regression model in the statistical analysis SAS 10.0.0 software is compared with the actual value obtained by the experiment, the conclusion is obtained, the experimental result is shown in FIG. 2, and the result can be shown in FIG. 3 that the predicted value and the actual value have better fitting degree.
TABLE 4-2 Box-Behnken test design and results
Amount of X1 Enzyme added, Enzyme dosage (%)
TABLE 4-3 regression model analysis of variance
Table 4-3 Variance Analysis ofRegression Models
TABLE 4-4 two-dimensional multiple equation Effect test
Table 4-4 Binary Multiple Equation Effect Test
Tables 4-4 are statistical analysis tables of a quadratic regression model, and regression fitting is performed on the response values and various factors to obtain a regression expression of the walnut cake protein yield:
Y＝1.0985X1 2-0.0066X2 2-0.01878X3 2+16.27069X4 2-0.03538X1X3+0.1113X1X2-0.0055X2X3+0.809 X1X4+0.41079X2X4+0.7738X3X4-5.89125X1+0.241X2-1.314574X3-98.836X4+23.6924
response surface optimization
According to the regression expression of the protein yield, JMP software is used for making a response surface and contour lines, the maximum walnut cake protein yield is used as an index for optimization, and the influence of each single factor on the walnut cake protein yield is shown in figures 4 to 9.
Meanwhile, the regression equation is subjected to mathematical processing for further solving the optimal parameters of all the factors. Through JMP software analysis, the optimal extraction process parameters of the walnut cake protein are as follows: the enzyme addition amount is 2.22%, the particle size is 89.22 meshes, the feed-liquid ratio is 1:30.280(g/ml), and the extraction time is 0.98 h. Under the parameter, the yield of the walnut cake protein reaches 27.96%, and in consideration of actual conditions, the parameters are corrected as follows: the enzyme addition amount is 2%, the particle size is 80 meshes, the extraction time is 1h, and the feed-liquid ratio is 1:30. And 3 times of verification tests are carried out under the parameters to verify the influence of buffer solution parameters obtained by JMP software and artificial correction on the walnut cake protein yield.
As can be seen from fig. 10, within the range of factors to be considered, the walnut cake protein yield shows a trend of decreasing first and then increasing as the extraction time is prolonged; the protein yield is basically kept unchanged along with the increase of the feed-liquid ratio; the increase of the enzyme addition amount can lead the walnut protein yield to have more detailed increase, the particle size has larger influence on the yield, and the yield tends to increase firstly and then decrease along with the expansion of the particle size.
(II) walnut cake protein antioxidation experiment
(1) DPPH radical scavenging Capacity determination
100 μ L of walnut cake protein solution was measured and 2ml of 0.1mM/L DPPH solution and 900 μ L of 50mM/L TRIS-HCL solution were added. After being mixed evenly, the mixture is reacted for 30min at room temperature in a dark place. Absorbance A was measured at 517nm using a spectrophotometerSAbsorbance A was measured using 3ml of deionized water instead of the sample and TRIS-HCL solution0Removing the error of the pigment of the sample by using 3ml of sample and 2ml of absolute ethyl alcohol to obtain a light absorption value AX. And finally, obtaining the DPPH free radical clearance rate of the walnut cake protein through calculation, wherein the calculation formula is as follows:
Y＝A0-(AS-AX)/A0×100％
(2) measurement of reducing Power
Weighing 2ml of walnut cake protein liquid sample into a 10ml test tube, adding 2ml of phosphate buffer solution with the concentration of 0.2mM/L and 2ml of 1% scholar salt, reacting for 20min in a constant-temperature water bath at 50 ℃, and then rapidly cooling. Cooling the reaction solution, adding 2ml of 10% trichloroacetic acid, mixing uniformly, and centrifuging for 5-10 min. After the centrifugation, 1.5ml of the supernatant was added to 1.5ml of distilled water and 0.3ml of 0.1% ferric chloride solution and reacted for 10 min. The absorbance was measured at 700nm using a spectrophotometer, the higher the absorbance, the stronger the reducing power was demonstrated.
(3) Membrane filtration treatment
Filtering most residues of the extracting solution by using common filter paper, removing residual solids by using an air-suction filtering device, and finally filtering by using 3kDa and 30kDa acetate fiber membranes by using a scavenging filtering system to obtain the micromolecule walnut cake protein.
(III) results of the experiment
Alkali extraction anti-oxidation experimental results
The results of the antioxidant capacity test using orthogonal experimental parameters of the alkali extraction experiment in the walnut cake protein extraction experiment are shown in table 4-2. Research shows that the DPPH free radical clearance rate of the alkali extraction walnut cake protein is 16.61-50.05%. The light absorption value range of the total reducing force of the walnut cake protein is 0.19-0.54.
Influence of membrane filtration treatment on walnut cake protein antioxidant activity
The obtained walnut cake protein liquid is subjected to antioxidant activity detection by using an experimental method for extracting walnut cake protein by using alkali, and after the DPPH free radical scavenging capacity and reducing power of the walnut cake protein liquid are measured, the difference of the antioxidant activity of the walnut cake protein before and after filtration is compared by filtering through an acetate fiber membrane. In the experiment, the optimal extraction process parameters of the alkali-extracted walnut cake protein obtained by response surface analysis are used for protein extraction, the antioxidant activity of the obtained walnut cake protein liquid is measured, and the experimental result is shown in fig. 11.
Buffer solution extraction anti-oxidation experimental result
The antioxidant capacity test was performed using orthogonal experimental parameters of the buffer solution extraction experiment in the walnut cake protein extraction experiment, and the results are shown in tables 3-2. Research shows that DPPH free radical clearance rate of walnut cake protein extracted by buffer solution is 26.26-68.72%. The light absorption value range of the total reducing force of the walnut cake protein is 1.02-1.37.
TABLE 3-2 Box-Behnken test design and results
Table 3-2 Box-Behnken design matrixand responsevalues
X1Buffer pH, Buffer pH value; x2Walnut cake grain size walnut oil cake grain size (mesh);
X3the ratio of feed to liquid, solid-liquidatio (g/mL); x4Extraction time, extraction time/(h)
FICP ferrous ion chelating ability, ferrous ion chelating power RP reducing power
Effect of Membrane filtration treatment on antioxidant Activity of extracted proteins
The obtained walnut cake protein liquid is subjected to antioxidant activity detection by using an experimental method for extracting walnut cake protein by using alkali, and after the DPPH free radical scavenging capacity and reducing power of the walnut cake protein liquid are measured, the difference of the antioxidant activity of the walnut cake protein before and after filtration is compared by filtering through an acetate fiber membrane. In the experiment, the optimal extraction process parameters of the alkali-extracted walnut cake protein obtained by response surface analysis are used for protein extraction, the antioxidant activity of the obtained walnut cake protein liquid is measured, and the experimental result is shown in fig. 12.
Results of enzyme-assisted extraction antioxidant experiments
The DPPH free radical scavenging capacity test is carried out by utilizing orthogonal experiment parameters of an enzyme-assisted walnut cake protein extraction experiment in the walnut cake protein extraction experiment, the result is shown in table 4-2, and the research shows that the DPPH free radical scavenging rate range of the enzyme-assisted walnut cake protein extraction is 46.49-60.94%.
Claims (8)
1. A method for improving the antioxidant activity of walnut cake protein is characterized by comprising the following steps:
s1: testing the influence of the enzyme variety on the walnut cake protein yield;
s2: testing the influence of the enzyme addition amount on the walnut cake protein yield;
s3: testing the influence of the feed-liquid ratio on the yield of the walnut cake protein;
s4: testing the influence of the particle size on the yield of the walnut cake protein;
s5: testing the influence of the extraction time on the yield of the walnut cake protein;
s6: measuring the protein content of the extracting solution;
s7: an enzyme-assisted walnut cake protein extraction response surface experiment;
s8: influence of enzyme types on the yield of walnut cake protein;
s9: analyzing a single-factor experiment result;
s10: obtaining a response surface analysis result;
s11: optimizing a response surface;
s12: performing an anti-oxidation experiment on the walnut cake protein;
s13: and obtaining the test result.
2. The method for improving the antioxidant activity of walnut cake protein according to claim 1, wherein the S1 comprises the following steps:
s1-1: weighing about 1g of walnut cake meal powder with the grain size of 60 meshes, adding the walnut cake meal powder into a 50ml glass conical flask, adding buffer solutions with different pH values according to the optimal pH values of different types of enzymes according to the feed liquid ratio of 1:30, setting the extraction time to be 4h, and stirring the walnut cake meal powder on a magnetic stirrer at the rotating speed of 500r/min for 4h at room temperature to fully dissolve the walnut cake meal powder;
s1-2: after the specified time, the mixture was transferred to a 1.5ml centrifuge tube and centrifuged in a centrifuge. Each sample was repeated 3 times, centrifuged at 4 ℃ and 12000rpm, sampled 5min later, and 0.2ml of supernatant was extracted and prepared for further experiments.
3. The method for improving the antioxidant activity of walnut cake protein according to claim 1, wherein the S2 comprises the following steps:
s2-1: weighing about 1g of walnut cake meal powder with the grain size of 60 meshes, adding the walnut cake meal powder into a 50ml glass conical flask, respectively adding 30ml of buffer solution with the pH value of 7.5 according to the material-liquid ratio of 1:30, placing the conical flask into a water bath kettle for constant temperature heating at 40 ℃, then respectively adding 1%, 2%, 3%, 4% and 5% of alpha-amylase, setting the extraction time to be 1, 2, 4, 6 and 8 hours, and placing the conical flask on a magnetic stirrer for stirring at the rotating speed of 500r/min for 4 hours at room temperature to fully dissolve the alpha-amylase;
s2-2: after the specified time, the mixture was transferred to a 1.5ml centrifuge tube and centrifuged in a centrifuge. Each sample was repeated 3 times, centrifuged at 4 ℃ and 12000rpm, sampled 5min later, and 0.2ml of supernatant was extracted and prepared for further experiments.
4. The method for improving the antioxidant activity of walnut cake protein according to claim 1, wherein the S3 comprises the following steps:
s3-1: weighing about 1g of walnut cake meal powder with the grain size of 60 meshes, adding the walnut cake meal powder into a 50ml glass conical flask, respectively adding 30ml of buffer solution with the pH value of 7.5 according to corresponding material-liquid ratio, putting the conical flask into a water bath kettle, heating at the constant temperature of 40 ℃, then respectively adding 3% of alpha-amylase, setting the extraction time to be 1, 2, 4, 6 and 8 hours, and stirring on a magnetic stirrer at the rotating speed of 500r/min for 4 hours at room temperature to fully dissolve the alpha-amylase;
s3-2: after the specified time, the mixture was transferred to a 1.5ml centrifuge tube and centrifuged in a centrifuge. Each sample was repeated 3 times, centrifuged at 4 ℃ and 12000rpm, sampled 5min later, and 0.2ml of supernatant was extracted and prepared for further experiments.
5. The method for improving the antioxidant activity of walnut cake protein according to claim 1, wherein the S5 comprises the following steps:
s5-1: weighing about 1g of walnut cake meal powder with the grain size of 60 meshes, adding the walnut cake meal powder into a 50ml glass conical flask, respectively adding 30ml of buffer solution with the pH value of 7.5 according to the material-to-liquid ratio of 1:30, placing the conical flask into a water bath kettle for constant temperature heating at 40 ℃, then respectively adding 3% of alpha-amylase, setting the extraction time to be 1, 2, 4, 6 and 8 hours, and stirring on a magnetic stirrer at the rotating speed of 500r/min at room temperature to fully dissolve the alpha-amylase;
s5-2: after the specified time, the mixture was transferred to a 1.5ml centrifuge tube and centrifuged in a centrifuge. Each sample was repeated 3 times, centrifuged at 4 ℃ and 12000rpm, sampled 5min later, and 0.2ml of supernatant was extracted and prepared for further experiments.
6. The method for improving the antioxidant activity of walnut cake protein according to claim 1, wherein the protein yield calculation formula of S6 is as follows:
7. the method for improving the antioxidant activity of walnut cake protein according to claim 1, wherein the enzyme species include alkaline protease, cellulase, alpha-amylase, pectinase and beta-amylase.
8. The method for improving the antioxidant activity of walnut cake protein according to claim 1, wherein the S12 comprises the following steps:
s12-1: measuring DPPH free radical scavenging ability;
s12-2: measuring the reducing power;
s12-3: and (4) treating membrane filtration.
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