CN110904176A - Preparation method of Astragalus membranaceus mushroom protein source antioxidant peptide - Google Patents
Preparation method of Astragalus membranaceus mushroom protein source antioxidant peptide Download PDFInfo
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Abstract
The invention discloses a preparation method of Astragalus membranaceus mushroom protein source antioxidant peptide, which comprises the following steps: s1: extracting protein of the astragalus membranaceus mushroom by an alkali-dissolving acid-precipitation method; s2: enzymolysis of the protein of the astragalus membranaceus mushroom; s3: screening protease; s4: selecting an enzyme adding mode; s5: selecting the enzyme adding proportion; s6: single factor test; s7: testing a response surface; s8: measuring DPPH free radical clearance rate; s9, measuring the hydrolysis degree; s10: ABTS and clearance assays; s11: testing the total reducing force; and S12 analyzing and calculating. According to the invention, the Astragalus membranaceus mushroom protein is used as a raw material, the antioxidative polypeptide is efficiently obtained in a compounding manner of two proteases, the optimal process conditions for preparing the Astragalus membranaceus mushroom protein antioxidative polypeptide are explored, and a foundation is provided for deep processing of the Astragalus membranaceus mushroom protein and development of the antioxidative peptide.
Description
Technical Field
The invention relates to the technical field of Astragalus membranaceus mushroom protein, and particularly relates to a preparation method of Astragalus membranaceus mushroom protein source antioxidant peptide.
Background
The astragalus mushroom contains most amino acids, proteins, organic acids, saccharides, alkaloids, phytosterols, flavonoids, glycosides, triterpene and the like, and the astragalus mushroom contains protein-derived antioxidant peptide which is an active peptide in organisms. In organisms, various polypeptides are obtained by hydrolyzing proteins through various chemical reactions, and some polypeptides have an antioxidant effect and can reduce the number of free radicals and reduce the influence of the free radicals on the bodies. The antioxidant peptide has high safety, no harm to human body, strong functionality, and can effectively prevent lipid oxidation. Through research, free radicals can cause cell aging and death, further cause color spots, wrinkles and certain skin diseases, and also cause food to be oxidized and rancid, so that the antioxidant peptide is applied to food processing, the shelf life of the food can be prolonged, and the damage of the free radicals to the body can be reduced.
A process method for preparing the Astragalus membranaceus mushroom protein source antioxidant peptide is lacked, the DPPH free radical clearance rate of antioxidant polypeptide in the traditional preparation method is not ideal, the processing process condition is poor, and the deep processing research and development of Astragalus membranaceus mushroom protein are influenced.
Disclosure of Invention
The invention aims to provide a preparation method of Astragalus membranaceus mushroom protein source antioxidant peptide, which aims to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a preparation method of Astragalus membranaceus mushroom protein source antioxidant peptide comprises the following steps:
s1: the method for extracting the protein of the astragalus membranaceus mushroom by an alkali-dissolving acid-precipitation method comprises the following specific steps:
1) putting the dried astragali mushroom into a grinder to grind into powder, weighing 5g of the dried astragali mushroom powder, adding 60ml of distilled water into a 100ml test tube to fully dissolve, and adjusting the pH value to 8.0 by using 0.5mol/L sodium hydroxide solution;
2) transferring the solution into two 50ml centrifuge tubes with the same specification, placing into a centrifuge, adjusting the centrifugation speed to 2700r/min, starting centrifugation, taking out after 5min, transferring the supernatant into a 100ml test tube, and adding hydrochloric acid to adjust the pH value to 4.5;
3) adjusting the centrifugation speed to 5000r/min again, beginning centrifugation, taking out after 15min, and removing the supernatant to obtain protein precipitate;
s2: the enzymolysis of the protein of the astragalus membranaceus mushroom comprises the following specific steps:
1) preparing 2% protein solution of Astragalus membranaceus mushroom, adding protease according to enzyme dosage under the conditions of pH of 7-8 and temperature of 35-45 deg.C, and hydrolyzing in a constant temperature water bath for 1-2 h;
2) inactivating enzyme in water bath at 95 deg.C for 15min, and centrifuging at 5000r/min for 15min to obtain supernatant, which is protein hydrolysate of Astragalus membranaceus mushroom;
s3: screening the protease, which comprises the following steps:
1) selecting papain, alkaline protease, neutral protease and trypsin, performing enzymolysis test at a concentration of 2% and an enzymolysis time of 2h and with an enzyme amount of 5000U/g;
2) setting the detection indexes as DPPH free radical scavenging rate and hydrolysis degree, and screening to obtain two optimal proteases;
s4: the selection of the enzyme adding mode comprises the following specific steps:
1) after the optimal two proteases are screened out, the two proteases are hydrolyzed under the respective optimal temperature and pH value, and the two proteases are mixed according to the ratio of 1: 1 and are respectively hydrolyzed under the respective optimal temperature and pH value of the two proteases under the hydrolysis condition of 2 percent concentration, 5000U/g enzyme dosage and 2 hours;
2) measuring and recording the hydrolysis degree index, and measuring and recording the DPPH free radical clearance index of the antioxidant peptide to obtain the optimal enzyme adding mode;
s5: the selection of the enzyme adding proportion comprises the following specific steps:
1) after the enzyme adding mode is determined, selecting the enzyme adding proportion of two enzymes, wherein the other conditions are that the concentration is 2%, the enzyme dosage is 5000U/g, and the time is 2 hours;
2) measuring and recording the hydrolysis degree index, and measuring and recording the DPPH free radical clearance index of the antioxidant peptide to obtain the most appropriate enzyme adding proportion;
s6: the single factor test comprises the following specific steps:
1) after determining two optimal proteases, an enzyme adding mode and an enzyme adding proportion, preparing a 2% concentration protein solution of the astragalus membranaceus, and performing a single-factor test;
2) under the conditions of temperature of 40 ℃, pH7.5 and time of 2h, researching the fluctuation situation of enzyme addition amount to DPPH free radical clearance rate and recording the fluctuation situation of hydrolysis degree and recording the fluctuation situation;
3) under the conditions of 40 ℃ of temperature, 5000U/g of enzyme amount and 2h of time, researching the fluctuation condition of pH value to DPPH free radical clearance rate and recording the fluctuation condition of the hydrolysis degree and recording the fluctuation condition;
4) under the conditions that the pH value is 7.5, the enzyme amount is 5000U/g and the time is 2h, the fluctuation condition of the temperature to the DPPH free radical clearance rate is researched and recorded, and the fluctuation condition of the hydrolysis degree is recorded and recorded;
5) under the conditions of pH value of 7.5, enzyme amount of 5000U/g and temperature of 40 ℃, researching and recording the fluctuation condition of time to DPPH free radical clearance and the fluctuation condition to hydrolysis degree;
s7: the response surface test comprises the following specific steps:
1) making statistical analysis on the single-factor test result, determining an optimal range, taking DPPH free radical clearance as a dependent variable, performing four-factor three-level tests on four factors, namely enzyme addition, temperature, pH and time, and determining a response surface test scheme according to response surface simulation software;
s8: the DPPH free radical clearance rate determination method comprises the following specific steps:
1) adding absolute ethyl alcohol into a 100ml volumetric flask, and weighing a DPPH reagent to prepare a DPPH solution with the concentration of 6.6 x 10-4 mol/L;
2) storing at low temperature and in dark environment, and diluting with anhydrous ethanol to concentration of 6.6 x 10-5mol/L, adding samples, shaking, uniformly mixing, and storing for 30min in a dark environment;
3) measuring the absorbance at 517nm once every three minutes until the absorbance value is stable, and repeating the test for three times to reduce the error;
s9: the method for measuring the hydrolysis degree comprises the following specific steps:
1) the degree of hydrolysis is determined by a pH-stat titration method, and when the protein is hydrolyzed under alkaline conditions, peptide bonds in the protein are broken, so that the pH value is reduced;
2) the degree of hydrolysis can be calculated from the amount of alkali used to adjust the pH of the solution back to the initial pH with a dilute alkali solution
S10: ABTS and clearance test, the specific steps are as follows:
1) preparing a phosphate buffer solution with pH 7.2: dissolving 1.975g of sodium chloride, 0.05g of potassium chloride, 0.06g of potassium dihydrogen phosphate and 0.45g of dipotassium hydrogen phosphate in 200ml of distilled water, uniformly stirring, measuring the pH value by using a pH meter to obtain the pH value of 7.2, and storing in a refrigerator at 4 ℃;
2) preparing a 2.45mmol/L potassium persulfate solution: taking 26.4914mg of potassium persulfate, dissolving the potassium persulfate in 40ml of phosphate buffer solution, and uniformly stirring;
3) preparing a 7mmol/L ABTS +. solution: 153.6304mg of 2, 2' -diamino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS + ·) is dissolved by 40ml of phosphate buffer solution and stirred uniformly;
4) mixing the prepared potassium persulfate solution and ABTS +. solution at a ratio of 1: 1, storing in a dark environment for 12-16h, diluting with phosphate buffer solution until the absorbance value is 0.70 + -0.02, and measuring the absorbance value at 734 nm;
s11: the total reducing force test comprises the following specific steps:
1) preparation of PBS buffer at pH 6.6: weighing 1.74g of monopotassium phosphate, 1.7g of sodium chloride and 2.7g of dipotassium phosphate, and adding distilled water to dissolve into 400ml to obtain the product;
2) measuring pH with a pH meter to obtain pH of 6.6, storing in a refrigerator at 4 deg.C, adding 2ml of solution to be measured into 2.5ml of PBS buffer solution with pH of 6.6 and 2.5ml of 1% potassium ferricyanide solution, mixing, standing in water bath at 50 deg.C for 20min, cooling, adding 1ml of 10% trichloroacetic acid solution, centrifuging at 5000r/min for 10min, taking 2.5ml of supernatant, adding 2.5ml of distilled water and 0.5ml of 0.1% ferric trichloride solution, and mixing;
3) standing for 10min, measuring absorbance at 700nm, taking 80% ethanol solution as blank group control, taking the difference between the absorbance of the solution to be measured and the blank group as the reduction capacity of the sample, and taking ascorbic acid as standard control;
s12: analysis calculation, and analysis of variance was performed on the data using Design-expert8.0.6.1 software.
Preferably, the enzyme adding ratio in the step S5 is 1: 1 respectively; 1: 2; 1: 3; 2: 1; 3: 1.
Preferably, the enzyme adding amount in the step S6 is 3000U/g, 4000U/g, 5000U/g, 6000U/g and 7000U/g respectively; the pH values are 7, 7.5, 8, 8.5 and 9 respectively; the temperatures are 30 ℃, 35 ℃, 40 ℃, 45 ℃ and 50 ℃ respectively; the time is 1h, 1.5h, 2h, 2.5h and 3h respectively.
Preferably, the DPPH radical clearance measurement and calculation formula in step S8 is represented by the following formula:
DPPH-clearance (%) {1- (a1-a2)/a0 }. 100% (1).
Preferably, the calculation formula for the determination of the degree of hydrolysis in step S9 is:
DH＝{(V*N)/(α*m*h)}*100％ (2)
wherein V (ml) ═ the volume of base consumed in the test, N (mol/L) ═ the concentration of base consumed by titration, m (g) ═ the total amount of protein involved in hydrolysis, h [20] ═ the number of gram equivalents of peptide bonds per gram of protein (7.52mmol/g), α is the average degree of dissociation of α -amino acids, formula α ═ 10pH-PK/(1+10pH-pK), pH is the pH at the beginning of the reaction, pK ═ 7.8+2400 (298-T)/298T, T ═ 273.15+ T, T is the temperature of the reaction environment.
Preferably, the formula of ABTS +. clearance rate in step S10 is:
ABTS +. clearance (%) {1- (a1-a2)/a0 }. 100% (3).
Preferably, in the step S12, a regression equation is used for calculation and analysis in the analysis of variance of the data, and the formula is:
Y(％)＝57.20-2.49A+1.89B-4.49C-0.84D-3.38AB+5.25AC+0.36AD+1.23BC-1.54BD+1.79CD-8.07A2-13.85B2-17.68C2-13.41D2 (4)
wherein Y is DPPH free radical scavenging rate, A is time, B is enzyme dosage, C is temperature, and D is pH.
Compared with the prior art, the invention has the beneficial effects that: the invention strictly controls the process steps of the preparation method of the Astragalus membranaceus mushroom protein source antioxidant peptide, takes the hydrolysis degree and the DPPH free radical clearance rate as detection indexes, screens out two optimal proteases, and further determines the enzyme adding mode and the enzyme adding proportion. Selecting four factors of enzymolysis time, enzyme addition amount, temperature and pH value to perform single-factor test to obtain three level values in the most appropriate range, performing four-factor three-level response surface test, and optimizing to obtain the most efficient process conditions: the time is 1.5h, the enzyme dosage is 5100U/g, the temperature is 40 ℃, and the pH value is 7.5. Under these conditions, the DPPH free radical clearance of the antioxidant polypeptide is 57.7%. According to the invention, the Astragalus membranaceus mushroom protein is used as a raw material, the antioxidative polypeptide is efficiently obtained in a compounding manner of two proteases, the optimal process conditions for preparing the Astragalus membranaceus mushroom protein antioxidative polypeptide are explored, a basis is provided for deep processing of the Astragalus membranaceus mushroom protein and development of the antioxidative peptide, 2% of Astragalus membranaceus mushroom protein liquid is used as a substrate, two proteases with the best effect are firstly screened by measuring the DPPH free radical clearance rate of the antioxidative peptide and the hydrolysis degree of the Astragalus membranaceus mushroom protein, the enzyme adding manner and proportion of the complex enzyme are determined, and then a single-factor test is carried out to determine the optimal range value of each factor. On the basis, the response surface method optimization analysis is carried out by taking the free radical clearance of DPPH as a response value, and the optimal process for determining the composite enzymolysis is as follows: the enzyme dosage is 5100U/g, the pH value is 7.5, the temperature is 40 ℃, the time is 1.5h, the hydrolysis is carried out under the process condition, and the DPPH free radical clearance rate detection index reaches 57.7 percent. The ABTS + scavenging ability of the antioxidant peptide prepared under the optimal process condition reaches 81.1%, the total reducing ability reaches 0.3393, and although the ABTS + scavenging ability and the total reducing ability are all weaker than the effect of ascorbic acid under the same concentration, the obtained polypeptide is also proved to have antioxidant property.
Drawings
FIG. 1 is a graph showing the effect of the type of enzyme of the present invention on the degree of hydrolysis and DPPH radical clearance;
FIG. 2 is a graph showing the effect of the enzymatic mode of the present invention on the degree of hydrolysis and DPPH radical clearance;
FIG. 3 is a graph showing the effect of enzyme addition ratio on degree of hydrolysis and DPPH radical scavenging rate in accordance with the present invention;
FIG. 4 is a graph of the effect of time on degree of hydrolysis and DPPH radical clearance according to the invention;
FIG. 5 is a graph showing the effect of enzyme dosage on degree of hydrolysis and DPPH radical clearance according to the present invention;
FIG. 6 is a graph of the effect of temperature on the degree of hydrolysis and DPPH radical scavenging rate according to the present invention;
FIG. 7 is a graph of the effect of pH on the degree of hydrolysis and DPPH radical scavenging rate in accordance with the present invention;
FIG. 8 is a graph of the response of the present invention in time and enzyme dosage interaction;
FIG. 9 is a line contour plot of the interaction of time and enzyme dosage according to the present invention;
FIG. 10 is a graph of the response of the time and temperature interaction of the present invention;
FIG. 11 is a line contour plot of the time and temperature interaction of the present invention;
FIG. 12 is a graph of the response of the time and pH interaction of the present invention;
FIG. 13 is a line contour plot of the time and pH interaction of the present invention;
FIG. 14 is a graph of the response of the interaction of enzyme dosage and temperature in accordance with the present invention;
FIG. 15 is a line contour diagram showing the interaction between enzyme addition and temperature in accordance with the present invention;
FIG. 16 is a graph of the response of the interaction between enzyme dosage and pH according to the present invention;
FIG. 17 is a line contour diagram showing the interaction between enzyme dosage and pH according to the present invention;
FIG. 18 is a graph of the response of the temperature and pH interaction of the present invention;
FIG. 19 is a line contour plot of the temperature and pH interaction of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Please refer to fig. 1-19:
a preparation method of Astragalus membranaceus mushroom protein source antioxidant peptide comprises the following steps:
s1: the method for extracting the protein of the astragalus membranaceus mushroom by an alkali-dissolving acid-precipitation method comprises the following specific steps:
1) putting the dried astragali mushroom into a grinder to grind into powder, weighing 5g of the dried astragali mushroom powder, adding 60ml of distilled water into a 100ml test tube to fully dissolve, and adjusting the pH value to 8.0 by using 0.5mol/L sodium hydroxide solution;
2) transferring the solution into two 50ml centrifuge tubes with the same specification, placing into a centrifuge, adjusting the centrifugation speed to 2700r/min, starting centrifugation, taking out after 5min, transferring the supernatant into a 100ml test tube, and adding hydrochloric acid to adjust the pH value to 4.5;
3) adjusting the centrifugation speed to 5000r/min again, beginning centrifugation, taking out after 15min, and removing the supernatant to obtain protein precipitate;
s2: the enzymolysis of the protein of the astragalus membranaceus mushroom comprises the following specific steps:
1) preparing 2% protein solution of Astragalus membranaceus mushroom, adding protease according to enzyme dosage under the conditions of pH of 7-8 and temperature of 35-45 deg.C, and hydrolyzing in a constant temperature water bath for 1-2 h;
2) inactivating enzyme in water bath at 95 deg.C for 15min, and centrifuging at 5000r/min for 15min to obtain supernatant, which is protein hydrolysate of Astragalus membranaceus mushroom;
s3: screening the protease, which comprises the following steps:
1) selecting papain, alkaline protease, neutral protease and trypsin, performing enzymolysis test at a concentration of 2% and an enzymolysis time of 2h and with an enzyme amount of 5000U/g;
2) setting the detection indexes as DPPH free radical scavenging rate and hydrolysis degree, and screening to obtain two optimal proteases;
two optimal proteases are screened through tests, as shown in figure 1, the hydrolysis capacity of neutral protease and alkaline protease on the protein of the astragalus membranaceus mushroom and the antioxidant capacity of the obtained polypeptide are higher than those of the other two proteases, so the neutral protease and the alkaline protease are selected to be compounded and continuously tested.
S4: the selection of the enzyme adding mode comprises the following specific steps:
1) after the optimal two proteases are screened out, the two proteases are hydrolyzed under the respective optimal temperature and pH value, and the two proteases are mixed according to the ratio of 1: 1 and are respectively hydrolyzed under the respective optimal temperature and pH value of the two proteases under the hydrolysis condition of 2 percent concentration, 5000U/g enzyme dosage and 2 hours;
2) measuring and recording the hydrolysis degree index, and measuring and recording the DPPH free radical clearance index of the antioxidant peptide to obtain the optimal enzyme adding mode;
the neutral alkali addition is that two proteases are mixed according to the proportion of 1: 1 and then hydrolyzed under the optimum temperature and pH value of the neutral protease, the alkaline alkali addition is that two proteases are mixed according to the proportion of 1: 1 and then hydrolyzed under the optimum temperature and pH value of the alkaline protease, as shown in figure 2, the alkaline alkali addition is higher than the neutral alkali addition in the hydrolysis degree index, but the neutral alkali addition is higher than the alkaline alkali addition in the DPPH free radical clearance index, the experiment is to seek the optimum process for preparing the antioxidant peptide, so the complex enzyme is selected to be hydrolyzed under the conditions of the optimum temperature of the neutral protease of 40 ℃ and the pH value of 7.5.
S5: the selection of the enzyme adding proportion comprises the following specific steps:
1) after the enzyme adding mode is determined, selecting the enzyme adding proportion of two enzymes, wherein the other conditions are that the concentration is 2%, the enzyme dosage is 5000U/g, and the time is 2 hours;
2) measuring and recording the hydrolysis degree index, and measuring and recording the DPPH free radical clearance index of the antioxidant peptide to obtain the most appropriate enzyme adding proportion;
the neutral protease and the alkaline protease have different protein hydrolysis abilities, so the ratio of the neutral protease to the alkaline protease when the neutral protease and the alkaline protease are compounded has a large influence on the test result, and as shown in fig. 3, the neutral protease to the alkaline protease is obviously higher than that of other groups when the neutral protease to the alkaline protease is 1: 1 in the index of the degree of hydrolysis, and the other groups are basically kept unchanged. In the index of DPPH free radical scavenging rate, the ratio of neutral protease to alkaline protease is 1: 1 and 2: 1, and the ratio of neutral protease to alkaline protease is 1: 1
S6: the single factor test comprises the following specific steps:
1) after determining two optimal proteases, an enzyme adding mode and an enzyme adding proportion, preparing a 2% concentration protein solution of the astragalus membranaceus, and performing a single-factor test;
2) under the conditions of temperature of 40 ℃, pH7.5 and time of 2h, researching the fluctuation situation of enzyme addition amount to DPPH free radical clearance rate and recording the fluctuation situation of hydrolysis degree and recording the fluctuation situation;
3) under the conditions of 40 ℃ of temperature, 5000U/g of enzyme amount and 2h of time, researching the fluctuation condition of pH value to DPPH free radical clearance rate and recording the fluctuation condition of the hydrolysis degree and recording the fluctuation condition;
4) under the conditions that the pH value is 7.5, the enzyme amount is 5000U/g and the time is 2h, the fluctuation condition of the temperature to the DPPH free radical clearance rate is researched and recorded, and the fluctuation condition of the hydrolysis degree is recorded and recorded;
5) under the conditions of pH value of 7.5, enzyme amount of 5000U/g and temperature of 40 ℃, researching and recording the fluctuation condition of time to DPPH free radical clearance and the fluctuation condition to hydrolysis degree;
as the reaction time was prolonged, the degree of hydrolysis increased first and then remained constant, peaking and settling at reaction time 2 h. Probably because the substrate and the protease fully react, the hydrolysis degree is basically kept unchanged after 2h of reaction, the DPPH free radical clearance rate is increased and then slowly reduced, the DPPH free radical clearance rate reaches a maximum value at 1.5h of reaction, and the DPPH free radical clearance rate is gradually reduced after 1.5h, so that the antioxidant polypeptide is further decomposed into amino acid or small molecular polypeptide, the antioxidant polypeptide is reduced, and the DPPH free radical clearance rate is reduced. Finally, the selection time is between 1h and 2h, see fig. 4.
With the increase of the enzyme dosage, the hydrolysis degree is increased and then kept stable basically, and the addition reaches the maximum value at 4000U/g. It is possible that the amount of substrate is constant and that the excess enzyme does not contribute much to the hydrolysis of the protein. The DPPH free radical clearance curve is increased and then decreased, a peak value is reached at the position of 5000U/g enzyme adding amount, more protein is hydrolyzed into polypeptide with oxidation resistance along with the increase of the enzyme adding amount in the period of 3000 to 5000U/g enzyme adding amount, and the Astragalus membranaceus mushroom protein is enzymolyzed into amino acid and polypeptide with small molecular weight along with the increase of the enzyme adding amount in the period of 5000 to 7000U/g enzyme adding amount, so that the quantity of the antioxidant peptide is gradually reduced. Thus, the index of DPPH scavenging free radicals decreases. Finally, the enzyme dosage was selected in the range of 4000 to 6000U/g, see FIG. 5.
The hydrolysis degree and the DPPH free radical scavenging rate are increased and then decreased along with the increase of the temperature, the hydrolysis degree and the DPPH free radical scavenging rate reach peak values when the temperature reaches 40 ℃, different proteases have the optimum temperatures, the temperature is too low, the activity of the protease is too low, the hydrolysis degree index is small, the enzymatic reaction is accelerated along with the increase of the temperature, the hydrolysis degree is increased, more antioxidant polypeptides are obtained, the temperature continues to increase, the efficiency of the enzyme is reduced, even the enzyme is inactivated, the hydrolysis degree is reduced, and the amount of the antioxidant polypeptides is small. For general consideration, the temperature is selected to be between 35-45 deg.C, see FIG. 6.
The pH value is gradually increased, the hydrolysis degree and the DPPH free radical scavenging rate are increased and then decreased, and the hydrolysis degree reaches the maximum value when the pH value is 8. The clearance of DPPH radicals reaches a maximum at pH 7.5. Since the protease has the most suitable pH, the hydrolysis ability is lowered by the distance from the optimum pH. The degree of hydrolysis is reduced, the amount of hydrolyzed protein is reduced, and the amount of antioxidant polypeptide obtained is also reduced, thereby reducing DPPH free radical scavenging rate. For general consideration, the pH range was selected to be 7-8, as shown in FIG. 7.
S7: the response surface test comprises the following specific steps:
1) making statistical analysis of the single-factor test result, determining the optimal range, taking DPPH free radical clearance as a dependent variable, performing four-factor three-level tests on four factors, namely enzyme addition, temperature, pH and time, and determining a response surface test scheme according to response surface simulation software, wherein the following table 1 shows that:
s8: the DPPH free radical clearance rate determination method comprises the following specific steps:
1) adding absolute ethyl alcohol into a 100ml volumetric flask, and weighing a DPPH reagent to prepare a DPPH solution with the concentration of 6.6 x 10-4 mol/L;
2) storing at low temperature and in dark environment, and diluting with anhydrous ethanol to concentration of 6.6 x 10-5mol/L, adding samples, shaking, uniformly mixing, and storing for 30min in a dark environment;
3) measuring the absorbance at 517nm once every three minutes until the absorbance value is stable, and repeating the test for three times to reduce the error;
DPPH clearance assay loading table 2 is as follows:
s9: the method for measuring the hydrolysis degree comprises the following specific steps:
1) the degree of hydrolysis is determined by a pH-stat titration method, and when the protein is hydrolyzed under alkaline conditions, peptide bonds in the protein are broken, so that the pH value is reduced;
2) the degree of hydrolysis can be calculated from the amount of alkali used to adjust the pH of the solution back to the initial pH with a dilute alkali solution
S10: ABTS and clearance test, the specific steps are as follows:
1) preparing a phosphate buffer solution with pH 7.2: dissolving 1.975g of sodium chloride, 0.05g of potassium chloride, 0.06g of potassium dihydrogen phosphate and 0.45g of dipotassium hydrogen phosphate in 200ml of distilled water, uniformly stirring, measuring the pH value by using a pH meter to obtain the pH value of 7.2, and storing in a refrigerator at 4 ℃;
2) preparing a 2.45mmol/L potassium persulfate solution: taking 26.4914mg of potassium persulfate, dissolving the potassium persulfate in 40ml of phosphate buffer solution, and uniformly stirring;
3) preparing a 7mmol/L ABTS +. solution: 153.6304mg of 2, 2' -diamino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS + ·) is dissolved by 40ml of phosphate buffer solution and stirred uniformly;
4) mixing the prepared potassium persulfate solution and ABTS +. solution at a ratio of 1: 1, storing in a dark environment for 12-16h, diluting with phosphate buffer solution until the absorbance value is 0.70 + -0.02, and measuring the absorbance value at 734 nm;
ABTS loading table 3 is as follows:
s11: the total reducing force test comprises the following specific steps:
1) preparation of PBS buffer at pH 6.6: weighing 1.74g of monopotassium phosphate, 1.7g of sodium chloride and 2.7g of dipotassium phosphate, and adding distilled water to dissolve into 400ml to obtain the product;
2) measuring pH with a pH meter to obtain pH of 6.6, storing in a refrigerator at 4 deg.C, adding 2ml of solution to be measured into 2.5ml of PBS buffer solution with pH of 6.6 and 2.5ml of 1% potassium ferricyanide solution, mixing, standing in water bath at 50 deg.C for 20min, cooling, adding 1ml of 10% trichloroacetic acid solution, centrifuging at 5000r/min for 10min, taking 2.5ml of supernatant, adding 2.5ml of distilled water and 0.5ml of 0.1% ferric trichloride solution, and mixing;
3) standing for 10min, measuring absorbance at 700nm, taking 80% ethanol solution as blank group control, taking the difference between the absorbance of the solution to be measured and the blank group as the reduction capacity of the sample, and taking ascorbic acid as standard control;
s12: analysis calculation, and analysis of variance was performed on the data using Design-expert8.0.6.1 software.
In order to obtain the optimal process for preparing the Astragalus membranaceus mushroom protein antioxidant peptide, the DPPH free radical clearance is taken as an index, four factors, namely enzymolysis time, enzyme addition amount, temperature and pH, which have great influence on the preparation of the antioxidant peptide are selected to perform four-factor three-level response surface analysis, the optimal extraction process for preparing the antioxidant peptide is explored, and the test data and results are shown in the following table 4:
specifically, the enzyme adding ratio in the step S5 is 1: 1 respectively; 1: 2; 1: 3; 2: 1; 3: 1.
Specifically, the enzyme adding amount in the step S6 is 3000U/g, 4000U/g, 5000U/g, 6000U/g and 7000U/g respectively; the pH values are 7, 7.5, 8, 8.5 and 9 respectively; the temperatures are 30 ℃, 35 ℃, 40 ℃, 45 ℃ and 50 ℃ respectively; the time is 1h, 1.5h, 2h, 2.5h and 3h respectively.
Specifically, the DPPH radical clearance measurement and calculation formula in step S8 is represented by the following formula:
DPPH-clearance (%) {1- (a1-a2)/a0 }. 100% (1).
Specifically, the calculation formula for the determination of the degree of hydrolysis in step S9 is:
DH＝{(V*N)/(α*m*h)}*100％ (2)
wherein V (ml) ═ the volume of base consumed in the test, N (mol/L) ═ the concentration of base consumed by titration, m (g) ═ the total amount of protein involved in hydrolysis, h [20] ═ the number of gram equivalents of peptide bonds per gram of protein (7.52mmol/g), α is the average degree of dissociation of α -amino acids, formula α ═ 10pH-PK/(1+10pH-pK), pH is the pH at the beginning of the reaction, pK ═ 7.8+2400 (298-T)/298T, T ═ 273.15+ T, T is the temperature of the reaction environment.
Specifically, the formula of ABTS +. clearance rate in step S10 is as follows:
ABTS +. clearance (%) {1- (a1-a2)/a0 }. 100% (3).
Specifically, in the step S12, a regression equation is used for calculation and analysis in the analysis of variance of the data, and the formula is as follows:
Y(％)＝57.20-2.49A+1.89B-4.49C-0.84D-3.38AB+5.25AC+0.36AD+1.23BC-1.54BD+1.79CD-8.07A2-13.85B2-17.68C2-13.41D2 (4)
wherein Y is DPPH free radical scavenging rate, A is time, B is enzyme dosage, C is temperature, and D is pH.
Regression model and analysis of variance table 5 is as follows:
TABLE 5 regression model and analysis of variance
Note: significant differences (p < 0.05); differences (p < 0.01) were very significant.
As can be seen from Table 5, p < 0.0001 for the model indicates that the model reaches a very significant level, while the mismatch term > 0.05, which is not significant, indicates that no mismatch factor exists, and the model is suitable. The determination coefficient R2 ═ 0.9943[28], indicating that the model has a high degree of fit to the data obtained by the test, and R2Adj ═ 0.9887, indicating that this model can account for a response value change of 98.87%, and that only a change of 1.13% cannot be accounted for by this model. CV is less than 10%, which shows that the model has high reliability and good precision. Therefore, the model can be used for predicting and explaining the DPPH free radical clearance rate of the astragalus membranaceus mushroom proteolytic liquid.
In this model, the effects of A, B, C were all very significant, with D being significant. Interactive items AB and AC are very significant, BD and CD are significant, and AD and BC are not significant. The quadratic terms are extremely remarkable, the F value in the variance analysis of the regression equation can be used for detecting the influence of independent variables on dependent variables, the larger the numerical value is, the larger the influence is, and according to the table 4, the influence level of each factor is known to be C > A > B > D.
Results of the Oxidation resistance test
The ABTS +. clearance rate of the obtained protein hydrolysate of the astragalus membranaceus mushroom is 81.1 percent, and the ABTS +. clearance rate of the ascorbic acid is 100 percent; the total reducing power of the protein hydrolysate of the astragalus membranaceus mushroom is 0.3393, and the total reducing power of the ascorbic acid is 2.8721. The polypeptide obtained from the protein hydrolysate of Astragalus membranaceus mushroom has antioxidant property, as shown in Table 6.
4 kinds of common proteases are used for hydrolyzing the protein of the astragalus membranaceus mushroom, the hydrolysis degree and the DPPH free radical clearance rate are used as detection indexes, two kinds of best proteases are firstly screened out, the enzyme adding mode and the enzyme adding proportion of the compound enzyme are further determined, then four factors of enzymolysis time, enzyme adding quantity, enzymolysis temperature and pH value are selected for single factor test, the most appropriate range value of the single factor test is obtained, finally, a four-factor three-level response surface test is carried out by taking the DPPH free radical clearance rate as a response value, and the most efficient process conditions are obtained through optimization: the enzymolysis time is 1.5h, the enzyme dosage is 5100U/g, the enzymolysis temperature is 40 ℃, and the pH value is 7.5. Under the condition, the DPPH free radical clearance rate of the obtained antioxidant polypeptide is 57.7%. The experiment provides the best process condition for preparing the Astragalus membranaceus mushroom protein antioxidant peptide and provides a theoretical basis for further researching the antioxidant property of the Astragalus membranaceus mushroom protein antioxidant peptide
In summary, the following steps: the invention strictly controls the process steps of the preparation method of the Astragalus membranaceus mushroom protein source antioxidant peptide, takes the hydrolysis degree and the DPPH free radical clearance rate as detection indexes, screens out two optimal proteases, and further determines the enzyme adding mode and the enzyme adding proportion. Selecting four factors of enzymolysis time, enzyme addition amount, temperature and pH value to perform single-factor test to obtain three level values in the most appropriate range, performing four-factor three-level response surface test, and optimizing to obtain the most efficient process conditions: the time is 1.5h, the enzyme dosage is 5100U/g, the temperature is 40 ℃, and the pH value is 7.5. Under these conditions, the DPPH free radical clearance of the antioxidant polypeptide is 57.7%. According to the invention, the Astragalus membranaceus mushroom protein is used as a raw material, the antioxidative polypeptide is efficiently obtained in a compounding manner of two proteases, the optimal process conditions for preparing the Astragalus membranaceus mushroom protein antioxidative polypeptide are explored, a basis is provided for deep processing of the Astragalus membranaceus mushroom protein and development of the antioxidative peptide, 2% of Astragalus membranaceus mushroom protein liquid is used as a substrate, two proteases with the best effect are firstly screened by measuring the DPPH free radical clearance rate of the antioxidative peptide and the hydrolysis degree of the Astragalus membranaceus mushroom protein, the enzyme adding manner and proportion of the complex enzyme are determined, and then a single-factor test is carried out to determine the optimal range value of each factor. On the basis, the response surface method optimization analysis is carried out by taking the free radical clearance of DPPH as a response value, and the optimal process for determining the composite enzymolysis is as follows: the enzyme dosage is 5100U/g, the pH value is 7.5, the temperature is 40 ℃, the time is 1.5h, the hydrolysis is carried out under the process condition, and the DPPH free radical clearance rate detection index reaches 57.7 percent. The ABTS + scavenging ability of the antioxidant peptide prepared under the optimal process condition reaches 81.1%, the total reducing ability reaches 0.3393, and although the ABTS + scavenging ability and the total reducing ability are all weaker than the effect of ascorbic acid under the same concentration, the obtained polypeptide is also proved to have antioxidant property.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (7)
1. A preparation method of Astragalus membranaceus mushroom protein source antioxidant peptide is characterized by comprising the following steps: the method comprises the following steps:
s1: the method for extracting the protein of the astragalus membranaceus mushroom by an alkali-dissolving acid-precipitation method comprises the following specific steps:
1) putting the dried astragali mushroom into a grinder to grind into powder, weighing 5g of the dried astragali mushroom powder, adding 60ml of distilled water into a 100ml test tube to fully dissolve, and adjusting the pH value to 8.0 by using 0.5mol/L sodium hydroxide solution;
2) transferring the solution into two 50ml centrifuge tubes with the same specification, placing into a centrifuge, adjusting the centrifugation speed to 2700r/min, starting centrifugation, taking out after 5min, transferring the supernatant into a 100ml test tube, and adding hydrochloric acid to adjust the pH value to 4.5;
3) adjusting the centrifugation speed to 5000r/min again, beginning centrifugation, taking out after 15min, and removing the supernatant to obtain protein precipitate;
s2: the enzymolysis of the protein of the astragalus membranaceus mushroom comprises the following specific steps:
1) preparing 2% protein solution of Astragalus membranaceus mushroom, adding protease according to enzyme dosage under the conditions of pH of 7-8 and temperature of 35-45 deg.C, and hydrolyzing in a constant temperature water bath for 1-2 h;
2) inactivating enzyme in water bath at 95 deg.C for 15min, and centrifuging at 5000r/min for 15min to obtain supernatant, which is protein hydrolysate of Astragalus membranaceus mushroom;
s3: screening the protease, which comprises the following steps:
1) selecting papain, alkaline protease, neutral protease and trypsin, performing enzymolysis test at a concentration of 2% and an enzymolysis time of 2h and with an enzyme amount of 5000U/g;
2) setting the detection indexes as DPPH free radical scavenging rate and hydrolysis degree, and screening to obtain two optimal proteases;
s4: the selection of the enzyme adding mode comprises the following specific steps:
1) after the optimal two proteases are screened out, the two proteases are hydrolyzed under the respective optimal temperature and pH value, and the two proteases are mixed according to the ratio of 1: 1 and are respectively hydrolyzed under the respective optimal temperature and pH value of the two proteases under the hydrolysis condition of 2 percent concentration, 5000U/g enzyme dosage and 2 hours;
2) measuring and recording the hydrolysis degree index, and measuring and recording the DPPH free radical clearance index of the antioxidant peptide to obtain the optimal enzyme adding mode;
s5: the selection of the enzyme adding proportion comprises the following specific steps:
1) after the enzyme adding mode is determined, selecting the enzyme adding proportion of two enzymes, wherein the other conditions are that the concentration is 2%, the enzyme dosage is 5000U/g, and the time is 2 hours;
2) measuring and recording the hydrolysis degree index, and measuring and recording the DPPH free radical clearance index of the antioxidant peptide to obtain the most appropriate enzyme adding proportion;
s6: the single factor test comprises the following specific steps:
1) after determining two optimal proteases, an enzyme adding mode and an enzyme adding proportion, preparing a 2% concentration protein solution of the astragalus membranaceus, and performing a single-factor test;
2) under the conditions of temperature of 40 ℃, pH7.5 and time of 2h, researching the fluctuation situation of enzyme addition amount to DPPH free radical clearance rate and recording the fluctuation situation of hydrolysis degree and recording the fluctuation situation;
3) under the conditions of 40 ℃ of temperature, 5000U/g of enzyme amount and 2h of time, researching the fluctuation condition of pH value to DPPH free radical clearance rate and recording the fluctuation condition of the hydrolysis degree and recording the fluctuation condition;
4) under the conditions that the pH value is 7.5, the enzyme amount is 5000U/g and the time is 2h, the fluctuation condition of the temperature to the DPPH free radical clearance rate is researched and recorded, and the fluctuation condition of the hydrolysis degree is recorded and recorded;
5) under the conditions of pH value of 7.5, enzyme amount of 5000U/g and temperature of 40 ℃, researching and recording the fluctuation condition of time to DPPH free radical clearance and the fluctuation condition to hydrolysis degree;
s7: the response surface test comprises the following specific steps:
1) making statistical analysis on the single-factor test result, determining an optimal range, taking DPPH free radical clearance as a dependent variable, performing four-factor three-level tests on four factors, namely enzyme addition, temperature, pH and time, and determining a response surface test scheme according to response surface simulation software;
s8: the DPPH free radical clearance rate determination method comprises the following specific steps:
1) adding absolute ethyl alcohol into a 100ml volumetric flask, and weighing a DPPH reagent to prepare a DPPH solution with the concentration of 6.6 x 10-4 mol/L;
2) storing at low temperature and in dark environment, and diluting with anhydrous ethanol to concentration of 6.6 x 10-5mol/L, adding samples, shaking, uniformly mixing, and storing for 30min in a dark environment;
3) measuring the absorbance at 517nm once every three minutes until the absorbance value is stable, and repeating the test for three times to reduce the error;
s9: the method for measuring the hydrolysis degree comprises the following specific steps:
1) the degree of hydrolysis is determined by a pH-stat titration method, and when the protein is hydrolyzed under alkaline conditions, peptide bonds in the protein are broken, so that the pH value is reduced;
2) the degree of hydrolysis can be calculated from the amount of alkali used to adjust the pH of the solution back to the initial pH with a dilute alkali solution
S10: ABTS and clearance test, the specific steps are as follows:
1) preparing a phosphate buffer solution with pH 7.2: dissolving 1.975g of sodium chloride, 0.05g of potassium chloride, 0.06g of potassium dihydrogen phosphate and 0.45g of dipotassium hydrogen phosphate in 200ml of distilled water, uniformly stirring, measuring the pH value by using a pH meter to obtain the pH value of 7.2, and storing in a refrigerator at 4 ℃;
2) preparing a 2.45mmol/L potassium persulfate solution: taking 26.4914mg of potassium persulfate, dissolving the potassium persulfate in 40ml of phosphate buffer solution, and uniformly stirring;
3) preparing a 7mmol/L ABTS +. solution: 153.6304mg of 2, 2' -diamino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS + ·) is dissolved by 40ml of phosphate buffer solution and stirred uniformly;
4) mixing the prepared potassium persulfate solution and ABTS +. solution at a ratio of 1: 1, storing in a dark environment for 12-16h, diluting with phosphate buffer solution until the absorbance value is 0.70 + -0.02, and measuring the absorbance value at 734 nm;
s11: the total reducing force test comprises the following specific steps:
1) preparation of PBS buffer at pH 6.6: weighing 1.74g of monopotassium phosphate, 1.7g of sodium chloride and 2.7g of dipotassium phosphate, and adding distilled water to dissolve into 400ml to obtain the product;
2) measuring pH with a pH meter to obtain pH of 6.6, storing in a refrigerator at 4 deg.C, adding 2ml of solution to be measured into 2.5ml of PBS buffer solution with pH of 6.6 and 2.5ml of 1% potassium ferricyanide solution, mixing, standing in water bath at 50 deg.C for 20min, cooling, adding 1ml of 10% trichloroacetic acid solution, centrifuging at 5000r/min for 10min, taking 2.5ml of supernatant, adding 2.5ml of distilled water and 0.5ml of 0.1% ferric trichloride solution, and mixing;
3) standing for 10min, measuring absorbance at 700nm, taking 80% ethanol solution as blank group control, taking the difference between the absorbance of the solution to be measured and the blank group as the reduction capacity of the sample, and taking ascorbic acid as standard control;
s12: analysis calculation, and analysis of variance was performed on the data using Design-expert8.0.6.1 software.
2. The method for preparing the antioxidative peptide derived from the protein of Astragalus membranaceus mushroom according to claim 1, wherein the method comprises the following steps: the enzyme adding ratio in the step S5 is 1: 1 respectively; 1: 2; 1: 3; 2: 1; 3: 1.
3. The method for preparing the antioxidative peptide derived from the protein of Astragalus membranaceus mushroom according to claim 1, wherein the method comprises the following steps: the enzyme adding amount in the step S6 is 3000U/g, 4000U/g, 5000U/g, 6000U/g and 7000U/g respectively; the pH values are 7, 7.5, 8, 8.5 and 9 respectively; the temperatures are 30 ℃, 35 ℃, 40 ℃, 45 ℃ and 50 ℃ respectively; the time is 1h, 1.5h, 2h, 2.5h and 3h respectively.
4. The method for preparing the antioxidative peptide derived from the protein of Astragalus membranaceus mushroom according to claim 1, wherein the method comprises the following steps: the measurement and calculation formula of the DPPH free radical clearance in the step S8 is represented by the following formula:
DPPH-clearance (%) {1- (a1-a2)/a0 }. 100% (1).
5. The method for preparing the antioxidative peptide derived from the protein of Astragalus membranaceus mushroom according to claim 1, wherein the method comprises the following steps: the calculation formula for the determination of the degree of hydrolysis in step S9 is:
DH＝{(V*N)/(α*m*h)}*100％ (2)
where v (ml) is the volume of base consumed by the test; n (mol/L) ═ concentration of consumed base by titration; m (g) total amount of protein participating in hydrolysis; h is[20]The number of gram equivalents of peptide bonds per gram of protein (7.52mmol/g), α is the average degree of dissociation of α -amino acids, and the formula is α -10pH-PK/(1+10pH-pK) (ii) a The pH is the initial pH of the reaction, pK ═ 7.8+2400 (298-T)/298T, T ═ 273.15+ T, and T is the temperature of the reaction environment.
6. The method for preparing the antioxidative peptide derived from the protein of Astragalus membranaceus mushroom according to claim 1, wherein the method comprises the following steps: the formula of ABTS +. clearance rate in step S10 is:
ABTS +. clearance (%) {1- (a1-a2)/a0 }. 100% (3).
7. The method for preparing the antioxidative peptide derived from the protein of Astragalus membranaceus mushroom according to claim 1, wherein the method comprises the following steps: in the step S12, a regression equation is used for calculation and analysis in the analysis of variance of the data, and the formula is as follows:
Y(％)＝57.20-2.49A+1.89B-4.49C-0.84D-3.38AB+5.25AC+0.36AD+1.23BC-1.54BD+1.79CD-8.07A2-13.85B2-17.68C2-13.41D2 (4)
wherein Y is DPPH free radical scavenging rate, A is time, B is enzyme dosage, C is temperature, and D is pH.
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| CN114027494A (en) * | 2021-12-10 | 2022-02-11 | 上海市农业科学院 | Flavor base material and preparation method and application thereof |
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| CN111793666A (en) * | 2020-07-06 | 2020-10-20 | 安徽工程大学宣城产业技术研究院有限公司 | Tea seed polypeptide, enzymolysis preparation method thereof and antioxidant activity determination method |
| CN114027494A (en) * | 2021-12-10 | 2022-02-11 | 上海市农业科学院 | Flavor base material and preparation method and application thereof |
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