CN113774102B - Method for optimizing apostichopus japonicus essence enzymolysis technology and researching antioxidation activity of enzymolysis liquid - Google Patents
Method for optimizing apostichopus japonicus essence enzymolysis technology and researching antioxidation activity of enzymolysis liquid Download PDFInfo
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Abstract
The invention provides a processing technology for optimizing apostichopus japonicus refined enzymolysis by a response surface method, wherein the optimal hydrolysis condition of the processing technology for optimizing apostichopus japonicus refined enzymolysis by the response surface method is that the temperature is 70 ℃, the enzyme adding amount is 4.4%, and the hydrolysis time is 4 hours. The invention takes apostichopus japonicus essence in gonad maturation period as raw material, optimizes the enzymolysis process of apostichopus japonicus essence by using a response surface method, and analyzes the antioxidant activity of the extracted apostichopus japonicus essence enzymolysis liquid.
Description
Technical Field
The invention relates to the technical field of recycling of apostichopus japonicus byproducts, in particular to an optimization method of an apostichopus japonicus essence enzymolysis process and a research method of antioxidation activity of enzymolysis liquid.
Background
Apostichopus japonicus is a sea cucumber mainly distributed in China, russian, korea and Japan coastal, and is an important sea food and drug resource. The extraction and utilization of bioactive substances in apostichopus japonicus is a research field which is always concerned, and is mainly focused on the research of body wall active substances of apostichopus japonicus. Gonad and intestinal tissues belong to viscera of apostichopus japonicus and are often discarded as byproducts in the processing process of the apostichopus japonicus and are not effectively utilized, so that resource waste and environmental pollution are caused.
The apostichopus japonicus gonad comprises ovum and sperm, and the ovum and the sperm are easy to mix together in the collecting process, and researches show that the apostichopus japonicus gonad is not only rich in polysaccharide, but also rich in protein, fatty acid and other active ingredients, wherein the protein is a source of high-quality polypeptide. At present, the research on apostichopus japonicus mainly focuses on the composition components and functional activities of body walls, the research on the gonads of apostichopus japonicus is very little, and the activity research on apostichopus japonicus essence is not reported at all.
Related studies have shown that polypeptides of different molecular weights generally exhibit significantly different antioxidant activity. O (O) 2 -· Is the first free radical in all oxygen free radicals, can generate other oxygen free radicals through a series of reactions, and has strong oxidizing capability; OH is the most chemically active species of active oxygen, which is the most harmful among the active oxygen, and reacts with almost all substances in the living body. Thus, we tested three polypeptide pairs O collected 2 -· And OH scavenging ability, thereby examining their antioxidant activity.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for optimizing the apostichopus japonicus essence enzymolysis process and researching the antioxidant activity of enzymolysis liquid.
The invention solves the technical problems by adopting the following technical scheme:
the invention provides a processing technology for optimizing apostichopus japonicus refined enzymolysis by a response surface method, wherein the optimal hydrolysis condition of the processing technology for optimizing apostichopus japonicus refined enzymolysis by the response surface method is that the temperature is 70 ℃, the enzyme adding amount is 4.4%, and the hydrolysis time is 4 hours.
Preferably, the operation steps of the specific processing technology for optimizing the apostichopus japonicus essence enzymolysis by the response surface method are as follows:
step one, enzymolysis of apostichopus japonicus essence: cleaning apostichopus japonicus essence, draining water, grinding and homogenizing by a colloid mill, accurately weighing 10g of apostichopus japonicus essence homogenate, adding 0.5-0.7g of papain, uniformly mixing, performing enzymolysis in a constant-temperature water bath, immediately taking out the apostichopus japonicus essence after enzymolysis is completed, and placing the enzymolysis liquid in the water bath at 100 ℃ to inactivate enzymes for 10min;
step two, measuring the degree of hydrolysis: measuring the hydrolysis degree according to a trichloroacetic acid (TCA) method, adding 1mL of enzymolysis liquid into 1mL of 10g/100mL of TCA, mixing and oscillating, standing for 10min, centrifuging for 10min at 10000r/min, taking supernatant, measuring the content of soluble protein by a biuret method, measuring the content of total protein by a Kjeldahl nitrogen method, and calculating the hydrolysis Degree (DH) according to the following formula:
DH＝(ρ 1 -ρ 2) /(ρ 0 -ρ 2 )×100 (1)
step three, single factor research: in order to examine the influence of three factors of enzymolysis temperature, enzyme adding amount and enzymolysis time on the hydrolysis degree, firstly, a single factor experiment is carried out, and basic conditions of enzymolysis are formulated as follows: the enzymolysis temperature is 65 ℃, the enzyme adding amount (E/S) is 3 percent, and the time is 5 hours; fixing two conditions, changing the other condition, and respectively observing different influencing factors;
step four, optimizing hydrolysis conditions by a response surface method: on the basis of a single-factor experiment, the hydrolysis degree is taken as a measurement index, three factors of enzymolysis temperature, enzyme adding amount and time are selected, a three-factor and three-level response surface experiment is designed, papain is selected for optimizing enzymolysis conditions, and according to the single-factor experiment result, the influence of three factors on the hydrolysis degree is analyzed by adopting a Box-Behnken design experiment, the experiment times are 17, the factor analysis part experiment times are 12, and the center point repeated experiment times are 5.
Preferably, the calculated Degree of Hydrolysis (DH) is of the formula: ρ 1 The content of soluble protein (mg/mL) in the enzymolysis liquid after the reaction; ρ 2 Is the content (mg/mL) of soluble protein in the apostichopus japonicus selenka before reaction; ρ 0 Is the total protein content (mg/mL) in the apostichopus japonicus essence.
Preferably, the range of values of each factor in the single factor study is as follows: the enzymolysis temperatures are respectively 50, 60, 65, 70 and 80 ℃, the enzyme adding amounts are respectively 1%, 2%, 3%, 4% and 5%, and the time is respectively 3, 4, 5, 6 and 7 hours.
A method for preparing polypeptides with three different molecular weight ranges by separating from enzymolysis liquid by using Pall minimum ultrafiltration system.
Preferably, the specific preparation method comprises the following steps:
step one, preparation of a step peptide: diluting the apostichopus japonicus essence enzymatic hydrolysate with distilled water twice, centrifuging at 10000r/min for 10min, collecting supernatant, filtering with common filter paper, and sequentially filtering with microporous filter membranes of 0.45 μm and 0.22 μm; then a Pall minimum ultrafiltration system is adopted, the filtered apostichopus japonicus essence enzymatic hydrolysate is added into an ultrafiltration cup, a tangential flow membrane package is arranged, ultrafiltration is carried out under the control of the pressure of 20-30psig, and filtrate and trapped fluid are respectively collected;
step two, selecting a membrane package with the molecular weight cutoff of 10kDa for ultrafiltration, collecting filtrate when the enzymatic hydrolysate in an ultrafiltration cup is left for 1/5 after the ultrafiltration begins, supplementing distilled water in the ultrafiltration cup to the initial volume, ultrafiltering again, stopping ultrafiltration after repeating for three times, and collecting filtrate (polypeptide with the molecular weight of less than 10 kDa) and retentate (protein with the molecular weight of more than 10 kDa) each time; and then respectively selecting tangential flow membrane bags with the molecular weight cut-off of 5kDa and 1kDa, finally preparing three polypeptides with different molecular weight ranges, and carrying out vacuum freeze-drying treatment on the three collected polypeptide solutions.
Antioxidant activity research of the peptide ladder extracted from the enzymolysis liquid, namely, the peptide ladder eliminates O 2 -· And OH capability.
The antioxidant activity research of the step peptide extracted from the enzymolysis liquid is characterized by comprising the following specific steps:
step one, clean O 2 -· Capacity measurement:
simulating xanthine and xanthine oxidase reaction system in organism to produce O 2 -· Adding electron transfer material and Griess developer to make the reaction system appear purple, measuring its absorbance at 550nm with spectrophotometer, and calculating polypeptide pair O 2 -· In the reaction system, O is inhibited by reacting for 40min at 37 ℃ per liter of sample 2 -· Equivalent to 1mg of vitamin C inhibited O 2 -· The change value is a vitality unit; calculated according to formula (2):
step two, measuring the OH removal capacity:
generating OH, H by Fenton reaction 2 O 2 In proportion to the amount of OH generated by the reaction, and after the electron acceptor is given, the color is developed by Griess reagent to form red substance, and the color is in proportion to the amount of OH; sequentially adding samples to a spectrophotometer 55Measuring the absorbance value of each tube at 0 nm; every milliliter of sample is reacted for 1min at 37 ℃ to ensure H in a reaction system 2 O 2 The concentration is reduced by 1mmol/L to be one inhibition-OH capacity unit; calculated according to formula (3):
compared with the prior art, the invention has the following beneficial effects:
the invention obtains the enzymolysis temperature, enzyme adding amount and time through a series of operation processes, and has obvious influence on the hydrolysis degree of apostichopus japonicus essence. The optimal hydrolysis condition is determined by a single factor experiment and a response surface method, the temperature is 70 ℃, the enzyme adding amount is 4.4%, and the hydrolysis time is 4 hours. Three polypeptides with different molecular weight ranges separated from enzymolysis liquid, and the polypeptides are used for preparing O 2 -· And OH show a relatively good scavenging ability. Among them, polypeptide P1 with molecular weight less than 1kDa has the best antioxidant activity. The research can provide a certain reference basis for the high value of the apostichopus japonicus byproduct.
Drawings
FIG. 1 is a graph showing the effect of the enzyme hydrolysis temperature, (b) the enzyme addition amount and (c) the time on the degree of hydrolysis;
FIG. 2 (a) influence of temperature and enzyme addition, (b) influence of temperature and time (c) enzyme addition and time interaction on hydrolysis degree;
FIG. 3 (a) Apostichopus japonicus selenka essence polypeptide clearing O 2 -· Capability;
FIG. 3 (b) apostichopus japonicus selenka polypeptide scavenging OH capacity.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1:
in the processing technology for the apostichopus japonicus refined enzymolysis by using the response surface method, the optimal hydrolysis condition of the processing technology for the apostichopus japonicus refined enzymolysis by using the response surface method is that the temperature is 70 ℃, the enzyme adding amount is 4.4%, and the hydrolysis time is 4 hours.
The operation steps of the specific processing technology of the response surface method optimized apostichopus japonicus refined enzymolysis are as follows:
step one, enzymolysis of apostichopus japonicus essence: cleaning apostichopus japonicus essence, draining water, grinding and homogenizing by a colloid mill, accurately weighing 10g of apostichopus japonicus essence homogenate, adding 0.5-0.7g of papain, uniformly mixing, performing enzymolysis in a constant-temperature water bath, immediately taking out the apostichopus japonicus essence after enzymolysis is completed, and placing the enzymolysis liquid in the water bath at 100 ℃ to inactivate enzymes for 10min;
step two, measuring the degree of hydrolysis: measuring the hydrolysis degree according to a trichloroacetic acid (TCA) method, adding 1mL of enzymolysis liquid into 1mL of 10g/100mL of TCA, mixing and oscillating, standing for 10min, centrifuging for 10min at 10000r/min, taking supernatant, measuring the content of soluble protein by a biuret method, measuring the content of total protein by a Kjeldahl nitrogen method, and calculating the hydrolysis Degree (DH) according to the following formula:
DH＝(ρ 1 -ρ 2) /(ρ 0 -ρ 2 )×100 (1)
step three, single factor research: in order to examine the influence of three factors of enzymolysis temperature, enzyme adding amount and enzymolysis time on the hydrolysis degree, firstly, a single factor experiment is carried out, and basic conditions of enzymolysis are formulated as follows: the enzymolysis temperature is 65 ℃, the enzyme adding amount (E/S) is 3 percent, and the time is 5 hours; fixing two conditions, changing the other condition, and respectively observing different influencing factors;
step four, optimizing hydrolysis conditions by a response surface method: on the basis of a single-factor experiment, the hydrolysis degree is taken as a measurement index, three factors of enzymolysis temperature, enzyme adding amount and time are selected, a three-factor and three-level response surface experiment is designed, papain is selected for optimizing enzymolysis conditions, and according to the single-factor experiment result, the influence of three factors on the hydrolysis degree is analyzed by adopting a Box-Behnken design experiment, the experiment times are 17, the factor analysis part experiment times are 12, and the center point repeated experiment times are 5.
This embodimentIn the formula (DH): ρ 1 The content of soluble protein (mg/mL) in the enzymolysis liquid after the reaction; ρ 2 Is the content (mg/mL) of soluble protein in the apostichopus japonicus selenka before reaction; ρ 0 Is the total protein content (mg/mL) in the apostichopus japonicus essence.
The range of values of each factor in the single factor study in this embodiment is as follows: the enzymolysis temperatures are respectively 50, 60, 65, 70 and 80 ℃, the enzyme adding amounts are respectively 1%, 2%, 3%, 4% and 5%, and the time is respectively 3, 4, 5, 6 and 7 hours.
In this embodiment, a Pall minimum ultrafiltration system is used to separate polypeptides in three different molecular weight ranges from the enzymatic hydrolysate.
The specific preparation method of the embodiment is as follows:
step one, preparation of a step peptide: diluting the apostichopus japonicus essence enzymatic hydrolysate with distilled water twice, centrifuging at 10000r/min for 10min, collecting supernatant, filtering with common filter paper, and sequentially filtering with microporous filter membranes of 0.45 μm and 0.22 μm; then a Pall minimum ultrafiltration system is adopted, the filtered apostichopus japonicus essence enzymatic hydrolysate is added into an ultrafiltration cup, a tangential flow membrane package is arranged, ultrafiltration is carried out under the control of the pressure of 20-30psig, and filtrate and trapped fluid are respectively collected;
step two, selecting a membrane package with the molecular weight cutoff of 10kDa for ultrafiltration, collecting filtrate when the enzymatic hydrolysate in an ultrafiltration cup is left for 1/5 after the ultrafiltration begins, supplementing distilled water in the ultrafiltration cup to the initial volume, ultrafiltering again, stopping ultrafiltration after repeating for three times, and collecting filtrate (polypeptide with the molecular weight of less than 10 kDa) and retentate (protein with the molecular weight of more than 10 kDa) each time; and then respectively selecting tangential flow membrane bags with the molecular weight cut-off of 5kDa and 1kDa, finally preparing three polypeptides with different molecular weight ranges, and carrying out vacuum freeze-drying treatment on the three collected polypeptide solutions.
The antioxidant activity of the peptide ladder extracted from the enzymatic hydrolysate is studied in this example, namely, the peptide ladder eliminates O 2 -· And OH capability.
The antioxidant activity research of the step peptide extracted from the enzymolysis liquid is characterized by comprising the following specific steps:
step one, clean O 2 -· Capacity measurement:
simulating xanthine and xanthine oxidase reaction system in organism to produce O 2 -· Adding electron transfer material and Griess developer to make the reaction system appear purple, measuring its absorbance at 550nm with spectrophotometer, and calculating polypeptide pair O 2 -· In the reaction system, O is inhibited by reacting for 40min at 37 ℃ per liter of sample 2 -· Equivalent to 1mg of vitamin C inhibited O 2 -· The change value is a vitality unit; calculated according to formula (2):
step two, measuring the OH removal capacity:
generating OH, H by Fenton reaction 2 O 2 In proportion to the amount of OH generated by the reaction, and after the electron acceptor is given, the color is developed by Griess reagent to form red substance, and the color is in proportion to the amount of OH; sequentially adding samples, and measuring the absorbance value of each tube at 550nm of a spectrophotometer; every milliliter of sample is reacted for 1min at 37 ℃ to ensure H in a reaction system 2 O 2 The concentration is reduced by 1mmol/L to be one inhibition-OH capacity unit; calculated according to formula (3):
example 2:
the specific operation procedure is the same as in example 1, except that the basic conditions for enzymolysis are as follows: the enzymolysis temperature is 65 ℃, the enzyme amount is added, the enzymolysis time is 3 hours, and finally the hydrolysis degree of the apostichopus japonicus extract under the enzymolysis condition is measured to be 37.45%.
Example 3:
the specific operation procedure is the same as in example 1, except that the basic conditions for enzymolysis are as follows: the enzymolysis temperature is 65 ℃, the enzyme amount is added, the enzymolysis time is 5 hours, and finally the hydrolysis degree of the apostichopus japonicus extract under the enzymolysis condition is 32.60 percent.
Example 4:
the specific operation procedure is the same as in example 1, except that the basic conditions for enzymolysis are as follows: the enzymolysis temperature is 65 ℃, the enzyme amount is 5 percent, the enzymolysis time is 4 hours, and finally the hydrolysis degree of the apostichopus japonicus extract under the enzymolysis condition is 32.60 percent.
Example 5:
the specific operation procedure is the same as in example 1, except that the basic conditions for enzymolysis are as follows: the enzymolysis temperature is 70 ℃, the enzyme amount is 3 percent, the enzymolysis time is 3 hours, and finally the hydrolysis degree of the apostichopus japonicus extract under the enzymolysis condition is 30.52 percent.
Example 6:
the specific operation procedure is the same as in example 1, except that the basic conditions for enzymolysis are as follows: the enzymolysis temperature is 70 ℃, the enzyme amount is 3 percent, the enzymolysis time is 5 hours, and finally the hydrolysis degree of the apostichopus japonicus extract under the enzymolysis condition is 27.64 percent.
Example 7:
the specific operation procedure is the same as in example 1, except that the basic conditions for enzymolysis are as follows: the enzymolysis temperature is 70 ℃, the enzyme amount is added for 4 percent, the enzymolysis time is 4 hours, the experiment is repeated for 5 times, and finally, the hydrolysis degrees of the apostichopus japonicus essence under the enzymolysis condition are respectively 41.21 percent, 42.00 percent, 43.70 percent and 43.92 percent.
Example 8:
the specific operation procedure is the same as in example 1, except that the basic conditions for enzymolysis are as follows: the enzymolysis temperature is 75 ℃, the enzyme amount is 3 percent, the enzymolysis time is 4 hours, and finally the hydrolysis degree of the apostichopus japonicus extract under the enzymolysis condition is 31.18 percent.
Example 9:
the specific operation procedure is the same as in example 1, except that the basic conditions for enzymolysis are as follows: the enzymolysis temperature is 75 ℃, the enzyme amount is added, the enzymolysis time is 3 hours, and finally the hydrolysis degree of the apostichopus japonicus extract under the enzymolysis condition is measured to be 35.05 percent.
Example 10:
the specific operation procedure is the same as in example 1, except that the basic conditions for enzymolysis are as follows: the enzymolysis temperature is 75 ℃, the enzyme amount is added, the enzymolysis time is 5 hours, and finally the hydrolysis degree of the apostichopus japonicus extract under the enzymolysis condition is 34.09%.
Example 11:
the specific operation procedure is the same as in example 1, except that the basic conditions for enzymolysis are as follows: the enzymolysis temperature is 75 ℃, the enzyme amount is 5 percent, the enzymolysis time is 4 hours, and finally the hydrolysis degree of the apostichopus japonicus extract under the enzymolysis condition is 41.73 percent.
TABLE 1
Response surface optimization and analysis were performed for the above specific example process:
based on the single-factor experimental results, three variable enzymolysis temperatures (X 1 ) Enzyme addition amount (X) 2 ) Sum time (X) 3 ) The influence on the degree of hydrolysis, the coefficients of the second order polynomial equation are calculated. The experimental design and the result of the response surface are shown in table 1, and the regression equation corresponding to the degree of hydrolysis can be expressed by the following quadratic equation:
Y＝42.91-0.40X 1 +3.53X 2 -1.54X 3 +1.37X 1 X 2 +0.97X 1 X 3 -0.18X 2 X 3 -1.67X 1 2 -4.22X 2 2 -6.44X 3 2 (4)
the experimental results of table 1 were fitted by quadratic multiple regression using Design Expert software and analyzed by variance, the results are shown in table 2. The significance of the influence of each variable on the response value in the regression equation is determined by F test, the smaller the probability P value is, the higher the significance degree of the corresponding variable is, P is smaller than 0.05, the significance of the variable item is represented, and the significance of the variable item is represented by larger than 0.1.
TABLE 2
As can be seen from Table 2, the quadratic model F value selected in this experiment was 43.89, and it was highly significant (P<0.0001). In all variables, X 2 、X 3 、X 1 X 2 、X 1 2 、X 2 2 And X 3 2 The corresponding P values are smaller than 0.05, which shows that the effect on experimental results is obvious. An F value of 0.31 indicates that the effect of the mismatch term is insignificant. The complex correlation R2 is 0.9826, which indicates that 98.26% of the variation in hydrolysis degree is derived from the selected variable, and the predicted correlation of 0.9257 is matched to the corrected correlation 0.9602, so that the regression equation, which can be used to determine the optimal hydrolysis conditions, can be used to better describe the true relationship between the variables and the response values. In addition, the established model is reliable when the signal to noise ratio is greater than 4, and the signal to noise ratio 19.768 of the experiment is far greater than 4, so that the experimental model is further proved to be reliable, and the relation between the degree of hydrolysis and the temperature, the enzyme adding amount and the time can be well reflected. Within the selected factor level range, the influence of each variable on the experimental result is as follows in sequence: enzyme addition amount > time > temperature.
By fixing one variable value, the influence of the other two variables on the hydrolysis degree is examined to draw a response surface diagram, and the result of the influence of temperature, enzyme adding amount and time on the hydrolysis degree is shown in fig. 2. As the variables change, the degree of hydrolysis first assumes an upward trend and begins to decrease when it reaches the highest point. The decrease in the degree of hydrolysis caused by the simultaneous change in the temperature and the amount of enzyme added is attributable to the inhibition of the enzyme activity with the increase in the temperature, and the concentration of the enzyme and the substrate gradually reaches saturation after the increase in the amount of enzyme added. The decrease in hydrolysis degree due to the change in the amount of enzyme added and the time may be due to the degradation of the enzyme with the lapse of time, thereby decreasing the activity. The literature reports that the bottom of the response surface presents an elliptic contour line, which shows that the interaction of the variable is remarkable, and the experimental result shows that the interaction of the temperature and the enzyme adding amount is strongest. The response surface plot shows very high accuracy in predicting the degree of hydrolysis and significant interactions between the two combined variables, with results consistent with the regression model.
According to the analysis result, the optimal hydrolysis conditions are as follows: the predicted highest degree of hydrolysis was 43.75% at 70.10 ℃with an enzyme addition of 4.43% and a reaction time of 3.88 h. In view of convenience in practical operation, the optimum hydrolysis conditions were modified to a temperature of 70℃and an enzyme addition amount of 4% for 4 hours, and the degree of hydrolysis obtained under these conditions was 43.18%. The relative error compared to the predicted value is only 1.30%. The regression equation established can well reflect the influence of temperature, enzyme adding amount and time on the hydrolysis degree of apostichopus japonicus essence, and the hydrolysis degree can be effectively improved through the optimization model.
Then, the specific examples are subjected to the preparation of the riser peptide and the analysis of the polypeptide antioxidant activity results:
diluting the apostichopus japonicus essence enzymatic hydrolysate with distilled water twice, centrifuging at 10000r/min for 10min, collecting supernatant, filtering with common filter paper, and sequentially filtering with microporous filter membranes of 0.45 μm and 0.22 μm.
And then adding the filtered apostichopus japonicus essence enzymatic hydrolysate into an ultrafiltration cup by adopting a Pall minimum ultrafiltration system, installing a proper tangential flow membrane package, performing ultrafiltration under the control of the pressure of 20-30psig, and respectively collecting filtrate and trapped fluid.
Selecting a membrane package with a molecular weight cut-off of 10kDa for ultrafiltration, collecting filtrate when the enzymatic hydrolysate in the ultrafiltration cup remains 1/5 after the ultrafiltration begins, supplementing distilled water in the ultrafiltration cup to an initial volume, ultrafiltering again, repeating for three times, stopping ultrafiltration, and collecting filtrate (polypeptide with a molecular weight of less than 10 kDa) and retentate (protein with a molecular weight of more than 10 kDa) each time. And then respectively selecting tangential flow membrane bags with the molecular weight cut-off of 5kDa and 1kDa, finally preparing three polypeptides with different molecular weight ranges, and carrying out vacuum freeze-drying treatment on the three collected polypeptide solutions, wherein the three peptides are respectively named as P1, P2 and P3.
The three polypeptide solutions P1, P2 and P3 collected above were then assayed for chemical antioxidant activity.
(1) Anti-superoxide anion radical (O) 2 -· ) Measurement
Simulating xanthine and xanthine oxidase reaction system in organism to produce O 2 -· AddingAdding electron transfer material and Griess developer to make the reaction system appear purple, measuring its absorbance at 550nm with spectrophotometer, and calculating polypeptide pair O 2 -· Is a natural product of the inhibition ability of the above-mentioned compound. In the reaction system, each liter of sample was reacted at 37℃for 40min with suppressed O 2 -· Equivalent to 1mg of vitamin C inhibited O 2 -· The change value is one vitality unit. Calculated according to formula (2):
under simulated physiological conditions (pH 7.4, 37 ℃), the apostichopus japonicus selenka polypeptide pair O was tested 2 -· The results show (see FIG. 3 a) that the apostichopus japonicus selenka polypeptide scavenges O 2 -· Has strong concentration dependence in the experimental concentration range, and eliminates O with the increase of the concentration 2 -· Capability is continually increasing. Three polypeptide pairs O 2 -· Shows a relatively significant difference in the scavenging ability of the peptide fragment with lower molecular weight to scavenge O 2 -· The stronger the ability of (a) to be, the strength is in turn P1>P2>P3。
(2) Determination of OH removal Capacity
The Fenton reaction is utilized to generate OH, the reaction is the most common chemical reaction for generating OH, H 2 O 2 The amount of (2) is proportional to the amount of OH generated by the reaction, and when an electron acceptor is supplied, the color is developed by Griess reagent to form a red color substance, and the color development is proportional to the amount of OH. Samples were sequentially added and absorbance values were measured for each tube at 550nm in a spectrophotometer. Every milliliter of sample is reacted for 1min at 37 ℃ to ensure H in a reaction system 2 O 2 The concentration reduction of 1mmol/L is one inhibiting-OH capability unit. Calculated according to formula (3):
under simulated physiological conditions (pH 7.4, 37 ℃), the removal capability of the apostichopus japonicus selenka polypeptides to OH was tested, and the results show that the removal capability of the apostichopus japonicus selenka polypeptides with different molecular weights to OH is greatly different (see figure 3 b). In the low concentration range, the ability of three polypeptides to scavenge OH is closely related to their molecular weight. At a concentration of 2.5-5mg/mL, the activity of the polypeptide P1 with a molecular weight of < 1kDa is much lower than that of P2 and P3. P1 has a strong concentration dependence on the scavenging ability of OH, and as the concentration increases, the activity of P1 rapidly increases over P2 and P3 at concentrations of about 12 and 10mg/mL, respectively. And the activity of P2 and P3 is kept higher in the experimental concentration range, the activity of P2 is not changed greatly with the increase of the concentration, and the activity of P3 is slightly reduced. The research results show that P1, P2 and P3 have better capability of removing OH, and show that the compounds have better antioxidant activity.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Claims (7)
1. A processing technology for optimizing apostichopus japonicus refined enzymolysis by a response surface method is characterized in that the optimal hydrolysis condition of the apostichopus japonicus refined enzymolysis processing technology by the response surface method is 70 ℃, the enzyme addition amount is 4.4%, and the hydrolysis time is 4 h;
the operation steps of the specific processing technology for optimizing the apostichopus japonicus essence enzymolysis by the response surface method are as follows:
step one, enzymolysis of apostichopus japonicus essence: cleaning apostichopus japonicus essence, draining water, grinding and homogenizing by a colloid mill, accurately weighing 10g of apostichopus japonicus essence homogenate, adding 0.5-0.7-g papain, uniformly mixing, performing enzymolysis in a constant-temperature water bath, immediately taking out the enzymolysis solution after the enzymolysis is completed, and placing the enzymolysis solution in the water bath at 100 ℃ to inactivate enzymes for 10min;
step two, measuring the degree of hydrolysis: measuring the hydrolysis degree according to the trichloroacetic acid TCA method, adding 1mL enzymolysis liquid into 10g/100mL TCA of 1mL, mixing and oscillating, standing for 10min, centrifuging 10000r/min for 10min, taking supernatant, measuring the content of soluble protein by a biuret method, measuring the content of total protein by a Kjeldahl nitrogen method, and calculating the hydrolysis degree DH according to the following formula:
DH = (ρ 1 -ρ 2) /(ρ 0 -ρ 2 )×100 （1）
step three, single factor research: in order to examine the influence of three factors of enzymolysis temperature, enzyme adding amount and enzymolysis time on the hydrolysis degree, firstly, a single factor experiment is carried out, and basic conditions of enzymolysis are formulated as follows: the enzymolysis temperature is 65 ℃, the enzyme adding amount [ E/S ] is 3%, and the time is 5h; fixing two conditions, changing the other condition, and respectively observing different influencing factors;
step four, optimizing hydrolysis conditions by a response surface method: on the basis of a single-factor experiment, three factors including hydrolysis degree are taken as measurement indexes, enzymolysis temperature, enzyme adding amount and time are selected, a three-factor and three-level response surface experiment is designed, papain is selected, enzymolysis conditions are optimized, according to a single-factor experiment result, box-Behnken design experiment is adopted, the influence of three factors on the hydrolysis degree is analyzed, the experiment times are 17, the experiment times of a factor analysis part are 12, and the experiment times of a center point repeat are 5.
2. The process for optimizing the enzymolysis of apostichopus japonicus essence by using a response surface method according to claim 1, wherein the calculated hydrolysis degree DH is as follows:ρ 1 the content of soluble protein in the enzymolysis liquid after the reaction is mg/mL;ρ 2 is imitated thorn before reactionSoluble protein content mg/mL in ginseng extract;ρ 0 is mg/mL of total protein content in the apostichopus japonicus essence.
3. The processing technology for optimizing apostichopus japonicus essence enzymolysis by using a response surface method according to claim 1, wherein the range of values of each factor in the single factor research is as follows: the enzymolysis temperatures are respectively 50, 60, 65, 70 and 80 ℃, the enzyme adding amounts are respectively 1%, 2%, 3%, 4% and 5%, and the time is respectively 3, 4, 5, 6 and 7h.
4. The processing technology for optimizing apostichopus japonicus essence enzymolysis by using a response surface method according to claim 1, wherein the processing technology further comprises a preparation method for separating three polypeptides with different molecular weight ranges from enzymolysis liquid by using a Pall minimum ultrafiltration system.
5. The processing technology for optimizing apostichopus japonicus essence enzymolysis by using a response surface method according to claim 4, wherein the specific preparation method of the polypeptide is as follows:
step one, preparation of a step peptide: diluting the apostichopus japonicus refined enzymolysis liquid with distilled water twice, centrifuging 10000r/min for 10min, collecting supernatant, filtering with common filter paper, and sequentially filtering with microporous filter membranes of 0.45 μm and 0.22 μm; then a Pall minimum ultrafiltration system is adopted, the filtered apostichopus japonicus essence enzymatic hydrolysate is added into an ultrafiltration cup, a tangential flow membrane package is arranged, ultrafiltration is carried out under the control of the pressure of 20-30psig, and filtrate and trapped fluid are respectively collected;
step two, selecting a membrane package with the molecular weight cutoff of 10kDa for ultrafiltration, after ultrafiltration begins, collecting filtrate when 1/5 of enzymolysis liquid in an ultrafiltration cup remains, supplementing distilled water in the ultrafiltration cup to the initial volume, ultrafiltering again, stopping ultrafiltration after repeating for three times, and collecting filtrate, polypeptide with the molecular weight less than 10kDa and retentate with the molecular weight more than 10kDa each time; and then respectively selecting tangential flow membrane bags with the molecular weight cut-off of 5kDa and 1kDa, finally preparing three polypeptides with different molecular weight ranges, and carrying out vacuum freeze-drying treatment on the three collected polypeptide solutions.
6. The processing technology for optimizing the enzymolysis of apostichopus japonicus essence by using a response surface method according to claim 4, wherein the processing technology further comprises an antioxidant activity research method of the step peptide extracted from the enzymolysis liquid, namely, the step peptide is used for removing O 2 -· And OH capability.
7. The processing technology for optimizing the enzymolysis of apostichopus japonicus essence by using a response surface method according to claim 6, wherein the specific steps of the research on the antioxidant activity of the step peptide extracted from the enzymolysis liquid are as follows:
step one, clean O 2 -· Capacity measurement:
simulating xanthine and xanthine oxidase reaction system in organism to produce O 2 -· Adding electron transfer material and Griess developer to make the reaction system appear purple, measuring its absorbance at 550nm with spectrophotometer, and calculating the polypeptide pair O 2 -· In the reaction system, O is inhibited by reacting for 40min at 37 ℃ per liter of sample 2 -· Vitamin C-inhibited O corresponding to 1mg 2 -· The change value is a vitality unit; calculated according to formula (2):
step two, measuring the OH removal capacity:
generating OH, H by Fenton reaction 2 O 2 In proportion to the amount of OH generated by the reaction, and after the electron acceptor is given, the color is developed by Griess reagent to form red substance, and the color is in proportion to the amount of OH; sequentially adding samples, and measuring the absorbance value of each tube at a spectrophotometer 550 nm; every milliliter of sample is reacted for 1min at 37 ℃ to ensure H in a reaction system 2 O 2 The concentration is reduced by 1mmol/L to be one inhibition-OH capacity unit; calculated according to formula (3):
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| CN105368901A (en) * | 2015-10-24 | 2016-03-02 | 山东好当家海洋发展股份有限公司 | Method for extracting antibacterial polypeptide by utilizing apostichopus japonicus working fluid |
| CN111919801A (en) * | 2020-05-22 | 2020-11-13 | 福建省水产研究所 | Method for out-of-season seed production and cultivation of south-shifted apostichopus japonicus |
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| CN105368901A (en) * | 2015-10-24 | 2016-03-02 | 山东好当家海洋发展股份有限公司 | Method for extracting antibacterial polypeptide by utilizing apostichopus japonicus working fluid |
| CN111919801A (en) * | 2020-05-22 | 2020-11-13 | 福建省水产研究所 | Method for out-of-season seed production and cultivation of south-shifted apostichopus japonicus |
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