阅读说明：本技术 一种利用鱼糜废水回收蛋白制备抗氧化肽方法 (Method for preparing antioxidant peptide by utilizing protein recovered from minced fillet wastewater ) 是由 祁兴普 刘萍 姚芳 刘靖 张静 张军燕 于 2019-10-08 设计创作，主要内容包括：本发明公开了一种利用鱼糜废水回收蛋白制备抗氧化肽方法,其工艺流程如下：鱼糜清洗废水→过滤→pH值调节→离心沉淀→脱脂→回收蛋白冻干→酶解→膜过滤→真空浓缩→酶解产物冻干→抗氧化活性肽；其中鱼糜废水的回收蛋白质量百分比为2.9～3.2％,用复合风味蛋白酶与中性蛋白酶作为反应酶,并在酶总添加量为3000～5000U的条件下酶解反应3.5～4.2h。本发明以鱼糜加工洗涤用水回收蛋白为底物,通过酶解法制备抗氧化肽,一方面可减少废水排放造成的污染,另一方面可提高加工产品的附加值,并获得一种安全性较高的抗氧化活性肽。而且,本发明的鱼糜废水回收蛋白来源的抗氧化肽具有较强的抗氧化性和热稳定性。(The invention discloses a method for preparing antioxidant peptide by utilizing protein recovered from minced fillet wastewater, which comprises the following process flows of: minced fillet washing wastewater → filtration → pH value adjustment → centrifugal precipitation → degreasing → freeze-drying of recovered protein → enzymolysis → membrane filtration → vacuum concentration → freeze-drying of enzymolysis product → antioxidative peptide; wherein the mass percentage of the recovered protein of the minced fillet wastewater is 2.9-3.2%, the compound flavourzyme and the neutral proteinase are used as reaction enzymes, and the enzymolysis reaction is carried out for 3.5-4.2 h under the condition that the total addition amount of the enzymes is 3000-5000U. The invention uses the protein recovered from the water used for processing and washing minced fillet as the substrate to prepare the antioxidant peptide by an enzymolysis method, thereby reducing the pollution caused by the discharge of waste water, improving the added value of the processed product and obtaining the antioxidant active peptide with higher safety. In addition, the antioxidant peptide from the surimi wastewater recycled protein has stronger oxidation resistance and thermal stability.)

1. A method for preparing antioxidant peptide by utilizing protein recovered from minced fillet wastewater is characterized by comprising the following steps: the mass percentage of the protein recovered from the minced fillet wastewater is 2.9-3.2%, the compound flavourzyme and the neutral proteinase are used as reaction enzymes, and the enzymolysis reaction is carried out for 3.5-4.5 h under the condition that the total addition amount of the enzymes is 3000-5000U.

2. The method for preparing antioxidant peptides by using protein recovered from surimi wastewater according to claim 1, wherein the unit ratio of the enzyme activity of the compound flavourzyme to that of the neutral proteinase is 1.20: 1-1.25: 1.

3. The method for preparing antioxidant peptides by using the protein recovered from the surimi wastewater according to claim 2, wherein the enzymolysis conditions are pH 6.2-6.9 and temperature 48-55 ℃.

4. The method for preparing antioxidant peptide by using the protein recovered from the minced fillet waste water as claimed in any one of claims 1 to 3, which is characterized by comprising the following steps:

(1) and (3) filtering: filtering the minced fillet wastewater by using gauze to remove fish scales, fishbones and other solid impurities;

(2) adjusting the pH value: adjusting the pH value of the filtered minced fillet wastewater to 5.4-6.2 by using an acid-base solution, and standing for 45-60 min at the temperature of 0-10 ℃;

(3) and (3) centrifugal precipitation: removing fat component in the supernatant, and retaining the precipitated crude protein;

(4) degreasing: unfreezing the minced fillet precipitate treated in the step (3), mashing at a high speed, mixing the mashed fillet with n-hexane according to the volume ratio of 1: 3, oscillating at a constant temperature, centrifuging to remove supernatant, ventilating and volatilizing the degreased minced fillet, and removing the n-hexane;

(5) freeze-drying: prefreezing the precipitated protein treated in the step (4) to the central temperature of 25 ℃, then freeze-drying under the conditions of the absolute pressure of 80Pa and the sublimation temperature of-20 ℃, and crushing the freeze-dried recovered protein for later use;

(6) preparing an enzymolysis solution: preparing freeze-dried recovered protein into protein suspension with the mass percentage of 2.9-3.2%, and adjusting the pH value to 6.2-6.9;

(7) enzymolysis: carrying out enzymolysis for 3.5-4.5 h at the temperature of 48-55 ℃ under the conditions that the unit ratio of the enzyme activity of the compound flavor protease to the enzyme activity of the neutral protease is 1.20: 1-1.25: 1 and the total enzyme addition is 3000-5000U;

(8) after membrane filtration, taking filtrate for later use;

(9) concentration and freeze-drying: concentrating the filtrate at 75 deg.C and absolute pressure of-72 KPa to 1/10, and lyophilizing at sublimation temperature of-50 deg.C under absolute pressure of 80Pa to obtain lyophilized product.

5. The method for preparing antioxidant peptides from protein recovered from surimi wastewater according to claim 4, wherein in the step (2), the adjusted pH value is 5.8; after the pH of the minced fillet wastewater is adjusted, the temperature is rapidly reduced to 0-10 ℃ by adopting an indirect heat exchange method.

6. The method for preparing antioxidant peptides from protein recovered from minced fillet wastewater as claimed in claim 4, wherein in the step (4), the minced fillet processed in the step (3) is thawed to the central temperature of-4 ℃, mashed in a high-speed tissue masher, the mashed and normal hexane are uniformly mixed according to the volume ratio of 1: 3, the mixture is oscillated for 3h in a constant-temperature water bath oscillator at 35 ℃ and then is centrifuged for 10min at 6000r/min in a refrigerated centrifuge and 0 ℃, the supernatant is discarded, the operations are repeated for 2 times, and the defatted minced fillet is volatilized at 75 ℃ in a ventilation cabinet to remove the normal hexane.

Technical Field

The invention relates to a method for preparing biological antioxidant active peptide, in particular to a method for preparing antioxidant peptide by utilizing surimi wastewater recovered protein, belonging to the technical field of aquatic product processing.

Background

Since the establishment of a bulk freshwater fish industrial technology system, the national bulk freshwater fish culture yield is increased from 1553.18 ten thousand tons in 2009 to 2008.64 ten thousand tons in 2014, and the annual increase is 5.9 percent^[1]. At present, except fresh food, part of Chinese freshwater fishes such as silver carps, bighead carps, grass carps, black carps and other low-value freshwater fishes are processed into special minced fillet products such as fish cakes, fish balls, fish bean curds, fish sausages and the like, and are popular with consumers. However, during the processing of such surimi products, multiple rinses are required to remove components such as lipid and sarcoplasmic proteins to improve surimi quality, and the rinsing process can cause about 30% of soluble protein loss^[2]And after the minced fillet soaking wastewater is discharged, not only is the resource waste caused, but also the sewage treatment burden is increased, and the direct discharge can seriously pollute the environment.

At present, a precipitation method is adopted for recovering protein in fish processing wastewater^[3,4]And membrane separation process^[5]The protein is recycled and used as feed^[6]Minced fish product^[7]And bioactive peptide, wherein the bioactive peptide prepared by using aquatic product processing by-products has wider sources, higher safety and more novel structure compared with the antioxidant peptide of terrestrial animal protein, and is a functional factor with development prospect^[8]。

[1] Goxianping, the current situation of bulk freshwater fish culture in China and the construction of industrial technical system [ J ]. aquatic products in China, 2010, 2010(5):5-9

[2] Wewawaft, Zhang Meng, Huxiao, etc. recovery of protein from surimi rinse water and its reuse [ J ] Nuclear agriculture report 2015(11):2172-

[3] Study on the rules of isoelectric precipitation of proteins in minced fillet wastewater [ J ] food industry science and technology, 2012(18):93-95

[4]Iwashita K,SumidaM,Shirota K,et al.Recovery method for surimi wash-water protein bypH shift and heat treatment[J].Food Science and Technology Research,2016,22(6):743-749

[5]Afonso M D,Bórquez R.Review ofthe treatment ofseafoodprocess－ing wastewaters and recovery ofproteins therein by membrane sepa－rationprocesses-prospects ofthe ultrafiltration of wastewaters from the fish mealindustry[J].Desalination,2002,142(1):29-45

[6] A first study on the neutral protease hydrolysis technique of the waste juice from the production of Kaolin, Sun Jing, Jiangjun, Fish powder (J) food science, 2007(7):121-

[7] Property of recovered protein in water for washing minced fillet of hairtail [ J ] modern food technology 2016(6):321-

[8] Zhoudqing, Lina, Wangshan, etc. research status and prospect of antioxidant peptide as byproduct source of aquatic product processing [ J ] aquatic product science report 2019,43(01): 190-.

Disclosure of Invention

In order to overcome the defects and shortcomings in the prior art, the invention aims to provide a method for preparing antioxidant peptide by utilizing surimi wastewater recovered protein, which can reduce pollution caused by wastewater discharge, improve the added value of processed products and obtain antioxidant peptide with higher safety.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a method for preparing antioxidant peptide by utilizing protein recovered from minced fillet wastewater comprises the following steps: the mass percentage of the protein recovered from the minced fillet wastewater is 2.9-3.2%, the compound flavourzyme and the neutral proteinase are used as reaction enzymes, and the enzymolysis reaction is carried out for 3.5-4.5 h under the condition that the total addition amount of the enzymes is 3000-5000U.

Further, the unit ratio of the enzyme activity of the compound flavor protease to the enzyme activity of the neutral protease is 1.20: 1-1.25: 1.

Further, in the method, the enzymolysis condition is that the pH value is 6.2-6.9, and the temperature is 48-55 ℃.

Further, the method for preparing the antioxidant peptide by utilizing the protein recovered from the minced fillet wastewater comprises the following specific steps:

(1) and (3) filtering: filtering the minced fillet wastewater by using gauze to remove fish scales, fishbones and other solid impurities;

(2) adjusting the pH value: adjusting the pH value of the filtered minced fillet wastewater to 5.4-6.2 by using an acid-base solution, and standing for 45-60 min at the temperature of 0-10 ℃;

(3) and (3) centrifugal precipitation: removing fat component in the supernatant, and retaining the precipitated crude protein;

(4) degreasing: unfreezing the minced fillet precipitate treated in the step (3), mashing at a high speed, mixing the mashed fillet with n-hexane according to the volume ratio of 1: 3, oscillating at a constant temperature, centrifuging to remove supernatant, ventilating and volatilizing the degreased minced fillet, and removing the n-hexane;

(5) freeze-drying: prefreezing the precipitated protein treated in the step (4) to the central temperature of 25 ℃, then freeze-drying under the conditions of the absolute pressure of 80Pa and the sublimation temperature of-20 ℃, and crushing the freeze-dried recovered protein for later use;

(6) preparing an enzymolysis solution: preparing freeze-dried recovered protein into protein suspension with the mass percentage of 2.9-3.2%, and adjusting the pH value to 6.2-6.9;

(7) enzymolysis: carrying out enzymolysis for 3.5-4.5 h at the temperature of 48-55 ℃ under the conditions that the unit ratio of the enzyme activity of the compound flavor protease to the enzyme activity of the neutral protease is 1.20: 1-1.25: 1 and the total enzyme addition is 3000-5000U;

(8) after membrane filtration, taking filtrate for later use;

(9) concentration and freeze-drying: concentrating the filtrate at 75 deg.C and absolute pressure of-72 KPa to 1/10, and lyophilizing at sublimation temperature of-50 deg.C under absolute pressure of 80Pa to obtain lyophilized product.

Further, in the step (2), the adjusted pH value is 5.8; after the pH of the minced fillet wastewater is adjusted, the temperature is rapidly reduced to 0-10 ℃ by adopting an indirect heat exchange method.

Further, in the step (4), the minced fillet processed in the step (3) is unfrozen to the central temperature of-4 ℃, mashed in a high-speed tissue mashing machine, the mashed surimi and n-hexane are uniformly mixed according to the volume ratio of 1: 3, the mixture is oscillated for 3 hours in a thermostatic water bath oscillator at the temperature of 35 ℃, then the mixture is centrifuged for 10 minutes at 6000r/min in a refrigerated centrifuge at the temperature of 0 ℃, the supernatant is discarded, the above operations are repeated for 2 times, and the n-hexane is volatilized and removed from the defatted surimi under the condition of volatilizing 75 ℃ in a ventilation cabinet.

During the processing of minced fillet, a large amount of soluble protein is washed for many times and lost along with the discharge of waste water, thereby causing environmental pollution and edible protein loss. The invention has the advantages that: the invention uses surimi (without any limit to surimi, only the surimi can be used) to process washing water to recover protein as a substrate, and prepares the antioxidant peptide by an enzymolysis method. In addition, the antioxidant peptide from the protein recovered from the fish surimi wastewater has stronger oxidation resistance and thermal stability.

Drawings

FIG. 1 shows the effect of different proteases on the degree of minced fillet hydrolysis and DPPH clearance of enzymatic products (n-3);

FIG. 2 shows the effect of different complex enzyme ratios on the degree of hydrolysis and the product DPPH radical clearance (n-3);

FIG. 3 is a graph of the effect of different substrate concentrations on the degree of hydrolysis and product DPPH radical clearance (n-3);

FIG. 4 is a graph of the effect of enzyme addition on degree of hydrolysis and product DPPH radical clearance (n-3);

figure 5 is a graph of the effect of temperature and pH on the degree of hydrolysis and product DPPH radical clearance (n-3): (A) the influence of temperature on the degree of hydrolysis and the product DPPH radical clearance rate; (B) influence of pH value on hydrolysis degree and DPPH free radical clearance rate of product;

figure 6 is a graph of the effect of reaction time on the degree of hydrolysis and product DPPH radical clearance (n-3);

FIG. 7 is a response surface graph and contour plot of the effect of 2-factor interaction on DPPH clearance of enzymatic products: (a) a response surface curve chart of interaction influence of substrate concentration and enzyme proportion; (b) is a contour diagram of the interaction influence of the substrate concentration and the enzyme proportion; (c) a response surface curve graph of the interaction influence of the enzyme addition amount and the enzyme proportion is shown; (d) is a contour diagram of the interaction influence of the enzyme addition amount and the enzyme proportion; (e) a response surface curve graph of the interaction influence of enzymolysis time and enzyme proportion; (f) is a contour diagram of the interaction influence of enzymolysis time and enzyme proportion; (g) a response surface curve chart of the interaction influence of the enzyme addition amount and the substrate concentration; (h) is a contour diagram of the interaction influence of the enzyme addition amount and the substrate concentration; (i) a response surface curve graph of the interaction influence of enzymolysis time and substrate concentration; (j) is a contour diagram of the interaction influence of enzymolysis time and substrate concentration; (k) a response surface curve graph of the interaction influence of the enzyme addition amount and the enzymolysis time is shown; (l) Is a contour diagram of the interaction influence of the enzyme addition amount and the enzymolysis time.

Detailed Description

The invention is further described below with reference to the accompanying drawings and examples.

The process flow of the method for preparing the antioxidant peptide by utilizing the surimi wastewater recovered protein is as follows: minced fillet washing wastewater → filtration → pH value adjustment → centrifugal precipitation → degreasing → freeze-drying of recovered protein → enzymolysis → membrane filtration → vacuum concentration → freeze-drying of enzymolysis product → antioxidative peptide.

(1) Screening for proteases

Equal amounts (3000U/g) of pepsin, papain, flavourzyme, alkaline protease and neutral protease are respectively added into 100mL of 2% freeze-dried silver carp protein solution, the mixture reacts for 2 hours under respective optimal reaction temperature and pH conditions, enzyme deactivation is carried out for 10 minutes at 100 ℃, and influences of enzymatic hydrolysates prepared by different proteases on the enzymolysis degree and the DPPH free radical clearance rate are measured.

Research shows that the preparation of polypeptide with high antioxidant activity needs high hydrolysis degree, and the molecular weight of the peptide with antioxidant activity is usually less than 3.5 KD. As shown in figure 1, the compound flavor protease has the highest hydrolysis degree, the product has the best oxidation resistance, and the neutral protease is inferior. In the preparation process of the antioxidant polypeptide, a complex enzymolysis method is mostly adopted, and the enzymolysis efficiency is improved by utilizing the difference of protease enzyme cutting sites to prepare the antioxidant polypeptide. The compound flavor protease and the neutral protease with the optimal enzymolysis pH value and the temperature close to each other are selected for the experiment to carry out enzymolysis on the surimi recovered protein, so that on one hand, the enzymolysis efficiency is improved, the simplification of the production process is facilitated, and on the other hand, the potential safety hazard and the environmental pollution of products caused by the use of a buffer solution and a metal salt for regulating the pH value can be avoided.

(2) Influence of complex enzyme proportion on hydrolysis degree and DPPH free radical clearance rate of enzymolysis product

In 100mL of 2% freeze-dried silver carp protein solution, the ratio of 0.3:1, 0.6: 1. 0.9:1, 1.2:1, 1.5: 1. adding compound flavourzyme and neutral protease according to the mass ratio of 1.8:1, wherein the total addition amount is 3000U/g, reacting for 2h under the condition that the pH value is 7.0, inactivating enzyme for 10min under the condition of 100 ℃, and determining the influence of enzymolysis solutions prepared by different compound enzyme ratios on the hydrolysis degree and the DPPH free radical clearance rate.

As shown in figure 2, the degree of hydrolysis increased slowly with increasing proportion of composite flavourzyme, and did not change significantly between 0.9:1 and 1.5: 1, but began to decrease at 1.5: 1. DPPH clearance increases and then decreases with different ratios of flavourzyme to neutrase, reaching a maximum at 1.2: 1. Relevant studies have shown that: the antioxidant activity of the polypeptide is closely related to the composition of amino acids, and the difference of the enzyme cutting sites and the enzyme cutting sequence of the protease can cause the difference of the amino acid sequences of peptides obtained by hydrolyzing the proteases with different combinations, thereby influencing the DPPH free radical clearance of hydrolysate, which is not completely the same as the change rule of the hydrolysis degree.

(3) Influence of substrate concentration on the degree of hydrolysis and the DPPH radical clearance of enzymatic products

3000U of pepsin and papain (the enzyme activity unit ratio is 1.2: 1) are respectively added into 100mL of 0.5%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5% and 4.5% (by mass percentage) of lyophilized silver carp protein liquid, the mixture reacts for 2 hours at the temperature of 50 ℃ and at the pH value of 7.0, enzyme deactivation is carried out for 10 minutes at the temperature of 100 ℃, and the influence of enzymolysis liquid prepared by different substrate concentrations on the enzymolysis degree and the DPPH free radical removal rate is measured.

As shown in fig. 3, as the substrate concentration increases, the hydrolysis degree increases and then decreases, and the DPPH clearance rate increases and then does not change significantly, because as the substrate concentration increases, the binding between the enzyme molecules and the substrate reaches saturation, and at the same time, the viscosity of the system increases, which is not favorable for sufficient contact between the enzyme and the substrate, and the total amount of the enzymatic hydrolysate of the system does not increase significantly, thereby resulting in the decrease of the hydrolysis degree and no significant change in the DPPH clearance rate. Thus, a substrate concentration of around 2.5% was initially selected.

(4) Influence of enzyme addition on hydrolysis degree and DPPH free radical clearance of enzymolysis products

Adding pepsin and papain (enzyme activity unit ratio of 1.2: 1) of 1500, 2000, 2500, 3000, 3500, 4000 and 4500U into 100mL of 2.5% lyophilized silver carp protein solution, reacting at 50 deg.C and pH of 7.0 for 2h, inactivating enzyme at 100 deg.C for 10min, and determining the influence of enzymolysis solutions prepared with different enzyme addition on hydrolysis degree and DPPH free radical removal rate.

As shown in fig. 4, when the amount of the enzyme added is less than 3500U, the substrate is sufficient, the hydrolysis degree increases significantly with the increase in the amount of the enzyme added, and the DPPH clearance increases, and when the amount of the enzyme added exceeds 3500U, the hydrolysis degree of the hydrolysate and the DPPH clearance do not increase significantly because the substrate and the enzyme are sufficiently bound, and therefore, the amount of the enzyme added is initially set to about 3500U.

(5) Influence of temperature and pH value on hydrolysis degree and DPPH free radical clearance rate of enzymolysis product

3000U pepsin and papain (the enzyme activity unit ratio is 1.2: 1) are respectively added into 100mL of 2.5% freeze-dried silver carp protein solution, the mixture reacts for 2 hours at the conditions of pH 6.0, 6.2, 6.4, 6.6, 6.8, 7.0 and 7.2, temperature 42 ℃, 44 ℃, 46 ℃, 48 ℃, 50 ℃, 52 ℃ and 54 ℃ respectively, enzyme inactivation is carried out for 10min at the temperature of 100 ℃, and the influences of enzymolysis solutions prepared at different pH values and temperatures on the hydrolysis degree and the DPPH free radical removal rate are respectively measured.

As shown in FIGS. 5(A) and (B), when the hydrolysis pH is 6.6 and the temperature is 52 ℃, the degree of hydrolysis and DPPH clearance of the recovered surimi protein are maximized by the combined action of the compound flavourzyme and the neutral protease, and therefore, the pH 6.6 and the temperature 52 ℃ are selected as the optimum reaction pH and temperature.

(6) Influence of reaction time on degree of hydrolysis and DPPH radical clearance of enzymatic products

3000U of pepsin and papain (the enzyme activity unit ratio is 1.2: 1) are respectively added into 100mL of 2.5% freeze-dried silver carp protein solution, the mixture is respectively reacted for 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 hours at the temperature of 52 ℃ and at the pH value of 6.6, enzyme is inactivated for 10min at the temperature of 100 ℃, and the influence of enzymolysis liquid prepared in different reaction times on the enzymolysis degree and the DPPH free radical removal rate is respectively measured.

As shown in fig. 6, the degree of hydrolysis and DPPH radical clearance increased with the time of hydrolysis, and did not increase significantly after 3 hours of hydrolysis, probably because the substrate had been completely consumed or the substrate had decomposed out of products that inhibit the enzymatic process. Therefore, about 3.5h is selected as the optimal enzymolysis time.

(7) Response surface test

(7.1) response surface analysis protocol and results

And (3) synthesizing the single-factor test result, selecting an enzyme proportion (X1), a substrate concentration (X2), an enzyme addition amount (X3) and an enzymolysis time (X4) as independent variables and a DPPH clearance rate as a dependent variable according to a Box-Behnken design principle, designing 4-factor 3 horizontal response surface experiments, totaling 29 groups of test points, wherein 5 groups of central test points have the results shown in table 1, and the analysis of variance of a regression model is shown in table 2.

TABLE 1Box-Behnken test design and results

Analyzing the test data of the table 2 by using Design-Expert V8.0.6 software, and obtaining a regression model equation as follows: DPPH clearance rate of 83.98731X₁+114.6322X₂+0.04857X₃+22.19596X₄+3.30142X₁X₃-0.92671X₁X₄-0.00170284X₂X₃+0.41702X₂X₄+0.00038227X₃X₄-33.74783X₁X₂-18.01358X₂X₂-0.0000048166X₃X₂-2.88744X₄X₂-310.14595

Analysis of variance of the regression equation is shown in Table 2, with the regression model being extremely significant (P)<0.0001), no mismatching term was significant (P ═ 0.0656)>0.05), the error between the predicted value and the measured value of the model is small, and the model is reliable; model correlation coefficient R²0.9980, which shows that the model has good precision; coefficient of determination of model correction R² _Adj0.9960, the model can explain the change of 99.80% response value, the fitting degree of the model and an actual test is good, and the model can be used for optimizing the enzymolysis conditions of the enzymolysis activity antioxidant peptide of the surimi wastewater recovered protein; r² _Pred0.9890 shows that the model has good prediction and can well predict the influence of enzymolysis conditions on the activity of the enzymolysis antioxidant peptide of the protein recovered from the minced fillet wastewater.

TABLE 2 regression model analysis of variance

Note: "x" indicates extreme significance (P < 0.001).

(7.2) analysis of interaction between response surface factors

By performing response surface analysis on the regression model, as shown in fig. 7, a response surface graph and a corresponding contour graph of any 2 factors fixed at a zero level and the other 2 factors and their interaction effects are obtained. The gradient of the response surface represents the relationship of the influence of the factor on the dependent variable, and the steeper the curved surface is, the more remarkable the influence is; the contour line represents the interaction strength of each factor, the ellipse represents that the factor interaction is obvious, and the circle represents that the factor interaction is not obvious; in the same contour map, the factor with a larger density has a larger influence on the response value than the factor with a smaller contour density. As can be seen from fig. 7, X3> X2> X1> X4, i.e., enzyme addition amount > substrate concentration > enzyme ratio > enzymolysis time.