CN102978269A - Method for preparing soybean peptides from carrier-free immobilized enzyme - Google Patents
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Abstract
A method for preparing soybean peptides from carrier-free immobilized enzyme belongs to an extract technology of soybean peptides. The method comprises the following steps of: (1) dissolving alkaline protease into phosphate buffer, adding ethanol to precipitate and gather, and then adding glutaraldehyde to carry out crosslinking, so as to obtain cross-linked enzyme aggregate, namely immobilized alkaline protease; and (2) adding water into isolated soy protein solution to be mixed and prepared into the isolated soy protein solution, adjusting the pH and the temperature, and then adding the immobilized enzyme obtained from the step (1) to carry out enzymolysis, recovering the immobilized enzyme after enzymolysis, centrifugally separating the enzymatic hydrolysate to obtain clear solution, and drying the clear solution to obtain the soybean peptides by freezing and drying. The carrier-free immobilized alkaline protease prepared by the method is high in activity, and does not need a high-cost carrier, and meanwhile, the isolated soy protein is subjected to enzymolysis by the immobilized enzyme to prepare the soybean peptides; the method is simple in required processing equipment, and low in energy consumption; and the immobilized enzyme can be recycled and reused. Thus, waste of resources is reduced, and the cost is saved.
Description
Technical field
The invention belongs to the soybean polypeptide extractive technique, relate in particular to the method that a kind of Immobilized Enzymes Without Carriers prepares soybean polypeptide.
Background technology
Immobilized enzyme is the new technology that grows up the sixties in 20th century, generally be that enzyme and insoluble carrier are combined, make enzyme in certain space, be closed state, can continuous reacting, reacted enzyme reclaims by the method such as filter or centrifugal and reuses.Immobilized enzyme is as a main direction of studying in the enzyme Application Areas, mostly by enzyme being fixed on insoluble polymer or the inorganic carrier, the immobilized enzyme of formation greatly reduces binding capacity and the response capacity of enzyme owing to the existence of polymer support in macromolecule substrate transforms at present.In addition, the reagent that immobilization is used and carrier is with high costs, fixed efficiency is on the low side, the immobilized enzyme that really drops into industrial applications is few, thereby further easier, the more applicable process for fixation of exploitation is still the target that pursue in this field.
The cross-linked enzyme aggregate technology is a kind of Immobilized Enzymes Without Carriers technology, and the immobilization of enzyme is by realizing the covalent cross-linking of the protein example of basic purifying, high density.Compare with traditional immobilized enzyme, the immobilized enzyme that this process for fixation obtains has higher catalyst specific surface, unit volume is active greatly, space efficiency is high, higher enzymatic activity is arranged, be subjected to the impact of substrate diffusional limitation less, under extreme conditions reach in the organic solvent and proteolytic enzyme in operational stability, with low cost, equipment is simple, need not carrier, applied range.Yet with the cross-linked enzyme aggregate technology---the Immobilized Enzymes Without Carriers technology is applied to the research that enzymatic soybean protein isolate extracts soybean polypeptide and has no report, and the method for existing extraction soybean polypeptide exists enzyme not reclaim to reuse the high in cost of production problem.
Summary of the invention
Technical problem to be solved by this invention is to overcome above-mentioned the deficiencies in the prior art, and the method that provides a kind of Immobilized Enzymes Without Carriers to prepare soybean polypeptide reaches the repeating utilization factor of simplifying technique, raising enzyme and the purpose that reduces cost.
Technical problem to be solved by this invention is achieved through the following technical solutions:
A kind of Immobilized Enzymes Without Carriers prepares the method for soybean polypeptide, the method may further comprise the steps: (1) is dissolved in the protex-6L Sumizyme MP in the phosphate buffered saline buffer, adding the ethanol precipitation assembles, described alcohol concn 50-90%, gathering time 10-50min, adding glutaraldehyde after the gathering, to carry out crosslinked cross-linked enzyme aggregate be the immobilization Sumizyme MP again, described glutaraldehyde concentration 10-50%, crosslinking time 1-3h; (2) soybean protein isolate is added water and be hybridly prepared into soybean protein isolate solution, regulate pH and temperature, add again the immobilized enzyme that obtains in the step (1) and carry out enzymolysis, enzymolysis time 2-4h, enzyme concentration 3000-7000U/g, enzymolysis pH 8-10, hydrolysis temperature 40-80 ℃, reclaim immobilized enzyme behind the enzymolysis, enzymolysis solution gets supernatant liquor through centrifugation, and supernatant liquor namely gets soybean polypeptide through lyophilize.
The preferred parameter of described immobilization protex-6L Sumizyme MP preparation is: alcohol concn 80%, assemble time 30min, glutaraldehyde concentration 40%, crosslinking time 2.5h.
Described enzymolysis preferred parameter is: enzymolysis time 2.84h, enzyme concentration 5450U/g, enzymolysis pH 9.5,50 ℃ of hydrolysis temperatures.
The inventive method adopts the carrier-free immobilization Sumizyme MP, at first use the precipitation agent aggregation enzyme, adding linking agent makes enzyme aggregate crosslinked again, namely get cross-linked enzyme aggregate---the immobilization Sumizyme MP, therefore this immobilized enzyme greatly reduces cost owing to need not carrier with high costs, use this immobilized enzyme enzymatic soybean protein isolate and extract soybean polypeptide, extraction process is simple, and enzyme is recyclable reuses, and has reduced the waste of resource.
Description of drawings
Fig. 1 is the process route chart of the inventive method;
Fig. 2 alcohol concn is on the impact of enzymatic activity recovery;
Fig. 3 assembles the time to the impact of enzymatic activity recovery;
Fig. 4 glutaraldehyde concentration is on the impact of enzymatic activity recovery;
Fig. 5 crosslinking time is on the impact of enzymatic activity recovery;
Fig. 6 enzymolysis time and enzyme concentration are alternately to the response surface of polypeptide extraction yield;
Fig. 7 enzymolysis time and enzymolysis pH are alternately to the response surface of polypeptide extraction yield;
Fig. 8 enzyme concentration and hydrolysis temperature are alternately to the response surface of polypeptide extraction yield;
Fig. 9 enzymolysis pH and hydrolysis temperature are alternately to the response surface of polypeptide extraction yield.
Specific embodiments
Below in conjunction with accompanying drawing the specific embodiment of the invention is described in detail,
A kind of Immobilized Enzymes Without Carriers prepares the method for soybean polypeptide, the method may further comprise the steps: (1) is dissolved in the protex-6L Sumizyme MP in the phosphate buffered saline buffer, adding the ethanol precipitation assembles, described alcohol concn 50-90%, gathering time 10-50min, adding glutaraldehyde after the gathering, to carry out crosslinked cross-linked enzyme aggregate be the immobilization Sumizyme MP again, described glutaraldehyde concentration 10-50%, crosslinking time 1-3h; (2) soybean protein isolate is added water and be hybridly prepared into soybean protein isolate solution, regulate pH and temperature, add again the immobilized enzyme that obtains in the step (1) and carry out enzymolysis, enzymolysis time 2-4h, enzyme concentration 3000-7000U/g, enzymolysis pH 8-10, hydrolysis temperature 40-80 ℃, reclaim immobilized enzyme behind the enzymolysis, enzymolysis solution gets supernatant liquor through centrifugation, and supernatant liquor namely gets soybean polypeptide through lyophilize.
The preferred parameter of described immobilization protex-6L Sumizyme MP preparation is: alcohol concn 80%, assemble time 30min, glutaraldehyde concentration 40%, crosslinking time 2.5h.
Described enzymolysis preferred parameter is: enzymolysis time 2.84h, enzyme concentration 5450U/g, enzymolysis pH 9.5,50 ℃ of hydrolysis temperatures.
Embodiment 1: the shaker test of preparation carrier-free immobilization Sumizyme MP optimum parameter
1 materials and methods
1.1 material, reagent
| The protex-6L Alcalase | Denmark novo company |
| Sodium phosphate dibasic | Commercially available analytical pure |
| SODIUM PHOSPHATE, MONOBASIC | Commercially available analytical pure |
| Ethanol | Commercially available analytical pure |
| Glutaraldehyde | Commercially available analytical pure |
1.2 key instrument equipment
| PHS-25 type acidometer | Shanghai great achievement instrument plant |
| Electronic analytical balance | Mei Lete-Tuo benefit instrument (Shanghai) Co., Ltd. |
| Whizzer | Beijing Medical Centrifugal Machine Factory |
| Electric-heated thermostatic water bath | Yuyao City east electric instrument factory |
| Freeze drier | Shanghai Qianjian Instrument Co., Ltd. |
1.3 test method
1.3.1 the preparation of carrier-free immobilization Sumizyme MP
Taking by weighing a certain amount of Sumizyme MP, is that Sodium phosphate dibasic/phosphate sodium dihydrogen buffer solution of 7 mixes with pH, drips the dehydrated alcohol precipitation again and assemble under room temperature, and the simultaneously stirring while dripping precipitates rear centrifugation fully wait the proteolytic enzyme that dissociates and gets throw out.In this precipitation, drip certain density glutaraldehyde cross-linking agent, stirring makes it mix again, centrifuging and taking throw out behind 4 ℃ of lower crosslinked certain hours, with the phosphate buffered saline buffer washing for several times, final separation is precipitated freeze-drying and gets cross-linked enzyme aggregate, i.e. the carrier-free immobilization Sumizyme MP.
1.3.2 enzyme activity determination
The Sumizyme MP enzyme activity determination adopts standard SB/T13017-1999, and namely forint-phenol law mensuration Sumizyme MP enzyme is lived, and immobilized enzyme unit is U/g.Enzyme activity, take the enzyme activity maximum value as 100%, its remainder values and its contrast, namely enzyme activity is expressed as a percentage.
2 results and discussion
2.1 the suitableeest alcohol concn determines
As shown in Figure 2, be 7 when assembling pH, assembling temperature is 25 ℃, the gathering time is when being 30min, be 50%, 60%, 70%, 80%, 90% to precipitate aggreation take enzymatic activity recovery as investigating index, choosing respectively alcohol concn, determine that the suitableeest alcohol concn is 80%.
2.2 determining of the suitableeest gathering time
As shown in Figure 3, be 7 when assembling pH, assembling temperature is 25 ℃, when alcohol concn is 70%, be that 10min, 20min, 30min, 40min, 50min precipitate aggreation take enzymatic activity recovery as investigating index, choosing respectively the gathering time, determine that the suitableeest gathering time is 30min.
2.3 the suitableeest glutaraldehyde concentration determines
As shown in Figure 4, when crosslinked pH is 5, crosslinking temperature is 4 ℃, when crosslinking time is 2h, is 10%, 20%, 30%, 40%, 50% to carry out crosslinking reaction take enzymatic activity recovery as investigating index, choosing respectively glutaraldehyde concentration, determines that the suitableeest glutaraldehyde concentration is 40%.
2.4 the suitableeest crosslinking time determines
As shown in Figure 5, when crosslinked pH is 5, crosslinking temperature is 4 ℃, when glutaraldehyde concentration is 30%, is that 1h, 1.5h, 2h, 2.5h, 3h carry out crosslinking reaction take enzymatic activity recovery as investigating index, choosing respectively crosslinking time, determines that the suitableeest crosslinking time is 2.5h.
Embodiment 2: the shaker test of enzymolysis process optimum parameter
1 materials and methods
1.1 material, reagent
| Soybean protein isolate | Ha Gaoke |
| The protex-6L Alcalase | Denmark novo company |
| Sodium phosphate dibasic | Commercially available analytical pure |
| SODIUM PHOSPHATE, MONOBASIC | Commercially available analytical pure |
| Ethanol | Commercially available analytical pure |
| Glutaraldehyde | Commercially available analytical pure |
1.2 key instrument equipment
| PHS-25 type acidometer | Shanghai great achievement instrument plant |
| Electronic analytical balance | Mei Lete-Tuo benefit instrument (Shanghai) Co., Ltd. |
| Whizzer | Beijing Medical Centrifugal Machine Factory |
| The electric precise stirrer | Jintan City, Jiangsu Province high honour instrument manufacturing company limited |
| Electric-heated thermostatic water bath | Yuyao City east electric instrument factory |
| Constant incubator | Beijing is bright Medical Instruments factory forever |
| Freeze drier | Shanghai Qianjian Instrument Co., Ltd. |
1.3 test method
1.3.1 technical process
Soybean protein isolate → adding water mixes → regulates pH and temperature → adding immobilized enzyme enzymolysis → recovery immobilized enzyme → enzymolysis solution → centrifugation → supernatant liquor → lyophilize → soybean polypeptide
1.3.2 calculation formula
2 results and discussion
2.1 experimental factor level code table
On the basis of single factor research, choosing enzymolysis time, enzyme concentration, enzymolysis pH, 4 factors of hydrolysis temperature is independent variable(s), take the polypeptide extraction yield as response value, according to the center combination principle of design, the test of design response surface analysis, its level of factor coding schedule sees Table table 2-1.
Table 2-1 level of factor coding schedule
2.2 response surface experimental establishment and experimental result
This experimental applications response surface optimized method carries out process optimization.Take A, B, C, D as independent variable(s), take the polypeptide extraction yield as response value R, response surface experimental program and the results are shown in Table 2-2.Experiment 1-24 is factorial experiment, and 25-36 is 12 center tests, in order to the estimating experiment error.
Table 2-2 test arrangement and result
2.3 response surface test result analysis
Polypeptide extraction yield R carries out data analysis by statistical analysis software Design-Expert, and it is as follows to set up Quadratic response surface regression model:
R=64.98+2.84A+3.60B+2.67C-3.50D-5.27AB-2.92AC+1.12AD+0.62BC+1.83BD-4.04CD-0.16A
2-2.77B
2-1.37C
2-1.68D
2
Recurrence and the results of analysis of variance of polypeptide extraction yield R see Table 2-3, and significant response surface analysis is seen Fig. 6-Fig. 9 mutually alternately.
Recurrence and the results of analysis of variance of table 2-3 polypeptide extraction yield
| Variable | Degree of freedom | Sum of squares | All square | The F value | Pr>F |
| A | 1 | 193.23 | 193.23 | 25.19 | ＜0.0001 |
| B | 1 | 311.76 | 311.76 | 40.64 | ＜0.0001 |
| C | 1 | 171.20 | 171.20 | 22.32 | 0.0001 |
| D | 1 | 293.30 | 293.30 | 38.23 | ＜0.0001 |
| AB | 1 | 444.16 | 444.16 | 57.89 | ＜0.0001 |
| AC | 1 | 136.31 | 136.31 | 17.77 | 0.0004 |
| BD | 1 | 53.66 | 53.66 | 6.99 | 0.0152 |
| CD | 1 | 261.63 | 261.63 | 34.10 | ＜0.0001 |
| A 2 | 1 | 0.79 | 0.79 | 0.10 | 0.7512 |
| B 2 | 1 | 245.50 | 245.50 | 32.00 | ＜0.0001 |
| C 2 | 1 | 60.04 | 60.04 | 7.83 | 0.0108 |
| D 2 | 1 | 90.56 | 90.56 | 11.80 | 0.0025 |
| Return | 14 | 2288.29 | 163.45 | 21.31 | ＜0.0001 |
| Residue | 21 | 161.11 | 7.67 | ? | ? |
| Lose and intend | 10 | 75.05 | 7.50 | 0.96 | 0.5223 |
| Error | 11 | 86.06 | 7.82 | ? | ? |
| Summation | 35 | 2449.40 | ? | ? | ? |
By table 2-3 as can be known, the linear relationship between equation dependent variable and the independent variable(s) is obvious, and this model returns significantly (p＜0.0001), and lose and intend item not significantly (p〉0.05), and this model R
2=93.42%, R
2 Adj=89.04%, illustrate that this model is good with the test match, linear relationship is remarkable between independent variable(s) and the response value, and the theory that can be used for this reaction is inferred.Can obtain factor contribution rate by the F check is: B〉D〉A〉C, i.e. enzyme concentration〉hydrolysis temperature〉enzymolysis time〉enzymolysis pH.
Application responds face optimizing analytical procedure is analyzed regression model, seeks as a result enzymolysis time 2.84h of optimal response, enzyme concentration 5450U/g, and enzymolysis pH 9.5, it is about 73.79% that 50 ℃ of hydrolysis temperatures, response value polypeptide extraction yield have optimum value.
2.4 proof test and simultaneous test
Under the top condition that the response surface analysis method is tried to achieve, i.e. enzymolysis time 2.84h, enzyme concentration 5450U/g, enzymolysis pH 9.5,50 ℃ of hydrolysis temperatures carry out 3 parallel tests, and the mean value of 3 parallel test polypeptide extraction yields is 72.48%.Trial value and regression equation predictor that response value is described are coincide good.
Claims (3)
1. an Immobilized Enzymes Without Carriers prepares the method for soybean polypeptide, it is characterized in that, the method may further comprise the steps: (1) is dissolved in the protex-6L Sumizyme MP in the phosphate buffered saline buffer, adding the ethanol precipitation assembles, described alcohol concn 50-90% assembles time 10-50min, adds glutaraldehyde after the gathering again and carries out crosslinked that cross-linked enzyme aggregate is the immobilization Sumizyme MP, described glutaraldehyde concentration 10-50%, crosslinking time 1-3h; (2) soybean protein isolate is added water and be hybridly prepared into soybean protein isolate solution, regulate pH and temperature, add again the immobilized enzyme that obtains in the step (1) and carry out enzymolysis, enzymolysis time 2-4h, enzyme concentration 3000-7000U/g, enzymolysis pH 8-10, hydrolysis temperature 40-80 ℃, reclaim immobilized enzyme behind the enzymolysis, enzymolysis solution gets supernatant liquor through centrifugation, and supernatant liquor namely gets soybean polypeptide through lyophilize.
2. a kind of Immobilized Enzymes Without Carriers according to claim 1 prepares the method for soybean polypeptide, the preferred parameter that it is characterized in that described immobilization protex-6L Sumizyme MP preparation is: alcohol concn 80%, gathering time 30min, glutaraldehyde concentration 40%, crosslinking time 2.5h.
3. a kind of Immobilized Enzymes Without Carriers according to claim 1 prepares the method for soybean polypeptide, it is characterized in that described enzymolysis preferred parameter is: enzymolysis time 2.84h, enzyme concentration 5450U/g, enzymolysis pH 9.5,50 ℃ of hydrolysis temperatures.
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| CN103923900A (en) * | 2014-04-14 | 2014-07-16 | 江南大学 | Preparation method and application of cross-linked enzyme aggregate of bifunctional enzyme for rice wine |
| CN104543371A (en) * | 2015-01-17 | 2015-04-29 | 东北农业大学 | Method for enzymolysis of soybean meal by employing gas-liquid-solid magnetically stabilized fluidized bed |
| CN106520880A (en) * | 2016-12-05 | 2017-03-22 | 东北农业大学 | Method for removing bitter taste of aqueous enzymatic method soybean polypeptide by using immobilized enzyme |
| CN106520735A (en) * | 2016-09-23 | 2017-03-22 | 天津大学 | Crosslinking protease aggregate and application for preparing black soybean hypotensive activity peptide |
| CN108094674A (en) * | 2018-01-10 | 2018-06-01 | 安徽中森生物技术有限公司 | A kind of method that Freeze Drying Technique prepares soybean peptide |
| CN108949887A (en) * | 2018-09-04 | 2018-12-07 | 哈尔滨工业大学 | A kind of preparation method of the multi-functional incretin peptide of soybean |
| WO2021142617A1 (en) * | 2020-01-14 | 2021-07-22 | 吉林凯莱英医药化学有限公司 | Immobilized enzyme, and preparation method therefor and use thereof |
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| CN103923900A (en) * | 2014-04-14 | 2014-07-16 | 江南大学 | Preparation method and application of cross-linked enzyme aggregate of bifunctional enzyme for rice wine |
| CN103923900B (en) * | 2014-04-14 | 2016-03-23 | 江南大学 | A kind of preparation and application of yellow rice wine bifunctional enzyme cross-linked enzyme aggregate |
| CN104543371A (en) * | 2015-01-17 | 2015-04-29 | 东北农业大学 | Method for enzymolysis of soybean meal by employing gas-liquid-solid magnetically stabilized fluidized bed |
| CN106520735A (en) * | 2016-09-23 | 2017-03-22 | 天津大学 | Crosslinking protease aggregate and application for preparing black soybean hypotensive activity peptide |
| CN106520880A (en) * | 2016-12-05 | 2017-03-22 | 东北农业大学 | Method for removing bitter taste of aqueous enzymatic method soybean polypeptide by using immobilized enzyme |
| CN108094674A (en) * | 2018-01-10 | 2018-06-01 | 安徽中森生物技术有限公司 | A kind of method that Freeze Drying Technique prepares soybean peptide |
| CN108949887A (en) * | 2018-09-04 | 2018-12-07 | 哈尔滨工业大学 | A kind of preparation method of the multi-functional incretin peptide of soybean |
| CN108949887B (en) * | 2018-09-04 | 2022-03-01 | 哈尔滨工业大学 | Preparation method of soybean multifunctional hypoglycemic peptide |
| WO2021142617A1 (en) * | 2020-01-14 | 2021-07-22 | 吉林凯莱英医药化学有限公司 | Immobilized enzyme, and preparation method therefor and use thereof |
| CN116574521A (en) * | 2023-05-20 | 2023-08-11 | 广州宏度精细化工有限公司 | Preparation method of plant hydrolyzed protein amino acid derivative surfactant |
| CN116574521B (en) * | 2023-05-20 | 2023-10-27 | 广州宏度精细化工有限公司 | Preparation method of plant hydrolyzed protein amino acid derivative surfactant |
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