CN114606286B - Novel enzymolysis extraction method of soybean peptide powder with multifunctional characteristic - Google Patents
Novel enzymolysis extraction method of soybean peptide powder with multifunctional characteristic Download PDFInfo
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- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
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- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
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- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
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Abstract
The invention provides a novel enzymolysis extraction method of soybean peptide powder with multifunctional characteristics, which comprises the following steps: (1) microfluidizing the isolated soy protein; (2) synthesis of protease composite materials; (3) preparation of protease biocatalysis membrane; (4) deep enzymolysis of an enzyme membrane reactor; (5) spray drying to prepare soybean peptide powder. The extraction method of the soybean peptide provided by the invention has the advantages of high efficiency, repeated utilization, high yield of the prepared soybean peptide, excellent functional characteristics and extremely high development and utilization values.
Description
Technical Field
The invention relates to the technical field of food development, in particular to a novel enzymolysis extraction method of soybean peptide powder with multifunctional characteristics.
Background
The soybean peptide is a small-molecule protein, has a molecular weight range below 5000, and is very easy to be absorbed and utilized by human bodies. The small molecular peptides with the molecular weight of about 1000 are generally considered to be best, and the soybean peptide with the molecular weight can be well absorbed by human bodies and can ensure the functionality and activity of the peptide. The 2-10 peptide belongs to oligopeptides, and the 10-50 peptide belongs to polypeptides, and generally, the following decapeptides have medical and commercial practicability.
The soybean peptide has the effects of reducing blood fat, improving immunity, whitening skin, resisting cancer, resisting oxidation and the like, and certain low molecular peptides can provide nutrient substances required by the growth and development of human bodies and are easier to digest and absorb by the human bodies. However, the processing functional properties of the modified soybean protein product after the modification of the structure, such as the emulsifying property, have not been high-emulsifying soybean peptide products which have high commercial value and are comparable to macromolecular emulsifying agents such as sodium caseinate. In order to meet the requirements of consumers on natural and green foods and meet the requirements of people on pursuing nutrition, low fat and environmental protection, development of a healthy and safe multifunctional soybean peptide powder is needed.
With the development of the domestic enzymology industry and the more and more intensive research on soybean functional components, soybean peptide is taken as a novel soybean deep processing product, must be further developed and utilized, and has huge market potential. Functional properties of proteins include emulsifying, foaming, water and oil retention properties, and the like. The process conditions of the enzymatic preparation of the soybean polypeptide are generally mild, but the reaction conditions are limited due to the difference of physicochemical properties of the enzyme, so that the enzyme is easy to inactivate and the reaction is incomplete in the enzymolysis process of the conventional enzymatic preparation, the content of peptide in the obtained product is low, and the quality of the soybean polypeptide is affected, so that in actual production, a novel enzymatic preparation of the soybean polypeptide is required to be developed.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a novel enzymolysis extraction method of soybean peptide powder with multifunctional characteristics. The extraction method of the soybean peptide provided by the invention has the advantages of high efficiency, repeated utilization, high yield of the prepared soybean peptide, excellent functional characteristics and extremely high development and utilization values.
The technical scheme of the invention is as follows:
a novel enzymolysis extraction method of soybean peptide powder with multifunctional characteristics comprises the following steps:
(1) Microfluidization of soy protein isolate: dissolving soybean protein isolate in deionized water, and then microfluidizing the soybean protein isolate at 40-160MPa by using a microfluidizer; subsequently, collecting a sample of the microfluidized soy protein isolate;
(2) Synthesis of protease composite: mixing imidazole-2-formaldehyde and polyvinylpyrrolidone, and then adding deionized water to enable the mass concentration of the imidazole-2-formaldehyde and the polyvinylpyrrolidone to be 5% -15%; dissolving the mixture at 70-90 ℃ for 3-6 hours to obtain a clear solution;
cooling the solution to room temperature, rapidly adding neutral protease, flavourzyme, ficin and alkaline protease into the solution, and uniformly mixing;
then adding 1.5-3wt% zinc nitrate into the mixed solution, standing, centrifuging and collecting precipitate, and washing with deionized water to obtain the protease composite material;
(3) Preparation of protease biocatalytic membrane: firstly, processing bacterial cellulose into cellulose nanofiber slurry by using a homogenizer;
then mixing cellulose nanofiber slurry, carboxylated multiwall carbon nanotubes, the protease composite material prepared in the step (2) and cetyltrimethylammonium bromide into a solution;
finally, using filter paper as a supporting layer, treating the solution through vacuum filtration, and separating a membrane on the surface of the filter paper to finally obtain the protease biocatalysis membrane;
(4) Deep enzymolysis of an enzyme membrane reactor: the protease biocatalysis film obtained in the step (3) is combined with an enzyme film reaction tank, the microfluidized soybean protein isolate sample obtained in the step (1) is added into the enzyme film reaction tank, and is placed in a constant temperature water bath kettle to be heated, a peristaltic pump is started, and the circulation is carried out for 1-5min under normal pressure; then, regulating the pressure of the system to 0.1-0.5MPa, and closing the peristaltic pump after 2-6 hours of operation;
(5) Spray drying to prepare soybean peptide powder: and (3) centrifuging the enzymolysis liquid obtained in the step (4), and performing spray drying on the supernatant obtained after centrifugation to obtain the soybean peptide powder.
Preferably, the concentration of the isolated soy protein of step (1) is 10-20%.
Preferably, the microfluidizer in the step (1) adopts a continuous high shear fluid treatment homogenizer with the model number of M-110EH, and the treatment time is 10-60min.
Preferably, the mixing mass ratio of the oxazole-2-formaldehyde, ICA and polyvinylpyrrolidone in the step (2) is 8-12:1.
Preferably, the neutral protease, the flavourzyme, the ficin and the alkaline protease added in the step (2) are respectively 1-5wt%; mixing time is 5-20min after adding the protease.
Preferably, in the step (2), zinc nitrate is added to the mixed solution and then the mixed solution is kept stand for 8 to 15 minutes.
Preferably, the mass ratio of the cellulose nanofiber slurry in the step (3), the carboxylated multiwall carbon nanotubes, the protease composite material prepared in the step (1) and cetyltrimethylammonium bromide (CTAB) is 4-6:1-3:1:1.
Preferably, in the step (3), when the bacterial cellulose is processed into cellulose nanofiber slurry by using a homogenizer, the rotation speed of the homogenizer is 10000rmp-14000rpm, and the homogenization time is 1-5min;
the average diameter of the carboxylated multi-wall carbon nano-tube is 2-8nm, and the average length is 5-16 mu m.
Preferably, the reaction conditions in step (4) are pH:6-11, temperature: 40-60 ℃.
Preferably, in the step (5), the centrifugal rotation speed is 5000-10000rmp, the centrifugal rotation speed is 20-60min, the spray drying temperature is 140-200 ℃, and the spray pressure is 0.1-1MPa.
The beneficial technical effects of the invention are as follows:
1. the invention improves the solubility of the soybean protein isolate by utilizing the microfluidization technology, and the microfluidization can change the physicochemical and functional characteristics of the protein, thereby improving the solubility, the emulsifying property, the foamability and other characteristics of the protein. The microfluidization is used as an effective auxiliary enzymolysis pretreatment method, can change the protein structure and influence the cleavage site of enzyme, so that the hydrolysis efficiency and the product property are changed, and the microfluidization can greatly improve the hydrolysis degree of the isolated soy protein.
2. The composite material is used as a matrix for fixing biological macromolecules, so that the enzyme can be protected from the external environment, and the fixation of the organic framework is even beneficial to enhancing the biological activity of the enzyme. The invention prepares the organic framework into a continuous membrane material, can solve the recycling problem, adopts the environment-friendly fiber material bacterial cellulose, and has high flexibility, strength and excellent biocompatibility. The use of the biocatalysis membrane not only can make a plurality of proteases simultaneously perform enzymatic reaction, but also can improve the utilization rate of the proteases and reduce the production cost.
3. The enzyme membrane reactor is a device for coupling enzyme catalytic reaction and membrane separation, and a system or an operation unit for combining the membrane and biochemical reaction integrates biological reaction with in-situ separation, concentration and enzyme recycling of reaction products by means of specificity, catalysis and membrane specific functions of the enzyme, so that the reaction process can be changed, the reaction progress can be controlled, the generation of byproducts can be reduced, and the soybean peptide yield can be improved.
4. The invention adopts biological composite material to prepare various proteases into composite protease material, improves the enzymatic reaction efficiency and range, synthesizes the prepared composite protease material into a biological catalytic film, repeatedly utilizes the composite protease, reduces the cost, and finally carries out enzymolysis on soybean protein isolate by combining an enzyme film reactor, greatly improves the enzymatic reaction process, increases the enzymolysis on the soybean protein isolate, improves the conversion rate of soybean peptide, and finally obtains soybean peptide powder by spray drying.
Detailed Description
The present invention will be specifically described with reference to examples. It will be apparent that the described embodiments 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:
(1) Microfluidization of soy protein isolate: a 10% soy protein isolate dispersion was prepared using deionized water. Then, the mixture was subjected to microfluidization at 40MPa using an M-110EH microfluidizer for a treatment time of 60 minutes. Subsequently, a sample of the microfluidized soy protein isolate is collected.
(2) Synthesis of protease composite: mixing imidazole-2-formaldehyde and polyvinylpyrrolidone according to the proportion of 8:1, and then adding deionized water to enable the mass concentration of the imidazole-2-formaldehyde and the polyvinylpyrrolidone to be 15%. Dissolving the mixture at 70 ℃ for 3 hours to obtain a clear solution;
after the solution was cooled to room temperature, 1% neutral protease, flavourzyme, ficin and alkaline protease were added to the solution, respectively, and mixed for 5min.
Then, 1.5% zinc nitrate was added to the solution, and the mixture was allowed to stand for 10 minutes. Finally, the precipitate was collected by centrifugation and washed with deionized water. Finally obtaining the protease composite material.
(3) Preparation of protease biocatalytic membrane: first, bacterial cellulose is processed into cellulose nanofiber slurry using a homogenizer. And (2) mixing cellulose nanofiber slurry, carboxylated multi-wall carbon nanotubes, the protease composite material prepared in the step (2) and cetyltrimethylammonium bromide into a solution according to the mass ratio of 4:1:1:1.
Wherein, the model of the homogenizer is GRX2000/4, the rotating speed is 10000rmp, and the time is 5min; the carboxylated multiwall carbon nanotubes had an average diameter of 4nm and an average length of 10. Mu.m.
Finally, the filter paper is used as a supporting layer, the mixture solution is processed through vacuum filtration, and the membrane on the surface of the filter paper is separated, so that the protease biocatalysis membrane is finally obtained.
(4) Deep enzymolysis of an enzyme membrane reactor: combining the novel biocatalysis film obtained in the step (3) with an enzyme film reaction tank, adding the microfluidized soybean protein isolate solution processed in the step (1) into the enzyme film reaction tank, heating the solution in a constant-temperature water bath kettle, adjusting the pH to 6, heating to 40 ℃, opening a peristaltic pump, and circulating for 1min at normal pressure; then, regulating the pressure of the system to 0.1MPa, and closing the peristaltic pump after 2 hours of operation;
(5) Spray drying to prepare soybean peptide powder: and (3) centrifuging the enzymolysis liquid treated in the step (4) at the rotation speed of 5000rmp for 60min, and performing spray drying on the supernatant obtained after centrifugation at the drying temperature of 140 ℃ and the spray pressure of 0.1MPa.
Example 2
(1) Microfluidization of soy protein isolate: a 15% soy protein isolate dispersion was prepared using deionized water. Then, the mixture was subjected to microfluidization at 100MPa using an M-110EH microfluidizer for a treatment time of 30 minutes. Subsequently, a sample of the microfluidized soy protein isolate is collected.
(2) Synthesis of protease composite: mixing imidazole-2-formaldehyde and polyvinylpyrrolidone according to the proportion of 10:1, and then adding deionized water to enable the mass concentration of the imidazole-2-formaldehyde and the polyvinylpyrrolidone to be 10%. Dissolving the mixture at 80 ℃ for 4.5 hours to obtain a clear solution;
after the solution was cooled to room temperature, 2.5% neutral protease, flavourzyme, ficin and alkaline protease were added to the solution, respectively, and mixed for 15min.
Then, 2% zinc nitrate was added to the solution, and the mixture was allowed to stand for 10 minutes. Finally, the precipitate was collected by centrifugation and washed with deionized water. Finally obtaining the protease composite material.
(3) Preparation of protease biocatalytic membrane: first, bacterial cellulose is processed into cellulose nanofiber slurry using a homogenizer. And (2) mixing the cellulose nanofiber slurry, the carboxylated multi-wall carbon nanotubes, the protease composite material prepared in the step (2) and cetyltrimethylammonium bromide into a solution according to the mass ratio of 5:2:1:1.
Wherein, the model of the homogenizer is GRX2000/4, the rotating speed is 12000rmp, the average diameter of the multi-wall carbon nano tube is 5nm, and the average length is 12 μm.
Finally, the filter paper is used as a supporting layer, the mixture solution is processed through vacuum filtration, and the membrane on the surface of the filter paper is separated, so that the protease biocatalysis membrane is finally obtained.
(4) Deep enzymolysis of an enzyme membrane reactor: combining the novel biocatalysis membrane obtained in the step (3) with an enzyme membrane reaction tank, adding the microfluidized soybean protein isolate solution treated in the step (1) into the enzyme membrane reaction tank, heating the mixture in a constant-temperature water bath kettle, adjusting the pH to 9, heating to 50 ℃, opening a peristaltic pump, and circulating for 3min at normal pressure; then, regulating the pressure of the system to 0.25MPa, and closing the peristaltic pump after running for 4 hours;
(5) Spray drying to prepare soybean peptide powder: and (3) centrifuging the enzymolysis liquid treated in the step (4) at 8000rmp for 45min, and performing spray drying on the supernatant obtained after centrifugation at 180 ℃ under the spray pressure of 0.5MPa.
Example 3
(1) Microfluidization of soy protein isolate: a 20% soy protein isolate dispersion was prepared using deionized water. Then, the mixture was subjected to microfluidization using an M-110EH microfluidizer at 160MPa for a treatment time of 60 minutes. Subsequently, a sample of the microfluidized soy protein isolate is collected.
(2) Synthesis of protease composite: mixing imidazole-2-formaldehyde and polyvinylpyrrolidone according to a ratio of 12:1, and then adding deionized water to enable the mass concentration of the imidazole-2-formaldehyde and the polyvinylpyrrolidone to be 5%. Dissolving the mixture at 90 ℃ for 6 hours to obtain a clear solution;
after the solution was cooled to room temperature, 5% neutral protease, flavourzyme, ficin and alkaline protease were added to the solution, respectively, and mixed for 20min.
Then, 3% zinc nitrate was added to the solution, and the mixture was allowed to stand for 10 minutes. Finally, the precipitate was collected by centrifugation and washed with deionized water. Finally obtaining the protease composite material.
(3) Preparation of protease biocatalytic membrane: first, bacterial cellulose is processed into cellulose nanofiber slurry using a homogenizer. And (2) mixing the cellulose nanofiber slurry, the carboxylated multi-wall carbon nanotubes, the protease composite material prepared in the step (2) and cetyltrimethylammonium bromide into a solution according to the mass ratio of 6:3:1:1.
Wherein, the model of the homogenizer is GRX2000/4, the rotating speed is 14000rmp, and the time is 1min; the carboxylated multiwall carbon nanotubes had an average diameter of 6nm and an average length of 8. Mu.m.
Finally, the filter paper is used as a supporting layer, the mixture solution is processed through vacuum filtration, and the membrane on the surface of the filter paper is separated, so that the protease biocatalysis membrane is finally obtained.
(4) Deep enzymolysis of an enzyme membrane reactor: combining the novel biocatalysis membrane obtained in the step (3) with an enzyme membrane reaction tank, adding the microfluidized soybean protein isolate solution treated in the step (1) into the enzyme membrane reaction tank, heating the mixture in a constant-temperature water bath, adjusting the pH to 11, heating to 60 ℃, opening a peristaltic pump, and circulating for 5min at normal pressure; then, regulating the pressure of the system to 0.5MPa, and closing the peristaltic pump after 6 hours of operation;
(5) Spray drying to prepare soybean powder: and (3) centrifuging the enzymolysis liquid treated in the step (4) at the rotating speed of 10000rmp for 20min, and performing spray drying on the supernatant obtained after centrifugation at the drying temperature of 200 ℃ and the spray pressure of 1MPa.
Test example:
the emulsification activity, emulsification stability, foamability, foam stability, water holding ability, oil holding ability and peptide content of the soybean peptide prepared by the experimental group were measured, and the emulsification activity and emulsification stability of soybean phospholipid, foamability and foam stability of egg white, water holding ability of starch phosphate and oil holding ability of starch were used as standards (the functional characteristics of the standard were set to 100%), and the specific measurement methods were as follows, and the measurement results are shown in table 1.
(1) Determination of the emulsibility: preparing 1% (m/V) solution to be measured by using 0.2mol/L phosphate solution with pH of 7.0, taking 30mL of the solution into a 100mL beaker, adding 10mL of soybean oil, homogenizing the mixed solution for 2min at a speed of 5000r/min by using a homogenizer, and preparing emulsion for later use. 50. Mu.L of the bottom emulsion was aspirated with a micropipette at 0 and 10min, respectively, and 25mL of 0.1% (m/V) SDS solution was added and mixed well (500-fold dilution). The absorbance at 500nm was measured immediately with the same SDS solution as a blank. The Emulsion Activity (EAI) and Emulsion Stability (ESI) were calculated as follows:
a0 is the absorbance of the emulsion which is diluted rapidly after homogenization; c is the protein concentration (g/mL) in the aqueous protein solution before emulsion formation; phi is the volume fraction of oil in the emulsion (1/4).
A0 is the absorbance of the emulsion which is diluted rapidly after homogenization; a10 is absorbance of the emulsion after standing for 10min; t is time (10 min).
(2) Determination of foamability: preparing 1% solution to be measured with 0.2mol/L, pH as 7.0 phosphate solution, taking 40mL with a pipette into a 100mL small beaker, homogenizing at 5000r/min with a homogenizer for 2min, immediately transferring into a measuring cylinder, and recording foam volume V after homogenizing stop and 30min respectively 1 、V 2 . The foaming power (FAI) and Foam Stability (FSI) are calculated as follows:
v1 is the foam volume at the time of homogenization stop.
V1 is the foam volume at the time of homogenization stop; v2 is the foam volume after 30min of homogenization stop.
(3) Determination of Water and oil retention: weigh 0.5g (m) 0 ) 10mL of water or soybean oil is added to soybean peptide, shaking is carried out fully, centrifugation is carried out for 30min at 4000r/min, water or soybean oil is discarded, mass (m 1) of sediment after centrifugation is weighed, and water retention (h 1) or oil retention (h 2) is calculated according to formula (5).
(4) Peptide content (molecular weight below 5000 Da) = (peptide mass/total protein mass of 5000 Da) ×100%
TABLE 1
As can be seen from table 1, example 2 is most preferred. The prepared soybean peptide has higher emulsifying activity, emulsifying stability, foamability, foam stability, water-holding capacity and oil-holding capacity than the reference standard, and the prepared soybean peptide has the highest content.
Comparative example:
on the basis of example 2, a comparative example was set up:
comparative example 1: the microfluidization treatment was omitted and the other processing procedures were the same as in example 2.
Comparative example 2: the biological composite material is omitted, the composite protease is used instead, and other processing techniques are the same as those of the embodiment 2.
Comparative example 3: the biocatalytic film was omitted and the other processing was the same as in example 2.
Comparative example 4: the enzymatic hydrolysis of the enzyme membrane reactor was omitted and the other processing steps were the same as in example 2.
The functional properties and peptide content of each control group were measured, and the specific measurement methods were the same as those of the test examples, and the measurement results are shown in table 2.
TABLE 2
As can be seen from Table 2, the soybean peptide prepared in example 2 was significantly improved in emulsification activity, emulsification stability, foamability, foam stability, water-holding capacity, oil-holding capacity and peptide content.
Although the embodiments of the present invention have been disclosed in the foregoing description and drawings, it is not limited to the details of the embodiments and examples, but is to be applied to all the fields of application of the present invention, it will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the spirit and scope of the invention as defined by the appended claims and their equivalents.
Claims (1)
1. A novel enzymolysis extraction method of soybean peptide powder with multifunctional characteristics is characterized by comprising the following steps:
(1) Microfluidization of soy protein isolate: preparing a 15% soy protein isolate dispersion using deionized water; then, using an M-110EH microfluidizer to carry out microfluidization under 100MPa, wherein the treatment time is 30min; subsequently, collecting a sample of the microfluidized soy protein isolate;
(2) Synthesis of protease composite: mixing imidazole-2-formaldehyde and polyvinylpyrrolidone according to the proportion of 10:1, and then adding deionized water to ensure that the mass concentration of the imidazole-2-formaldehyde and the polyvinylpyrrolidone is 10%; dissolving the mixture at 80 ℃ for 4.5 hours to obtain a clear solution;
after the solution is cooled to room temperature, 2.5 percent of neutral proteinase, flavourzyme, ficin and alkaline proteinase are respectively added into the solution rapidly and mixed for 15 minutes;
then adding 2% zinc nitrate into the solution, and standing for 10min; finally, centrifugally collecting sediment and using deionized water; finally obtaining the protease composite material;
(3) Preparation of protease biocatalytic membrane: firstly, processing bacterial cellulose into cellulose nanofiber slurry by using a homogenizer; then mixing cellulose nanofiber slurry, carboxylated multi-wall carbon nanotubes, the protease composite material prepared in the step (2) and cetyltrimethylammonium bromide into a solution according to the mass ratio of 5:2:1:1;
wherein, the model of the homogenizer is GRX2000/4, the rotating speed is 12000rmp, the average diameter of the multi-wall carbon nano tube is 5nm, and the average length is 12 μm;
finally, using filter paper as a supporting layer, and vacuum filtering to treat the mixture solution, and separating a membrane on the surface of the filter paper to finally obtain the protease biocatalysis membrane;
(4) Deep enzymolysis of an enzyme membrane reactor: combining the novel biocatalysis membrane obtained in the step (3) with an enzyme membrane reaction tank, adding the microfluidized soybean protein isolate solution treated in the step (1) into the enzyme membrane reaction tank, heating the mixture in a constant-temperature water bath kettle, adjusting the pH to 9, heating to 50 ℃, opening a peristaltic pump, and circulating for 3min at normal pressure; then, regulating the pressure of the system to 0.25MPa, and closing the peristaltic pump after running for 4 hours;
(5) Spray drying to prepare soybean peptide powder: and (3) centrifuging the enzymolysis liquid treated in the step (4) at 8000rmp for 45min, and performing spray drying on the supernatant obtained after centrifugation at 180 ℃ under the spray pressure of 0.5MPa.
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