CN114395600B - Preparation method and application of multifunctional pea peptide - Google Patents
Preparation method and application of multifunctional pea peptide Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/011—Hydrolysed proteins; Derivatives thereof from plants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
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- C—CHEMISTRY; METALLURGY
- 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
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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Abstract
The invention discloses a preparation method and application of multifunctional pea peptide, which are applied to the technical field of biological medicine, and the preparation method of the multifunctional pea peptide comprises the following steps: s1, preparing pea protein feed liquid; s2, preparing compound protease: 1) Fermentation: inoculating bacillus natto into pea protein feed liquid for fermentation, centrifuging fermentation liquor, taking supernatant, and drying to obtain compound protease; 2) Compounding: compounding the compound protease with papain, alkaline protease and flavourzyme to prepare the compound protease; s3, enzymolysis; s4, membrane filtration; s5, drying. The method has the advantages of simple process, short period, high yield, low cost, high yield of the prepared pea peptide, high protein recovery rate, small relative molecular weight of protein hydrolysate, good product flavor, and high-quality pea peptide product, and has the functions of reducing blood pressure, reducing uric acid and resisting oxidation.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a preparation method and application of multifunctional pea peptide.
Background
The pea is a leguminous pea plant, also called netherlands, wheat beans, and Pisum sativum, and is rich in starch, protein, vitamins, minerals, and various amino acids essential to human body. Pea protein is an excellent plant protein, the essential amino acid composition of the pea protein is relatively close to that of the FAO/WHO recommended mode, the sensitization is low, the nutritive value is high, and the pea protein is widely paid attention to.
However, pea proteins cannot fully exert their biological activity in the human body due to poor solubility and digestibility of proteins, and thus, it is necessary to treat pea proteins by a certain means to improve their physiological activity and to utilize the nutritional value of pea proteins to a greater extent.
The existing advanced utilization treatment method for pea protein mainly comprises an enzymolysis treatment method and an enzymolysis and fermentation treatment method: the enzymatic hydrolysis method is to treat pea protein by adopting industrial protease, and has the advantages of low enzyme preparation specificity, low enzymolysis efficiency, low product yield, larger average relative molecular weight of pea peptide, small protein hydrolysate with molecular weight less than 1000u, low polypeptide content and heavier bitter taste; the enzymolysis and fermentation treatment method adopts industrial protease to carry out enzymolysis on pea protein, and then carries out fermentation on the enzymolysis liquid to prepare pea peptide, so that the production period is long, and the problem of low product yield also exists.
Therefore, how to provide a pea peptide preparation method which has simple process, short period, high yield and low cost and is more suitable for industrial production.
The pea peptide is prepared by adopting methods of enzymolysis, microbial fermentation, physical synergistic enzymolysis and the like, and is an effective method for deeply utilizing pea protein resources. The pea protein resources in China are rich, and great development potential exists. Therefore, how to fully utilize pea protein resources is of great significance to the development of the pea industry.
In recent years, with the rise of enzyme industry technology, the use of proteases to hydrolyze macromolecular proteins into readily absorbable small peptides has received widespread attention from various industries. Therefore, the pea protein is prepared into pea peptide by utilizing an enzymolysis technology, and is an effective means for improving the nutrition performance and high-value utilization of the pea protein. The prior report of preparing pea peptide mainly adopts an enzymolysis method, but the research on preparing pea peptide by combining fermentation and enzymolysis has not been reported yet. .
Therefore, how to provide a pea peptide preparation method which has simple process, short period, high yield and low cost and is more suitable for industrial production.
Disclosure of Invention
The invention discloses a preparation method of multifunctional pea peptide, which has the advantages of simple process, short period, high yield and low cost and is suitable for industrial production.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a preparation method of multifunctional pea peptide comprises the following steps:
s1, preparing pea protein feed liquid:
mixing pea protein powder with water, and preparing pea protein feed liquid;
s2, preparing compound protease:
1) Fermentation:
inoculating bacillus natto into pea protein feed liquid for fermentation, centrifuging fermentation liquor, taking supernatant, and drying to obtain compound protease;
2) Compounding:
compounding the compound protease with papain, alkaline protease and flavourzyme to prepare the compound protease;
s3, enzymolysis:
taking pea protein feed liquid as a substrate, adding compound protease, stirring for enzymolysis, and inactivating enzyme after the enzymolysis is finished to obtain pea peptide enzymolysis liquid;
s4, membrane filtration:
centrifuging pea peptide enzymolysis liquid to obtain supernatant, and sequentially performing microfiltration, ultrafiltration and nanofiltration membrane filtration to obtain nanofiltration components with relative molecular mass less than 1000 u;
s5, drying:
and (5) drying the nanofiltration component to obtain the pea peptide.
The invention firstly compounds the composite protease generated by fermenting pea protein by bacillus natto with three industrial enzymes, and utilizes the enzyme preparation after the compounding to carry out enzymolysis on the pea protein to prepare pea peptide; the bacillus natto takes pea protein as the sole carbon source for fermentation, the composite protease obtained by fermentation has strong enzymolysis effect specificity on pea protein and high enzymolysis efficiency, and the obtained product has stronger biological function activity.
The composite protease produced by fermenting pea protein by bacillus natto consists of a plurality of endo-enzyme and exo-enzyme, has a plurality of enzyme cutting sites, is compounded with papain, alkaline protease and flavourzyme, not only can improve the enzymolysis efficiency of pea protein and the product yield, but also can effectively improve the quality of pea peptide, has high polypeptide content, mainly comprises four-octapeptide, has a large proportion of protein hydrolysate with molecular weight less than 1000u, and has bitter taste peptide effectively hydrolyzed by a plurality of enzymes, so that the flavor of the product is improved. And the enzymolysis products of different enzyme cutting sites of the compound protease are rich, so that the pea peptide has multiple biological functional activities.
Compared with the prior enzymolysis technology, the compound protease is added at the same time without adding the protease step by step and time-sharing, so that the production process is simplified, the production time of each batch is shortened, the energy consumption is reduced, and the cost is low; in addition, the enzymolysis process of the compound protease does not need to add chemical reagents such as sodium hydroxide or hydrochloric acid to adjust the enzymolysis pH value, and does not add any chemical reagent, so that the safety of the final product is high, the quality of the product is good, and the qualification rate is high.
Compared with the method for producing the pea peptide by directly utilizing microbial fermentation, the method for producing the pea peptide by utilizing the microbial fermentation technology firstly prepares the composite protease, and then the composite protease and other three industrial proteases are compounded and subjected to enzymolysis to produce the pea peptide, so that the production period of the product is shortened, the production condition is mild and easy to control, the production cost is low, the time consumption of each batch in large-scale production is short, and the production period flexibility is strong.
Further, the existing separation and purification technology mainly adopts the methods of filtering enzymolysis liquid through a plate frame, directly filtering through an ultrafiltration membrane, separating through resin, separating through a dialysis bag and the like. However, if the pore diameter of the filter paper or the filter membrane is larger, the plate and frame filtration has no separation and purification effect, and only impurities in the filtered feed liquid can be filtered; filter paper or filter membrane with proper pore size is needed, but the filtering efficiency is low, and the filter paper or filter membrane is easy to be blocked. The material liquid after centrifugation contains macromolecular proteins and peptides, and also comprises a small amount of suspended matter impurities, and the material liquid is easy to cause ultrafiltration membrane blockage by directly treating with the ultrafiltration membrane, so that the filtration efficiency is low, the separation and purification effects are not obvious, the ultrafiltration membrane is difficult to clean after filtration, and the service life is shortened. The resin separation method cannot purify and separate the molecular weight of the peptide solution. Dialysis bag separation is more in laboratory application at present, and is consuming time long, requires the feed liquid concentration lower, increases the concentration cost of follow-up feed liquid. The method adopts the filtration technology of coupling microfiltration-ultrafiltration-nanofiltration, and the enzymolysis liquid is filtered by the microfiltration-ultrafiltration-nanofiltration membrane and then dried, so that decolorization such as active carbon and further treatments such as purification and concentration are not required, inorganic and organic reagents are not required to be used for extraction, the process is simple, a large amount of solid waste is avoided, the production efficiency is high, the cost is low, and the method is more suitable for the industrial production concept; the prepared pea peptide has pure color, white-like color, light bitter taste, slight bean flavor, no other peculiar smell, small peptide molecular weight and easy absorption by human body.
Preferably, in step S1, the process is performed,
the mass ratio of the pea protein powder to the water is 1:10-1:25;
the dry protein content of the pea protein powder is more than 70%.
Preferably, in step S2, the step of generating a signal,
the bacillus natto inoculation amount is 2-5%, pH7.0, temperature is 37 ℃, rotating speed is 200-250r/min, air flow is 20-25L/min, tank pressure is 0.03MPa, and fermentation is carried out for 34-40h.
Preferably, in step S2, the step of generating a signal,
the compound protease, papain, alkaline protease and flavourzyme are mixed according to the enzyme activity unit ratio of 0.5:1:3:0.3.
Preferably, in step S3, the step of generating a signal,
the addition amount of the compound protease is 0.3-0.5% of the mass of the pea protein powder.
Preferably, in step S3, the step of generating a signal,
performing enzymolysis at natural pH and 45-55deg.C for 3-5 hr;
after the enzymolysis is finished, the temperature is raised to more than 90 ℃ and the enzyme is inactivated for 10 to 15min.
Preferably, in step S5, the process of the present invention,
spray drying the nanofiltration component, wherein the air inlet temperature is 165-185 ℃ and the air outlet temperature is 65-85 ℃.
The multifunctional pea peptide prepared by the method is applied to the preparation of the medicine for treating hypertension and complications thereof.
The multifunctional pea peptide prepared by the method is applied to the preparation of uric acid-reducing or antioxidant stress products.
Uric acid reduction includes inhibition of XOD enzymes; antioxidant includes scavenging abts+ free radicals and scavenging DPPH free radicals.
In conclusion, the multifunctional pea peptide with the functions of reducing blood pressure, reducing uric acid and resisting oxidation is prepared by adopting the fermentation-enzymolysis combined technology for the first time, can be used for relieving hypertension and the accompanying hyperuricemia and oxidative stress, and is further applied to relieving hyperuricemia and oxidative stress caused by other diseases.
The composite protease has strong enzymolysis specificity on pea protein, high enzymolysis efficiency, high yield, high protein recovery rate and improved product flavor when being compounded with other enzymes, and the protein hydrolysate with the relative molecular mass of less than 500U in the prepared pea peptide accounts for more than 60 percent, the protein hydrolysate with the relative molecular mass of less than 1000U accounts for more than 90 percent, and the pea peptide is easy to be absorbed by human bodies and is a high-quality pea peptide product. The compound protease is subjected to simultaneous enzymolysis, the pH is not required to be manually adjusted in the whole enzymolysis process, and compared with the multi-enzyme step-by-step and time-division enzymolysis process, the pH adjustment process by adopting chemical reagents and the like in the prior art, the enzymolysis time is effectively shortened, and the production operation is simplified. The liquid material is separated, purified and concentrated by adopting the microfiltration-ultrafiltration-nanofiltration membrane coupling technology, the liquid material is operated at normal temperature, the treatments such as decolorization and further concentration are not needed, the process is simple, the pollution is not generated in the production process, the product quality stability is good, the production efficiency is high, the cost is low, and the industrial mass production is easy.
Drawings
FIG. 1 shows the inhibition of the XOD enzyme by allopurinol;
FIG. 2 shows the inhibition of the XOD enzyme by pea peptide.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. 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.
The Bacillus natto used in the examples was a commercially available strain, and papain, alkaline protease, and flavourzyme were purchased from Nanning Donghenghua Biotechnology Co., ltd.
Example 1
A preparation method of multifunctional pea peptide comprises the following steps:
s1, preparing pea protein feed liquid:
adding pea protein powder (the dry protein content is 83.20%) into pure water according to the mass ratio of 1:10, and stirring for 30min to obtain pea protein feed liquid.
S2, preparing compound protease:
(1) fermentation: inoculating bacillus natto into a liquid fermentation tank filled with pea protein feed liquid, adjusting the inoculum size to 2%, adjusting the pH to 7.0, the temperature to 37 ℃, the rotation speed of the fermentation tank to 200r/min, the air flow to 20L/min, controlling the tank pressure to 0.03MPa, culturing for 40h to obtain fermentation liquor, centrifuging the fermentation liquor by a tube type centrifuge of 16000r/min to obtain composite protease liquid, and freeze-drying to obtain the composite protease.
(2) Compounding: and (3) compounding the composite protease obtained by fermentation with papain, alkaline protease and flavourzyme according to the enzyme activity unit ratio of 0.5:1:3:0.3 in sequence to obtain the composite protease.
S3, enzymolysis:
taking the pea protein feed liquid in the step S1 as a substrate, adjusting the temperature of an enzymolysis tank to 45 ℃ under natural pH, adding compound protease with the mass of 0.3% of the pea protein powder, stirring and carrying out enzymolysis for 3 hours to obtain pea peptide enzymolysis liquid;
after the enzymolysis is finished, the temperature of the enzymolysis liquid is raised to 90 ℃ and kept for 10min to inactivate enzyme.
S4, membrane filtration:
cooling the enzymolysis liquid to below 30 ℃, and centrifuging by a tube type centrifuge of 16000 r/min.
The centrifuged feed liquid is sequentially subjected to microfiltration (pressure 0.2 MPa), ultrafiltration (pressure 0.6 MPa) and nanofiltration (pressure 1.5 MPa) membrane filtration to obtain nanofiltration components with relative molecular mass less than 1000 u.
S5, drying:
spray drying the nanofiltration component feed liquid, wherein the air inlet temperature is 170 ℃, and the air outlet temperature is 75 ℃, so as to obtain the pea peptide; the obtained pea peptide has pure color, white-like color, light bitter taste, slightly bean flavor, and no other peculiar smell.
The pea peptide is detected and the detection of the pea peptide is carried out,
product yield (%) = pea peptide product yield (kg)/enzymatic hydrolysis process pea protein powder raw material feed (kg) ×100%;
the yield of pea peptide product was 72.5%, the protein content (dry basis) was 88.3%, the polypeptide content was 82.1%, the relative molecular mass of protein hydrolysate less than 1000u was 90.6%, the moisture was 5.53%, and the ash content was 4.72%.
Example 2
A preparation method of multifunctional pea peptide comprises the following steps:
s1, preparing pea protein feed liquid:
adding pea protein powder (the dry protein content is 83.20%) into pure water according to the mass ratio of 1:10, and stirring for 30min to obtain pea protein feed liquid.
S2, preparing compound protease:
(1) fermentation: inoculating bacillus natto into a liquid fermentation tank filled with pea protein feed liquid, adjusting the inoculum size to 3%, adjusting the pH to 7.0, the temperature to 37 ℃, the rotation speed of the fermentation tank to 250r/min, the air flow to 20L/min, controlling the tank pressure to 0.03MPa, culturing for 36h to obtain fermentation liquor, centrifuging the fermentation liquor by a tube centrifuge of 16000r/min to obtain composite protease liquid, and freeze-drying to obtain the composite protease.
(2) Compounding: and (3) compounding the composite protease obtained by fermentation with papain, alkaline protease and flavourzyme according to the enzyme activity unit ratio of 0.5:1:3:0.3 in sequence to obtain the composite protease.
S3, enzymolysis: taking the pea protein feed liquid in the step S1 as a substrate, adjusting the temperature of an enzymolysis tank to 50 ℃ under natural pH, adding compound protease with the mass of 0.4% of the pea protein powder, stirring and carrying out enzymolysis for 4 hours to obtain pea peptide enzymolysis liquid;
after the enzymolysis is finished, the temperature of the enzymolysis liquid is raised to 90 ℃ and kept for 10min to inactivate enzyme.
S4, membrane filtration:
cooling the enzymolysis liquid to below 30 ℃, and centrifuging by a tube type centrifuge of 16000 r/min.
The centrifuged feed liquid is sequentially subjected to microfiltration (pressure 0.2 MPa), ultrafiltration (pressure 0.6 MPa) and nanofiltration (pressure 1.5 MPa) membrane filtration to obtain nanofiltration components with relative molecular mass less than 1000 u.
S5, drying:
spray drying the nanofiltration component feed liquid, wherein the air inlet temperature is 170 ℃, and the air outlet temperature is 75 ℃, so as to obtain the pea peptide.
The yield of pea peptide product was 75.0%, the protein content (dry basis) was 90.8%, the polypeptide content was 83.6%, the protein hydrolysate with a relative molecular mass of less than 1000u was 91.4%, the moisture was 5.43% and the ash content was 4.32%.
Example 3
A preparation method of multifunctional pea peptide comprises the following steps:
s1, preparing pea protein feed liquid:
adding pea protein powder (the dry protein content is 83.20%) into pure water according to the mass ratio of 1:15, and stirring for 30min to obtain pea protein feed liquid.
S2, preparing compound protease:
(1) fermentation: inoculating bacillus natto into a liquid fermentation tank filled with pea protein feed liquid, adjusting the inoculum size to 2%, adjusting the pH to 7.0, the temperature to 37 ℃, the rotation speed of the fermentation tank to 200r/min, the air flow to 25L/min, controlling the tank pressure to 0.03MPa, culturing for 40h to obtain fermentation liquor, centrifuging the fermentation liquor by a tube type centrifuge of 16000r/min to obtain composite protease liquid, and freeze-drying to obtain the composite protease.
(2) Compounding: and (3) compounding the composite protease obtained by fermentation with papain, alkaline protease and flavourzyme according to the enzyme activity unit ratio of 0.5:1:3:0.3 in sequence to obtain the composite protease.
S3, enzymolysis: taking the pea protein feed liquid in the step S1 as a substrate, adjusting the temperature of an enzymolysis tank to 55 ℃ under natural pH, adding compound protease with the mass of 0.5% of the pea protein powder, stirring and carrying out enzymolysis for 5 hours to obtain pea peptide enzymolysis liquid;
after the enzymolysis is finished, the temperature of the enzymolysis liquid is raised to 90 ℃ and kept for 10min to inactivate enzyme.
S4, membrane filtration:
cooling the enzymolysis liquid to below 30 ℃, and centrifuging by a tube type centrifuge of 16000 r/min.
The centrifuged feed liquid is sequentially subjected to microfiltration (pressure 0.2 MPa), ultrafiltration (pressure 0.6 MPa) and nanofiltration (pressure 1.5 MPa) membrane filtration to obtain nanofiltration components with relative molecular mass less than 1000 u.
S5, drying:
spray drying the nanofiltration component feed liquid, wherein the air inlet temperature is 170 ℃, and the air outlet temperature is 75 ℃, so as to obtain the pea peptide.
The yield of pea peptide product was 78.4%, the protein content (dry basis) was 91.3%, the polypeptide content was 82.1%, the protein hydrolysate with a relative molecular mass of less than 1000u was 91.6%, the moisture was 5.37% and the ash content was 4.41%.
Example 4
A preparation method of multifunctional pea peptide comprises the following steps:
s1, preparing pea protein feed liquid:
adding pea protein powder (the dry protein content is 83.20%) into pure water according to the mass ratio of 1:25, and stirring for 30min to obtain pea protein feed liquid.
S2, preparing compound protease:
(1) fermentation: inoculating bacillus natto into a liquid fermentation tank filled with pea protein feed liquid, adjusting the inoculum size to 5%, adjusting the pH to 7.0, the temperature to 37 ℃, the rotation speed of the fermentation tank to 200r/min, the air flow to 20L/min, controlling the tank pressure to 0.03MPa, culturing for 40h to obtain fermentation liquor, centrifuging the fermentation liquor by a tube type centrifuge of 16000r/min to obtain composite protease liquid, and freeze-drying to obtain the composite protease.
(2) Compounding: and (3) compounding the composite protease obtained by fermentation with papain, alkaline protease and flavourzyme according to the enzyme activity unit ratio of 0.5:1:3:0.3 in sequence to obtain the composite protease.
S3, enzymolysis: taking the pea protein feed liquid in the step S1 as a substrate, adjusting the temperature of an enzymolysis tank to 50 ℃ under natural pH, adding compound protease with the mass of 0.5% of the pea protein powder, stirring and carrying out enzymolysis for 5 hours to obtain pea peptide enzymolysis liquid;
after the enzymolysis is finished, the temperature of the enzymolysis liquid is raised to 90 ℃ and kept for 10min to inactivate enzyme.
S4, membrane filtration:
cooling the enzymolysis liquid to below 30 ℃, and centrifuging by a tube type centrifuge of 16000 r/min.
The centrifuged feed liquid is sequentially subjected to microfiltration (pressure 0.2 MPa), ultrafiltration (pressure 0.6 MPa) and nanofiltration (pressure 1.5 MPa) membrane filtration to obtain nanofiltration components with relative molecular mass less than 1000 u.
S5, drying:
spray drying the nanofiltration component feed liquid, wherein the air inlet temperature is 170 ℃, and the air outlet temperature is 75 ℃, so as to obtain the pea peptide.
The yield of pea peptide product was 77.2%, the protein content (dry basis) was 94.5%, the polypeptide content was 87.1%, the relative molecular mass of protein hydrolysate less than 1000u was 92.7%, the moisture was 5.63% and the ash content was 4.45%.
Example 5
Comparison of pea peptides prepared by enzymatic hydrolysis of different enzyme preparations:
the test enzymes include:
papain, alkaline protease, flavoured protease, complex protease one (complex protease prepared by the method of example 1), complex protease two (papain: alkaline protease: flavoured protease = 1:3:0.3), complex protease (complex protease prepared by the method of example 3).
The preparation process flow of pea peptide comprises the following steps: stirring and homogenizing pea protein powder and water according to a feed liquid ratio of 1:20, regulating the temperature to 50 ℃ under the condition of natural pH, adding enzyme to be 0.4% of the pea protein powder in mass, respectively carrying out enzymolysis for 4 hours, inactivating enzyme at 90 ℃ for 15min, centrifuging for 30min at 4000r/min, taking supernatant, and freeze-drying to obtain pea peptide. Detecting pea stations prepared by enzymolysis of each group of test enzymes, wherein the product yield (%) = pea peptide product yield (kg)/pea protein powder raw material feeding amount during enzymolysis (kg) ×100%; protein recovery (%) = total crude protein in pea peptide product (kg)/total crude protein in pea protein meal raw material (kg) ×100%;
the results are shown in Table 1.
TABLE 1
The experimental results in table 1 show that the product yield, protein recovery rate, protein content, polypeptide content and the ratio of protein hydrolysate with molecular weight smaller than 1000u of pea peptide obtained by hydrolyzing pea protein with compound protease are all larger than that of papain, alkaline protease, flavourzyme, compound protease I and compound protease II.
Example 6
The inhibition of XOD enzyme by pea peptide of example 1 was determined by uv spectrophotometry, as follows:
4 10mL centrifuge tubes, numbered OD1, OD2, OD3 and OD4, were used, and reagents were added to each tube according to the combinations of Table 2.
TABLE 2 addition amount of reagents
Note that: preparation of allopurinol/Pisi peptide solutions at different concentrations using PBS as needed
0.5mL of xanthine oxidase solution and 0.75mL of allopurinol solution were added to centrifuge tube 1 as OD1 group; 0.5mL xanthine oxidase solution was added to centrifuge tube number 2 as OD2 group; 0.75mL of allopurinol solution was added to centrifuge tube 3 as OD3 group; adding a certain amount of PBS solution shown in table 3 into each of the separation tubes 1-4, uniformly mixing, and fully reacting for 15min in a constant-temperature water bath kettle at 25 ℃; then adding 1.5mL of xanthine solution, uniformly mixing, fully reacting for 30min in a constant-temperature water bath kettle at 25 ℃, finally adding 1mL of HCl to terminate the reaction, and measuring the absorbance value (PBS solution zero setting calibration) by an ultraviolet spectrophotometer under the condition of the wavelength of 290 nm.
Experimental data processing:
concentration is plotted on the abscissa and inhibition ratio is plotted on the ordinate (fig. 1 and 2), and IC50 of the concentration of half inhibition ratio is calculated.
Wherein:
OD 1-refers to the absorbance value of a living assay system containing substrate, enzyme solution and inhibitor;
OD 2-refers to the absorbance value of a living assay system containing substrate and enzyme solution, without allopurinol/sample;
OD 3-refers to the absorbance value of a living assay system containing substrate and inhibitor, without enzyme solution;
OD4: refers to the absorbance value of a living assay system containing substrate, without allopurinol/sample and enzyme solution.
The in vitro XOD inhibition of 18mg/mL pea peptide solution was 74.24% with half inhibition IC50 of 12.79mg/mL determined as above; the median inhibition IC50 of allopurinol was 0.01mg/mL.
Example 7
Determination of the clearance of the pea peptide of example 1 to abts+ free radical:
preparing an ABTS solution: 200.0mg of ABTS, 34.4mg of potassium persulfate, dissolved in 50.0mL of distilled water, are uniformly shaken, and are placed at room temperature in a dark place for 24 hours to serve as an ABTS mother solution. Diluting appropriate amount of ABTS mother liquor with 95% ethanol until absorbance value is within 0.70+ -0.02 (OD 734 ) As an ABTS assay solution, this solution should be ready for use.
Sample preparation: and (3) preparing glutathione/pea peptide mother solutions with different concentrations by using distilled water according to the requirements, and diluting the mother solutions to different multiples by using 95% ethanol to obtain solutions to be measured with different concentrations.
Taking 2 branch separation tubes with the numbers of 1 and 2 respectively, and adding reagents into each branch tube according to the combination of the table 3.
TABLE 3 addition amount of reagents
Into centrifuge tube No. 1, 3.6mL of ABTS solution and 0.4mL of glutathione solution were added as an experimental group (A S ) The method comprises the steps of carrying out a first treatment on the surface of the 3.6mL of ABTS solution and 0.4mL of sample solvent solution were added to centrifuge tube number 2 as blank (Ab); after being fully and uniformly mixed, the materials react for 5 minutes at room temperature in a dark place, and the absorbance value (sample solvent zero setting calibration) is measured by an ultraviolet spectrophotometer under the condition of the wavelength of 734 nm.
Experimental data processing:
wherein:
p-clearance;
absorbance of the Ab-ABTS solution mixed with the sample solvent solution;
as-absorbance of the mixture of the solution to be measured and the ABTS solution.
The clearance of ABTS+ free radicals by 1.6mg/mL pea peptide was determined as above to be 63.38%.
Example 7
Determination of the clearance of the pea peptide of example 1 to DPPH-free radical:
preparing DPPH solution: weighing DPPH 5.0mg, dissolving with proper amount of absolute ethyl alcohol, fully dissolving with light-shielding ultrasonic, and then preparing into 50.0 mug/mL DPPH solution by using absolute ethyl alcohol to fix the volume to 100.0 mL. The solution should be ready to use.
Sample preparation: and (3) preparing glutathione/pea peptide mother solutions with different concentrations by using distilled water according to the requirements, and diluting the mother solutions to different multiples by using distilled water to obtain solutions to be measured with different concentrations.
3 branch separation tubes were taken and numbered 1, 2 and 3, and reagents were added to each branch according to the combinations shown in Table 4.
TABLE 4 addition amount of reagents
| Solution name | No. 1 (A) S ) | No. 2 (Ac) | No. 3 (Ab) |
| DPPH solution | 3.0mL | — | 3.0mL |
| Glutathione/pea peptide solution | 1.0mL | 1.0mL | — |
| Sample solvent solution (distilled water) | — | — | 1.0mL |
| Absolute ethyl alcohol | — | 3.0mL | — |
Note that: preparation of different concentrations of allopurinol/Pisi de solutions with PBS as needed
Into centrifuge tube No. 1, 3.0mL of DPPH solution and 1.0mL of glutathione solution were added as an experimental group (A) S ) The method comprises the steps of carrying out a first treatment on the surface of the 1.0mL of glutathione solution and 3.0mL of absolute ethanol solution are added into a centrifuge tube No. 2 to serve as a control group (Ac); a3.0 mL of PPH solution and 1.0mL of sample solvent solution were added to a centrifuge tube No. 3 as a blank (Ab). After being fully and uniformly mixed, the materials react for 30 minutes at room temperature in a dark place, and the absorbance value of the supernatant is measured by an ultraviolet spectrophotometer under the condition of the wavelength of 734nm (zero setting and calibration of a sample solvent).
Experimental data processing:
wherein:
p-clearance;
as-absorbance of mixed solution of solution to be measured and DPPH solution;
ac-absorbance of the mixed solution of the solution to be measured and the absolute ethyl alcohol solution;
absorbance of the mixture of Ab-DPPH solution and sample solvent solution.
The clearance of DPPH free radical by 30mg/mL pea peptide was determined as above to be 81.04%.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to the embodiments described above will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (3)
1. The preparation method of the multifunctional pea peptide is characterized by comprising the following steps of:
s1, preparing pea protein feed liquid:
mixing pea protein powder with water, and preparing pea protein feed liquid;
s2, preparing compound protease:
1) Fermentation:
inoculating bacillus natto into pea protein feed liquid for fermentation, centrifuging fermentation liquor, taking supernatant, and drying to obtain compound protease;
2) Compounding:
compounding the compound protease with papain, alkaline protease and flavourzyme to prepare the compound protease;
s3, enzymolysis:
taking pea protein feed liquid as a substrate, adding compound protease, stirring for enzymolysis, and inactivating enzyme after the enzymolysis is finished to obtain pea peptide enzymolysis liquid;
s4, membrane filtration:
centrifuging pea peptide enzymolysis liquid to obtain supernatant, and sequentially performing microfiltration, ultrafiltration and nanofiltration membrane filtration to obtain nanofiltration components with relative molecular mass less than 1000 u;
s5, drying:
the nanofiltration component is dried to obtain pea peptide;
in the step S1 of the process,
the mass ratio of the pea protein powder to the water is 1:10-1:25, a step of selecting a specific type of material;
the dry protein content of the pea protein powder is more than 70%;
in the step S2 of the process,
the bacillus natto inoculum size is 2-5%, pH7.0, temperature is 37 ℃, rotating speed is 200-250r/min, air flow is 20-25L/min, tank pressure is 0.03MPa, and fermentation is carried out for 34-40h;
in the step S2 of the process,
complex protease, papain, alkaline protease and flavourzyme according to 0.5:1:3: mixing enzyme activity unit proportion of 0.3;
in the step S3 of the process,
the addition amount of the compound protease is 0.3-0.5% of the mass of the pea protein powder;
in the step S3 of the process,
performing enzymolysis at natural pH and 45-55deg.C for 3-5 hr;
heating to above 90deg.C after enzymolysis, inactivating enzyme for 10-15min;
in the step S5 of the process,
spray drying the nanofiltration component, wherein the air inlet temperature is 165-185 ℃ and the air outlet temperature is 65-85 ℃.
2. The use of the multifunctional pea peptide prepared by the method of claim 1 in the preparation of a medicament for treating hypertension.
3. Use of the multifunctional pea peptide prepared by the method of claim 1 for the preparation of uric acid lowering or antioxidant stress products, said uric acid lowering comprising an inhibitory effect on XOD enzymes; the antioxidation comprises a scavenging action on ABTS+ free radicals and a scavenging action on DPPH free radicals.
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