CN114395600A - Preparation method and application of multifunctional pea peptide - Google Patents
Preparation method and application of multifunctional pea peptide Download PDFInfo
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- 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
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- 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, and belongs to the technical field of biological medicine, wherein 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 the pea protein feed liquid with bacillus natto for fermentation, centrifuging the fermentation liquid, taking the supernatant, and drying to obtain the composite protease; 2) compounding: compounding the compound protease with papain, alkaline protease and flavourzyme to prepare compound protease; s3, carrying out enzymolysis; s4, membrane filtration; and 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 mass of protein hydrolysate, good product flavor, and high activity of reducing blood pressure, uric acid and oxidation resistance, and is a high-quality pea peptide product.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a preparation method and application of multifunctional pea peptide.
Background
The pea is a plant belonging to the genus Pisum of the family Leguminosae, also called as Dutch bean, wheat bean, Bidou, etc., and is rich in starch, protein, vitamins, minerals and various amino acids necessary for human body. Pea protein is excellent plant protein, the essential amino acid composition of the pea protein is closer to the FAO/WHO recommended mode, the pea protein has low allergenicity and high nutritional value, and the pea protein is widely concerned.
However, since pea protein cannot fully exert its biological activity in human body due to poor solubility and digestibility of protein, it is necessary to treat pea protein by a certain means to improve its physiological activity and to utilize the nutritional value of pea protein to a greater extent.
The current deep utilization treatment method for pea protein mainly comprises an enzymolysis treatment method and an enzymolysis and fermentation treatment method: the enzymolysis method is characterized in that pea protein is treated by industrial protease, the enzyme preparation has low specificity, the enzymolysis efficiency is low, the product yield is not high, the average relative molecular weight of pea peptide is large, the ratio of protein hydrolysate with the molecular weight less than 1000u is small, the polypeptide content is low, and the bitter taste is heavy; the enzymolysis and fermentation treatment method firstly adopts industrial protease to carry out enzymolysis on pea protein, and then carries out fermentation on the enzymolysis liquid to prepare the pea peptide, so that the production period is long, and the problem of low product yield also exists.
Therefore, how to provide a preparation method of pea peptide, which has the advantages of simple process, short period, high yield and low cost and is more suitable for industrial production.
The method for preparing the pea peptide from the pea protein by adopting the methods of enzymolysis, microbial fermentation, physical synergistic enzymolysis and the like 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 make full use of pea protein resources has important significance for the development of the pea industry.
In recent years, with the rise of enzyme industrial technologies, hydrolysis of large molecular proteins into absorbable small molecular peptides by proteases has received much attention from various industries. Therefore, the method for preparing the pea peptides from the pea proteins by using the enzymolysis technology is an effective means for improving the nutritional performance and high-value utilization of the pea proteins. The existing reports for preparing the pea peptide mainly adopt an enzymolysis method, and no report exists on the research of preparing the pea peptide by combining fermentation and enzymolysis. .
Therefore, how to provide a preparation method of pea peptide, which has the advantages of 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 purpose, the 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 to prepare pea protein liquid;
s2, preparing compound protease:
1) fermentation:
inoculating the pea protein feed liquid with bacillus natto for fermentation, centrifuging the fermentation liquid, taking the supernatant, and drying to obtain the composite protease;
2) compounding:
compounding the compound protease with papain, alkaline protease and flavourzyme to prepare compound protease;
s3, enzymolysis:
adding compound protease into pea protein feed liquid as a substrate, stirring for enzymolysis, and inactivating enzyme after the enzymolysis is finished to obtain pea peptidase hydrolysate;
s4, membrane filtration:
centrifuging the pea peptidase hydrolysate to obtain supernatant, and sequentially performing microfiltration, ultrafiltration and nanofiltration membrane filtration to obtain a nanofiltration component with the relative molecular mass of less than 1000 u;
s5, drying:
and drying the nanofiltration component to obtain the pea peptide.
According to the invention, the composite protease generated by fermenting pea protein with bacillus natto is compounded with three industrial enzymes for the first time, and the pea peptide is prepared by utilizing the compounded enzyme preparation to carry out enzymolysis on the pea protein; the bacillus natto takes pea protein as a unique carbon source for fermentation, the enzymolysis specificity of the pea protein by the composite protease obtained by fermentation is strong, the enzymolysis efficiency is high, and the obtained product has strong biological functional activity.
The composite protease generated by fermenting pea protein with bacillus natto consists of various endonucleases and exonucleases, has multiple enzyme cutting sites, is compounded with papain, alkaline protease and flavourzyme, can improve the enzymolysis efficiency of the pea protein, improve the product yield, effectively improve the quality of the pea peptide, has high polypeptide content, is mainly composed of four-octapeptide and protein hydrolysate with the molecular weight less than 1000u, and effectively hydrolyzes the bitter peptide through various 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 various biological functional activities.
Compared with the prior enzymolysis technology, the compound protease is added simultaneously, and the step-by-step and time-by-time enzyme addition is not needed, 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, chemical reagents such as sodium hydroxide or hydrochloric acid are not required to be added in the enzymolysis process of the compound protease to adjust the enzymolysis pH, and no chemical reagent is added, so that the safety of the final product is high, the product quality is good, and the qualification rate is high.
Compared with the method for directly producing the pea peptide by using microbial fermentation, the method disclosed by the invention has the advantages that the compound protease is prepared by using the microbial fermentation technology, and then the compound 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 during large-scale production is short, and the flexibility of the production period is strong.
Furthermore, the existing separation and purification technology mainly adopts the methods of filtering the enzymatic hydrolysate by a plate frame, directly filtering by an ultrafiltration membrane, separating by resin, separating by a dialysis bag and the like. However, if the filter paper or filter membrane used in the plate-and-frame filtration has a large aperture, the plate-and-frame filtration has no separation and purification effect and can only filter impurities in the discharged liquid; the filter paper or filter membrane with proper pore size needs to be selected, but the filtering efficiency is low, and the filter paper or filter membrane is easy to block. The feed liquid after centrifugation contains macromolecular protein and peptide, and also contains a small amount of suspended impurities, the feed liquid is directly treated by the ultrafiltration membrane, so that the ultrafiltration membrane is easy to block, 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. Resin separation cannot purify and separate peptide liquid according to molecular weight. Dialysis bag separation is applied more at present in the laboratory, and is consuming time long, requires that the feed liquid concentration is lower, increases the concentrated cost of follow-up feed liquid. The method adopts a microfiltration-ultrafiltration-nanofiltration coupled filtration technology, the enzymatic hydrolysate is centrifuged, filtered by the microfiltration-ultrafiltration-nanofiltration membrane and then dried, decolorization, further purification, concentration and other treatment such as activated carbon and the like are not needed, inorganic and organic reagents are not needed 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 industrial production concepts; the prepared pea peptide has pure color, white-like color, light bitter taste, slight bean fragrance, no other peculiar smell, small peptide molecular weight and easy absorption by human body.
Preferably, in step S1,
the weight ratio of the pea protein powder to the water is 1:10-1: 25;
the content of the pea protein powder dry-based protein is more than 70 percent.
Preferably, in step S2,
the inoculation amount of the bacillus natto is 2-5 percent, the pH value is 7.0, the temperature is 37 ℃, the rotating speed is 200-.
Preferably, in step S2,
the compound protease, the papain, the alkaline protease and the flavor protease are mixed according to the enzyme activity unit proportion of 0.5:1:3: 0.3.
Preferably, in step S3,
the addition amount of the compound protease is 0.3-0.5% of the weight of the pea protein powder.
Preferably, in step S3,
performing enzymolysis at 45-55 deg.C at natural pH for 3-5 h;
heating to above 90 deg.C after enzymolysis, and inactivating enzyme for 10-15 min.
Preferably, in step S5,
and (3) carrying out spray drying on 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 preparing the medicine for treating hypertension and complications thereof.
The multifunctional pea peptide prepared by the method is applied to preparation of uric acid reducing or antioxidant stress products.
The antioxidant includes inhibition of XOD enzyme, scavenging ABTS + free radical, and scavenging DPPH free radical.
In conclusion, the multifunctional pea peptide with the functions of reducing blood pressure, reducing uric acid and resisting oxidation is prepared by adopting a fermentation-enzymolysis combined technology for the first time, can be used for relieving hypertension and hyperuricemia and oxidative stress accompanied with the hypertension, and is further applied to relieving hyperuricemia and oxidative stress caused by other diseases.
The composite protease has strong enzymolysis specificity to pea protein, high enzymolysis efficiency after being compounded with other enzymes, high yield, high protein recovery rate and improved product flavor, and the prepared pea peptide has the protein hydrolysate with the relative molecular mass less than 500U accounting for more than 60 percent and the protein hydrolysate with the relative molecular mass less than 1000U accounting for more than 90 percent, is easy to be absorbed by human bodies and is a high-quality pea peptide product. The compound protease is subjected to enzymolysis at the same time, the whole enzymolysis process does not need manual pH adjustment, and compared with the multi-enzyme step-by-step enzymolysis process, the time-phased enzymolysis process, the pH adjustment process by adopting a chemical reagent and the like in the prior art, the enzymolysis time is effectively shortened, and the production operation is simplified. The microfiltration-ultrafiltration-nanofiltration membrane coupling technology is adopted to separate, purify and concentrate the liquid material, the operation is carried out at normal temperature, the treatment such as decolorization and further concentration is not needed, the process is simple, the production process does not produce pollution, 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 is a graph showing inhibition of XOD enzyme by allopurinol;
FIG. 2 shows the inhibition curves of pea peptides against XOD enzymes.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Bacillus natto used in the examples is a commercially available strain, and papain, alkaline protease and flavourzyme are all purchased from Nanning Dong Henghuadao Biotech, Inc.
Example 1
A preparation method of multifunctional pea peptide comprises the following steps:
s1, preparing pea protein feed liquid:
adding pea protein powder (dry basis protein content is 83.20%) into pure water according to the mass ratio of 1:10, and stirring for 30min to obtain pea protein liquid.
S2, preparing compound protease:
fermentation: inoculating bacillus natto into a liquid fermentation tank filled with pea protein feed liquid, regulating the pH to be 7.0, the temperature to be 37 ℃, the rotation speed of the fermentation tank to be 200r/min, the air flow to be 20L/min, controlling the tank pressure to be 0.03MPa, culturing for 40h to obtain fermentation liquor, centrifuging the fermentation liquor by a tubular centrifuge of 16000r/min to obtain composite protease liquid, and freeze-drying to obtain the composite protease.
Compounding: and compounding the compound protease prepared by fermentation with papain, alkaline protease and flavourzyme in sequence according to the enzyme activity unit proportion of 0.5:1:3:0.3 to obtain the compound protease.
S3, enzymolysis:
taking the pea protein liquid obtained in the step S1 as a substrate, adjusting the temperature of an enzymolysis tank to 45 ℃ under natural pH, adding compound protease accounting for 0.3% of the mass of the pea protein powder, stirring and carrying out enzymolysis for 3 hours to obtain pea peptidase hydrolysate;
after the enzymolysis is finished, the temperature of the enzymolysis liquid is increased to 90 ℃, and the enzymolysis liquid is kept for 10min for enzyme deactivation.
S4, membrane filtration:
cooling the enzymolysis solution to below 30 deg.C, and centrifuging with tubular centrifuge at 16000 r/min.
And (3) sequentially carrying out microfiltration (pressure of 0.2MPa), ultrafiltration (pressure of 0.6MPa) and nanofiltration (pressure of 1.5MPa) on the centrifuged feed liquid to obtain a nanofiltration component with the relative molecular mass of 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 ℃ to obtain the pea peptide; the prepared pea peptide has pure color, similar white color, light bitter taste, slight bean flavor and no other peculiar smell.
The pea peptides are detected, and the detection is carried out,
the product yield (%) < pea peptide product yield (kg)/pea protein powder raw material dosage (kg) x 100% in the enzymolysis process;
the yield of the pea peptide product is 72.5%, the protein content (dry basis) is 88.3%, the polypeptide content is 82.1%, the ratio of the protein hydrolysate with the relative molecular mass of less than 1000u is 90.6%, the water content is 5.53%, and the ash content is 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 (dry basis protein content is 83.20%) into pure water according to the mass ratio of 1:10, and stirring for 30min to obtain pea protein liquid.
S2, preparing compound protease:
fermentation: inoculating bacillus natto into a liquid fermentation tank filled with pea protein feed liquid, regulating the pH to be 7.0, the temperature to be 37 ℃, the rotation speed of the fermentation tank to be 250r/min, the air flow to be 20L/min, controlling the tank pressure to be 0.03MPa, culturing for 36h to obtain fermentation liquor, centrifuging the fermentation liquor by a tubular centrifuge of 16000r/min to obtain composite protease liquid, and freeze-drying to obtain the composite protease.
Compounding: and compounding the compound protease prepared by fermentation with papain, alkaline protease and flavourzyme in sequence according to the enzyme activity unit proportion of 0.5:1:3:0.3 to obtain the compound protease.
S3, enzymolysis: taking the pea protein liquid obtained in the step S1 as a substrate, adjusting the temperature of an enzymolysis tank to 50 ℃ under natural pH, adding compound protease accounting for 0.4% of the mass of the pea protein powder, stirring and carrying out enzymolysis for 4 hours to obtain pea peptidase hydrolysate;
after the enzymolysis is finished, the temperature of the enzymolysis liquid is increased to 90 ℃, and the enzymolysis liquid is kept for 10min for enzyme deactivation.
S4, membrane filtration:
cooling the enzymolysis solution to below 30 deg.C, and centrifuging with tubular centrifuge at 16000 r/min.
And (3) sequentially carrying out microfiltration (pressure of 0.2MPa), ultrafiltration (pressure of 0.6MPa) and nanofiltration (pressure of 1.5MPa) on the centrifuged feed liquid to obtain a nanofiltration component with the relative molecular mass of less than 1000 u.
S5, drying:
and (3) carrying out spray drying on 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 is 75.0%, the protein content (dry basis) is 90.8%, the polypeptide content is 83.6%, the ratio of protein hydrolysate with relative molecular mass less than 1000u is 91.4%, the water content is 5.43%, and the ash content is 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 (dry basis protein content is 83.20%) into pure water according to the mass ratio of 1:15, and stirring for 30min to obtain pea protein liquid.
S2, preparing compound protease:
fermentation: inoculating bacillus natto into a liquid fermentation tank filled with pea protein feed liquid, regulating the pH to be 7.0, the temperature to be 37 ℃, the rotation speed of the fermentation tank to be 200r/min, the air flow to be 25L/min, controlling the tank pressure to be 0.03MPa, culturing for 40h to obtain fermentation liquor, centrifuging the fermentation liquor by a tubular centrifuge of 16000r/min to obtain composite protease liquid, and freeze-drying to obtain the composite protease.
Compounding: and compounding the compound protease prepared by fermentation with papain, alkaline protease and flavourzyme in sequence according to the enzyme activity unit proportion of 0.5:1:3:0.3 to obtain the compound protease.
S3, enzymolysis: taking the pea protein liquid obtained in the step S1 as a substrate, adjusting the temperature of an enzymolysis tank to 55 ℃ under natural pH, adding compound protease accounting for 0.5% of the mass of the pea protein powder, stirring and carrying out enzymolysis for 5 hours to obtain pea peptidase hydrolysate;
after the enzymolysis is finished, the temperature of the enzymolysis liquid is increased to 90 ℃, and the enzymolysis liquid is kept for 10min for enzyme deactivation.
S4, membrane filtration:
cooling the enzymolysis solution to below 30 deg.C, and centrifuging with tubular centrifuge at 16000 r/min.
And (3) sequentially carrying out microfiltration (pressure of 0.2MPa), ultrafiltration (pressure of 0.6MPa) and nanofiltration (pressure of 1.5MPa) on the centrifuged feed liquid to obtain a nanofiltration component with the relative molecular mass of less than 1000 u.
S5, drying:
and (3) carrying out spray drying on 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 the pea peptide product is 78.4%, the protein content (dry basis) is 91.3%, the polypeptide content is 82.1%, the ratio of the protein hydrolysate with the relative molecular mass of less than 1000u is 91.6%, the water content is 5.37%, and the ash content is 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 (dry basis protein content is 83.20%) into pure water according to the mass ratio of 1:25, and stirring for 30min to obtain pea protein liquid.
S2, preparing compound protease:
fermentation: inoculating the bacillus natto into a liquid fermentation tank filled with pea protein feed liquid, regulating the pH to be 7.0, the temperature to be 37 ℃, the rotation speed of the fermentation tank to be 200r/min, the air flow to be 20L/min, controlling the tank pressure to be 0.03MPa, culturing for 40h to obtain fermentation liquor, centrifuging the fermentation liquor by a tubular centrifuge of 16000r/min to obtain composite protease liquid, and freeze-drying to obtain the composite protease.
Compounding: and compounding the compound protease prepared by fermentation with papain, alkaline protease and flavourzyme in sequence according to the enzyme activity unit proportion of 0.5:1:3:0.3 to obtain the compound protease.
S3, enzymolysis: taking the pea protein liquid obtained in the step S1 as a substrate, adjusting the temperature of an enzymolysis tank to 50 ℃ under natural pH, adding compound protease accounting for 0.5% of the mass of the pea protein powder, stirring and carrying out enzymolysis for 5 hours to obtain pea peptidase hydrolysate;
after the enzymolysis is finished, the temperature of the enzymolysis liquid is increased to 90 ℃, and the enzymolysis liquid is kept for 10min for enzyme deactivation.
S4, membrane filtration:
cooling the enzymolysis solution to below 30 deg.C, and centrifuging with tubular centrifuge at 16000 r/min.
And (3) sequentially carrying out microfiltration (pressure of 0.2MPa), ultrafiltration (pressure of 0.6MPa) and nanofiltration (pressure of 1.5MPa) on the centrifuged feed liquid to obtain a nanofiltration component with the relative molecular mass of less than 1000 u.
S5, drying:
and (3) carrying out spray drying on 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 the pea peptide product is 77.2 percent, the protein content (dry basis) is 94.5 percent, the polypeptide content is 87.1 percent, the ratio of the protein hydrolysate with the relative molecular mass of less than 1000u is 92.7 percent, the water content is 5.63 percent, and the ash content is 4.45 percent.
Example 5
Comparison of pea peptides prepared by enzymatic hydrolysis of different enzyme preparations:
the test enzymes included:
papain, alkaline protease, flavourzyme, compound protease one (compound protease prepared by the method in example 1), compound protease two (papain: alkaline protease: flavourzyme ═ 1:3:0.3), and compound protease (compound protease prepared by the method in example 3).
The pea peptide preparation process flow comprises the following steps: stirring and homogenizing pea protein powder and water according to a material-liquid ratio of 1:20, adjusting the temperature to 50 ℃ under the natural pH condition, adding 0.4% of enzyme by mass of the pea protein powder, respectively carrying out enzymolysis for 4h, carrying out enzyme deactivation for 15min at 90 ℃, centrifuging for 30min at 4000r/min, taking supernatant, and freeze-drying into powder to obtain the pea peptide. Detecting each group of pea platforms prepared by enzymatic hydrolysis of the tested enzyme, wherein the product yield (%) is pea peptide product yield (kg)/pea protein powder raw material feeding amount (kg) multiplied by 100% in the enzymatic hydrolysis process; the protein recovery (%). percent is total crude protein (kg) in the pea peptide product/total crude protein (kg) x 100% in the pea protein powder material;
the results are shown in Table 1.
TABLE 1
The experimental results shown in table 1 show that the pea peptide obtained by hydrolyzing the pea protein by the compound protease has the product yield, the protein recovery rate, the protein content, the polypeptide content, the ratio of protein hydrolysate with the molecular weight of less than 1000u and the ACE inhibition rate indicating the blood pressure lowering activity which are all higher than those of papain, alkaline protease, flavourzyme, compound protease I and compound protease II.
Example 6
The inhibition rate of the pea peptide of example 1 on XOD enzyme is determined by adopting an ultraviolet spectrophotometry, which comprises the following steps:
4 10mL centrifuge tubes were numbered OD1, OD2, OD3, and OD4, respectively, and the reagents were added to each tube in the combinations shown in Table 2.
TABLE 2 amount of reagent added
Note: PBS is used for preparing allopurinol/pea peptide solutions with different concentrations according to requirements
To centrifuge tube # 1, 0.5mL xanthine oxidase solution and 0.75mL allopurinol solution were added as OD1 groups; 0.5mL xanthine oxidase solution was added to centrifuge tube No. 2 as OD2 group; 0.75mL of allopurinol solution was added to centrifuge tube No. 3 as OD3 group; adding a certain amount of PBS (phosphate buffer solution) shown in the table 3 into each centrifuge tube No. 1-4, uniformly mixing, and fully reacting for 15min in a constant-temperature water bath kettle at 25 ℃; then adding 1.5mL xanthine solution, mixing well, reacting in 25 deg.C water bath for 30min, adding 1mL HCl to terminate the reaction, and measuring its absorbance value with ultraviolet spectrophotometer (PBS solution zeroing calibration) at 290 nm.
And (3) processing experimental data:
the concentration is plotted on the abscissa and the inhibition ratio is plotted on the ordinate (fig. 1 and 2), and the concentration IC50 of the half inhibition ratio is calculated.
In the formula:
OD 1-refers to the absorbance value of the assay system containing substrate, enzyme solution and inhibitor;
OD 2-refers to the absorbance value of the assay system containing substrate and enzyme solution, without allopurinol/sample;
OD 3-refers to the absorbance value of the assay system containing substrate and inhibitor, and no enzyme solution;
OD 4: refers to the absorbance value of the assay system containing substrate, no allopurinol/sample and enzyme solution.
The in vitro XOD inhibition of the 18mg/mL pea peptide solution was 74.24% and its half-inhibition IC50 was 12.79mg/mL as determined by the above method; the half inhibition rate IC50 of allopurinol is 0.01 mg/mL.
Example 7
Determination of the clearance of ABTS + free radicals by pea peptides of example 1:
preparing an ABTS solution: ABTS 200.0mg and potassium persulfate 34.4mg are weighed, dissolved in 50.0mL of distilled water, shaken up and placed at room temperature in the dark for 24h to be used as ABTS mother liquor. Taking appropriate amount of ABTS mother liquor, diluting with 95% ethanol until absorbance value is within 0.70 + -0.02 (OD)734) As ABTS assay solution, the solution should be ready for use.
Sample preparation: preparing glutathione/pea peptide mother liquor with different concentrations by using distilled water according to needs, and diluting the mother liquor to different times by using 95% ethanol to obtain solutions to be measured with different concentrations.
2 centrifuge tubes were numbered 1 and 2, respectively, and reagents were added to each tube according to the combinations in Table 3.
TABLE 3 amount of reagent added
A3.6 mL LABTS solution and 0.4mL glutathione solution were added to No. 1 centrifuge tube as an experimental group (A)S) (ii) a Add 3.6mL of LABTS solution and 0.4mL of sample solvent solution to centrifuge tube No. 2 as a blank (Ab); after being mixed well, the mixture is reacted for 5min in a dark place at room temperature, and the absorbance value is measured by an ultraviolet spectrophotometer under the condition of 734nm wavelength (sample solvent is adjusted to zero and calibrated).
And (3) processing experimental data:
in the formula:
p-clearance;
the absorbance of the mixed solution of the Ab-ABTS solution and the sample solvent solution;
as is the absorbance of the mixed solution of the solution to be measured and the ABTS solution.
ABTS + free radical clearance was 63.38% for 1.6mg/mL pea peptide as determined above.
Example 7
Determination of DPPH.radical scavenging ratio of pea peptide of example 1:
preparing a DPPH solution: weighing DPPH 5.0mg, dissolving with appropriate amount of absolute ethyl alcohol, keeping out of the sun and carrying out ultrasonic treatment to fully dissolve, then adding absolute ethyl alcohol to the constant volume of 100.0mL, and preparing into DPPH solution with the volume of 50.0 mu g/mL. The solution should be ready for use.
Sample preparation: and preparing glutathione/pea peptide mother liquor with different concentrations by using distilled water according to requirements, and diluting the mother liquor to different times by using the distilled water to obtain solutions to be measured with different concentrations.
3 centrifuge tubes were numbered 1, 2, 3, respectively, and reagents were added to each tube according to the combinations in Table 4.
TABLE 4 amount of reagent added
| Name of solution | Number 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 |
| Anhydrous ethanol | — | 3.0mL | — |
Note: preparing allopurinol/pea peptide solutions with different concentrations by using PBS according to requirements
The experimental group (A) was prepared by adding 3.0mL of DPPH solution and 1.0mL of glutathione solution to No. 1 centrifuge tubeS) (ii) a Adding 1.0mL of glutathione solution and 3.0mL of absolute ethanol solution into a No. 2 centrifuge tube to serve as a control group (Ac); adding 3.0mL of the LDPPH solution and 1.0mL of the sample solvent solution into a No. 3 centrifuge tube,as blank group (Ab). After being mixed well, the mixture is reacted for 30min in a dark place at room temperature, and the absorbance value of the supernatant is measured by an ultraviolet spectrophotometer under the condition of 734nm wavelength (sample solvent is adjusted to zero and calibrated).
And (3) processing experimental data:
in the formula:
p-clearance;
as is the absorbance of the mixed solution of the solution to be measured and the DPPH solution;
ac-absorbance of a mixed solution of the solution to be detected and the absolute ethyl alcohol solution;
ab-absorbance of the mixture of DPPH solution and sample solvent solution.
The DPPH radical clearance of 30mg/mL pea peptide was 81.04% as determined above.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred 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 above-described embodiments 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 (10)
1. The preparation method of the multifunctional pea peptide is characterized by comprising the following steps:
s1, preparing pea protein feed liquid:
mixing pea protein powder with water to prepare pea protein liquid;
s2, preparing compound protease:
1) fermentation:
inoculating the pea protein feed liquid with bacillus natto for fermentation, centrifuging the fermentation liquid, taking the supernatant, and drying to obtain the composite protease;
2) compounding:
compounding the compound protease with papain, alkaline protease and flavourzyme to prepare compound protease;
s3, enzymolysis:
adding compound protease into pea protein feed liquid as a substrate, stirring for enzymolysis, and inactivating enzyme after the enzymolysis is finished to obtain pea peptidase hydrolysate;
s4, membrane filtration:
centrifuging the pea peptidase hydrolysate to obtain supernatant, and sequentially performing microfiltration, ultrafiltration and nanofiltration membrane filtration to obtain a nanofiltration component with the relative molecular mass of less than 1000 u;
s5, drying:
and drying the nanofiltration component to obtain the pea peptide.
2. The method for preparing a multifunctional pea peptide according to claim 1,
in the step S1, in the step S,
the weight ratio of the pea protein powder to the water is 1:10-1: 25;
the content of the pea protein powder dry-based protein is more than 70 percent.
3. The method for preparing a multifunctional pea peptide according to claim 1,
in the step S2, in the step S,
the inoculation amount of the bacillus natto is 2-5 percent, the pH value is 7.0, the temperature is 37 ℃, the rotating speed is 200-.
4. The method for preparing a multifunctional pea peptide according to claim 1,
in the step S2, in the step S,
the compound protease, the papain, the alkaline protease and the flavor protease are mixed according to the enzyme activity unit proportion of 0.5:1:3: 0.3.
5. The method for preparing a multifunctional pea peptide according to claim 1,
in the step S3, in the step S,
the addition amount of the compound protease is 0.3-0.5% of the weight of the pea protein powder.
6. The method for preparing a multifunctional pea peptide according to claim 1,
in the step S3, in the step S,
performing enzymolysis at 45-55 deg.C at natural pH for 3-5 h;
heating to above 90 deg.C after enzymolysis, and inactivating enzyme for 10-15 min.
7. The method for preparing a multifunctional pea peptide according to claim 1,
in the step S5, in the step S,
and (3) carrying out spray drying on the nanofiltration component, wherein the air inlet temperature is 165-185 ℃, and the air outlet temperature is 65-85 ℃.
8. Use of the multifunctional pea peptides prepared according to the method of any one of claims 1 to 7 for the preparation of a medicament for the treatment of hypertension and its complications.
9. Use of the multifunctional pea peptide prepared by the method according to any one of claims 1 to 7 in the preparation of a product for reducing uric acid or resisting oxidative stress.
10. The use according to claim 9,
the antioxidant includes inhibition of XOD enzyme, scavenging ABTS + free radicals, and scavenging DPPH free radicals.
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| 秦修远 等: "豌豆低聚肽的体外抗氧化作用", 食品工业, vol. 39, no. 12, pages 181 * |
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| WO2023133913A1 (en) | 2023-07-20 |
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